CANCER

Angiogenesis - pathological processes

excessive = - cancer - rheumatoid arthritis - blindness - AIDS complications - psoriasis insufficient = - infertility - scleroderma - Ulcers - Heart disease - Stroke

Telomere Shortening

Telomere shortens with each division cycle chromosome instability replicative senescence cell death Telomere length maintenance: essential for replicative immortality

Loss of Heterozygosity and Mechanisms of Tumor Suppressor Gene Inactivation

"Loss of heterozygosity" is a phrase often used to describe the process that leads to the inactivation of the second copy of a tumor suppressor gene. During this process, a heterozygous cell receives a second hit in its remaining functional copy of the tumor suppressor gene, thereby becoming homozygous for the mutated gene. Mutations that inactivate tumor suppressor genes, called loss-of-function mutations, are often point mutations or small deletions that disrupt the function of the protein that is encoded by the gene; chromosomal deletions or breaks that delete the tumor suppressor gene; or instances of somatic recombination during which the normal gene copy is replaced with a mutant copy.

Insensitivity to Antigrowth Signals

a tumor cell is missing the part of the nucleus that starts and stops cell growth (no brakes) Within a normal tissue, multiple antiproliferative signals operate to maintain cellular quiescence and tissue homeostasis; these signals include both soluble growth inhibitors and immobilized inhibitors embedded in the extracellular matrix and on the surfaces of nearby cells. These growth-inhibitory signals, like their positively acting counterparts, are received by transmembrane cell surface receptors coupled to intracellular signaling circuits. Antigrowth signals can block proliferation by two distinct mechanisms. Cells may be forced out of the active proliferative cycle into the quiescent (G0) state from which they may reemerge on some future occasion when extracellular signals permit. Alternatively, cells may be induced to permanently relinquish their proliferative potential by being induced to enter into postmitotic states, usually associated with acquisition of specific differentiation-associated traits. Incipient cancer cells must evade these antiproliferative signals if they are to prosper. Much of the circuitry that enables normal cells to respond to antigrowth signals is associated with the cell cycle clock, specifically the components governing the transit of the cell through the G1 phase of its growth cycle. Cells monitor their external environment during this period and, on the basis of sensed signals, decide whether to proliferate, to be quiescent, or to enter into a postmitotic state. At the molecular level, many and perhaps all antiproliferative signals are funneled through the retinoblastoma protein (pRb) and its two relatives, p107 and p130. When in a hypophosphorylated state, pRb blocks proliferation by sequestering and altering the function of E2F transcription factors that control the expression of banks of genes essential for progression from G1 into S phase (Weinberg 1995).Disruption of the pRb pathway liberates E2Fs and thus allows cell proliferation, rendering cells insensitive to antigrowth factors that normally operate along this pathway to block advance through the G1 phase of the cell cycle. The effects of the soluble signaling molecule TGFβ are the best documented, but we envision other antigrowth factors will be found to signal through this pathway as well. TGFβ acts in a number of ways, most still elusive, to prevent the phosphorylation that inactivates pRb; in this fashion, TGFβ blocks advance through G1. In some cell types, TGFβ suppresses expression of the c-myc gene, which regulates the G1 cell cycle machinery in still unknown ways (Moses et al. 1990). More directly, TGFβ causes synthesis of the p15INK4B and p21 proteins, which block the cyclin:CDK complexes responsible for pRb phosphorylation (42, 24).The pRb signaling circuit, as governed by TGFβ and other extrinsic factors, can be disrupted in a variety of ways in different types of human tumors (Fynan and Reiss 1993). Some lose TGFβ responsiveness through downregulation of their TGFβ receptors, while others display mutant, dysfunctional receptors (34, 65). The cytoplasmic Smad4 protein, which transduces signals from ligand-activated TGFβ receptors to downstream targets, may be eliminated through mutation of its encoding gene (Schutte et al. 1996). The locus encoding p15INK4B may be deleted (Chin et al. 1998). Alternatively, the immediate downstream target of its actions, CDK4, may become unresponsive to the inhibitory actions of p15INK4B because of mutations that create amino acid substitutions in its INK4A/B-interacting domain; the resulting cyclin D:CDK4 complexes are then given a free hand to inactivate pRb by hyperphosphorylation (Zuo et al. 1996). Finally, functional pRb, the end target of this pathway, may be lost through mutation of its gene. Alternatively, in certain DNA virus-induced tumors, notably cervical carcinomas, pRb function is eliminated through sequestration by viral oncoproteins, such as the E7 oncoprotein of human papillomavirus (Dyson et al. 1989). In addition, cancer cells can also turn off expression of integrins and other cell adhesion molecules that send antigrowth signals, favoring instead those that convey progrowth signals; these adherence-based antigrowth signals likely impinge on the pRb circuit as well. The bottom line is that the antigrowth circuit converging onto Rb and the cell division cycle is, one way or another, disrupted in a majority of human cancers, defining the concept and a purpose of tumor suppressor loss in cancer.Cell proliferation depends on more than an avoidance of cytostatic antigrowth signals. Our tissues also constrain cell multiplication by instructing cells to enter irreversibly into postmitotic, differentiated states, using diverse mechanisms that are incompletely understood; it is apparent that tumor cells use various strategies to avoid this terminal differentiation. One strategy for avoiding differentiation directly involves the c-myconcogene, which encodes a transcription factor. During normal development, the growth-stimulating action of Myc, in association with another factor, Max, can be supplanted by alternative complexes of Max with a group of Mad transcription factors; the Mad-Max complexes elicit differentiation-inducing signals (Foley and Eisenman 1999). However, overexpression of the c-Myc oncoprotein, as is seen in many tumors, can reverse this process, shifting the balance back to favor Myc-Max complexes, thereby impairing differentiation and promoting growth. During human colon carcinogenesis, inactivation of the APC/β-catenin pathway serves to block the egress of enterocytes in the colonic crypts into a differentiated, postmitotic state (Kinzler and Vogelstein 1996). Analogously, during the generation of avian erythroblastosis, the erbAoncogene acts to prevent irreversible erythrocyte differentiation (Kahn et al. 1986).While the components and interconnections between the various antigrowth and differentiation-inducing signals and the core cell cycle machinery are still being delineated, the existence of an antigrowth signaling circuitry is clear (Figure 2), as is the necessity for its circumvention by developing cancers.

Limitless replicative potential

Hallmark: activation of telomerase which maintains normal chromosome length and allows continued division Three acquired capabilities—growth signal autonomy, insensitivity to antigrowth signals, and resistance to apoptosis—all lead to an uncoupling of a cell's growth program from signals in its environment. In principle, the resulting deregulated proliferation program should suffice to enable the generation of the vast cell populations that constitute macroscopic tumors. However, research performed over the past 30 years indicates that this acquired disruption of cell-to-cell signaling, on its own, does not ensure expansive tumor growth. Many and perhaps all types of mammalian cells carry an intrinsic, cell-autonomous program that limits their multiplication. This program appears to operate independently of the cell-to-cell signaling pathways described above. It too must be disrupted in order for a clone of cells to expand to a size that constitutes a macroscopic, life-threatening tumor. The early work of Hayflick demonstrated that cells in culture have a finite replicative potential (reviewed in Hayflick 1997). Once such cell populations have progressed through a certain number of doublings, they stop growing—a process termed senescence. The senescence of cultured human fibroblasts can be circumvented by disabling their pRb and p53 tumor suppressor proteins, enabling these cells to continue multiplying for additional generations until they enter into a second state termed crisis. The crisis state is characterized by massive cell death, karyotypic disarray associated with end-to-end fusion of chromosomes, and the occasional emergence of a variant (1 in 107) cell that has acquired the ability to multiply without limit, the trait termed immortalization (Wright et al. 1989).Provocatively, most types of tumor cells that are propagated in culture appear to be immortalized, suggesting that limitless replicative potential is a phenotype that was acquired in vivo during tumor progression and was essential for the development of their malignant growth state (Hayflick 1997). This result suggests that at some point during the course of multistep tumor progression, evolving premalignant cell populations exhaust their endowment of allowed doublings and can only complete their tumorigenic agenda by breaching the mortality barrier and acquiring unlimited replicative potential.Observations of cultured cells indicate that various normal human cell types have the capacity for 60-70 doublings. Taken at face value, these numbers make little sense when attempting to invoke cell mortality as an impediment to cancer formation: 60-70 doublings should enable clones of tumor cells to expand to numbers that vastly exceed the number of cells in the human body. If clues from evaluation of proliferation and apoptotic rates in certain human tumors (Wyllie et al. 1980) and transgenic mouse models (88, 80, 4) prove generalizable, the paradox can be resolved: evolving premalignant and malignant cell populations evidence chronic, widespread apoptosis and consequently suffer considerable cell attrition concomitant with cell accumulation. Thus, the number of cells in a tumor greatly underrepresents the cell generations required to produce it, raising the generational limit of normal somatic cells as a barrier to cancer.The counting device for cell generations has been discovered over the past decade: the ends of chromosomes, telomeres, which are composed of several thousand repeats of a short 6 bp sequence element. Replicative generations are counted by the 50-100 bp loss of telomeric DNA from the ends of every chromosome during each cell cycle. This progressive shortening has been attributed to the inability of DNA polymerases to completely replicate the 3′ ends of chromosomal DNA during each S phase. The progressive erosion of telomeres through successive cycles of replication eventually causes them to lose their ability to protect the ends of chromosomal DNA. The unprotected chromosomal ends participate in end-to-end chromosomal fusions, yielding the karyotypic disarray associated with crisis and resulting, almost inevitably, in the death of the affected cell (Counter et al. 1992).Telomere maintenance is evident in virtually all types of malignant cells (Shay and Bacchetti 1997); 85%-90% of them succeed in doing so by upregulating expression of the telomerase enzyme, which adds hexanucleotide repeats onto the ends of telomeric DNA (Bryan and Cech 1999), while the remainder have invented a way of activating a mechanism, termed ALT, which appears to maintain telomeres through recombination-based interchromosomal exchanges of sequence information (Bryan et al. 1995). By one or the other mechanism, telomeres are maintained at a length above a critical threshold, and this in turn permits unlimited multiplication of descendant cells. Both mechanisms seem to be strongly suppressed in most normal human cells in order to deny them unlimited replicative potential.The role of telomerase in immortalizing cells can be demonstrated directly by ectopically expressing the enzyme in cells, where it can convey unlimited replicative potential onto a variety of normal early passage, presenescent cells in vitro (7, 92). Further, late passage cells poised to enter crisis continue to proliferate without giving any evidence of crisis when supplied with this enzyme (20, 40, 101). Additional clues into the importance of telomere maintenance for cancer comes from analysis of mice lacking telomerase function. For example, mice carrying a homozygous knockout of the cell cycle inhibitor p16INK4A are tumor prone, particularly when exposed to carcinogens; the tumors that arise show comparatively elevated telomerase activity. When carcinogens were applied to p16INK4A-null mice that also lacked telomerase, tumor incidence was reduced, concomitant with substantial telomere shortening and karyotypic disarray in those tumors that did appear (Greenberg et al. 1999).While telomere maintenance is clearly a key component of the capability for unlimited replication, we remain uncertain about another one, the circumvention of cellular senescence. The phenomenon of senescence was originally observed as a delayed response of primary cells to extended propagation in vitro and has thus been associated with mechanisms of divisional counting (Hayflick 1997). More recently, the senescent state has been observed to be inducible in certain cultured cells in response to high level expression of genes such as the activated ras oncogene (Serrano et al. 1997).The above-cited observations might argue that senescence, much like apoptosis, reflects a protective mechanism that can be activated by shortened telomeres or conflicting growth signals that forces aberrant cells irreversibly into a G0-like state, thereby rendering them incapable of further proliferation. If so, circumvention of senescence in vivo may indeed represent an essential step in tumor progression that is required for the subsequent approach to and breaching of the crisis barrier. But we consider an alternative model equally plausible: senescence could be an artifact of cell culture that does not reflect a phenotype of cells within living tissues and does not represent an impediment to tumor progression in vivo. Resolution of this quandary will be critical to completely understand the acquisition of limitless replicative potential.

Evading apoptosis

-Normally apoptosis triggered in response to normal development and excess growth -Pathway to apoptosis is disabled (mutation of p53) The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation but also by the rate of cell attrition. Programmed cell death—apoptosis—represents a major source of this attrition. The evidence is mounting, principally from studies in mouse models and cultured cells, as well as from descriptive analyses of biopsied stages in human carcinogenesis, that acquired resistance toward apoptosis is a hallmark of most and perhaps all types of cancer. Observations accumulated over the past decade indicate that the apoptotic program is present in latent form in virtually all cell types throughout the body. Once triggered by a variety of physiologic signals, this program unfolds in a precisely choreographed series of steps. Cellular membranes are disrupted, the cytoplasmic and nuclear skeletons are broken down, the cytosol is extruded, the chromosomes are degraded, and the nucleus is fragmented, all in a span of 30-120 min. In the end, the shriveled cell corpse is engulfed by nearby cells in a tissue and disappears, typically within 24 hr (Wyllie et al. 1980).The apoptotic machinery can be broadly divided into two classes of components—sensors and effectors. The sensors are responsible for monitoring the extracellular and intracellular environment for conditions of normality or abnormality that influence whether a cell should live or die. These signals regulate the second class of components, which function as effectors of apoptotic death. The sentinels include cell surface receptors that bind survival or death factors. Examples of these ligand/receptor pairs include survival signals conveyed by IGF-1/IGF-2 through their receptor, IGF-1R, and by IL-3 and its cognate receptor, IL-3R (63, 12). Death signals are conveyed by the FAS ligand binding the FAS receptor and by TNFα binding TNF-R1 (Ashkenazi and Dixit 1999). Intracellular sensors monitor the cell's well-being and activate the death pathway in response to detecting abnormalities, including DNA damage, signaling imbalance provoked by oncogene action, survival factor insufficiency, or hypoxia (Evan and Littlewood 1998). Further, the life of most cells is in part maintained by cell-matrix and cell-cell adherence-based survival signals whose abrogation elicits apoptosis (50, 36). Both soluble and immobilized apoptotic regulatory signals likely reflect the needs of tissues to maintain their constituent cells in appropriate architectural configurations.Many of the signals that elicit apoptosis converge on the mitochondria, which respond to proapoptotic signals by releasing cytochrome C, a potent catalyst of apoptosis (Green and Reed 1998). Members of the Bcl-2 family of proteins, whose members have either proapoptotic (Bax, Bak, Bid, Bim) or antiapoptotic (Bcl-2, Bcl-XL, Bcl-W) function, act in part by governing mitochondrial death signaling through cytochrome C release. The p53 tumor suppressor protein can elicit apoptosis by upregulating expression of proapoptotic Bax in response to sensing DNA damage; Bax in turn stimulates mitochondria to release cytochrome C.The ultimate effectors of apoptosis include an array of intracellular proteases termed caspases (Thornberry and Lazebnik 1998). Two "gatekeeper" caspases, −8 and −9, are activated by death receptors such as FAS or by the cytochrome C released from mitochondria, respectively. These proximal caspases trigger the activation of a dozen or more effector caspases that execute the death program, through selective destruction of subcellular structures and organelles, and of the genome.The possibility that apoptosis serves as a barrier to cancer was first raised in 1972, when Kerr, Wyllie, and Currie described massive apoptosis in the cells populating rapidly growing, hormone-dependent tumors following hormone withdrawal (Kerr et al. 1972). The discovery of the bcl-2 oncogene by its upregulation via chromosomal translocation in follicular lymphoma (reviewed in Korsmeyer 1992) and its recognition as having antiapoptotic activity (Vaux et al. 1988) opened up the investigation of apoptosis in cancer at the molecular level. When coexpressed with a myc oncogene in transgenic mice, the bcl-2 gene was able to promote formation of B cell lymphomas by enhancing lymphocyte survival, not by further stimulating their myc-induced proliferation (Strasser et al. 1990); further, 50% of the infrequent lymphomas arising in bcl-2 single transgenic transgenic mice had somatic translocations activating c-myc, confirming a selective pressure during lymphomagenesis to upregulate both Bcl-2 and c-Myc (McDonnell and Korsmeyer 1991).Further insight into the myc-bcl-2 interaction emerged later from studying the effects of a myc oncogene on fibroblasts cultured in low serum. Widespread apoptosis was induced in myc-expressing cells lacking serum; the consequent apoptosis could be abrogated by exogenous survival factors (e.g., IGF-1), by forced overexpression of Bcl-2 or the related Bcl-XL protein, or by disruption of the FAS death signaling circuit (Hueber et al. 1997). Collectively, the data indicate that a cell's apoptotic program can be triggered by an overexpressed oncogene. Indeed, elimination of cells bearing activated oncogenes by apoptosis may represent the primary means by which such mutant cells are continually culled from the body's tissues.Other examples strengthen the consensus that apoptosis is a major barrier to cancer that must be circumvented. Thus, in transgenic mice where the pRb tumor suppressor was functionally inactivated in the choroid plexus, slowly growing microscopic tumors arose, exhibiting high apoptotic rates; the additional inactivation of the p53 tumor suppressor protein, a component of the apoptotic signaling circuitry, led to rapidly growing tumors containing low numbers of apoptotic cells (Symonds et al. 1994). The role of extracellular survival factors is illustrated by disease progression in transgenic mice prone to pancreatic islet tumors. If IGF-2 gene expression, which is activated in this tumorigenesis pathway, was abrogated using gene knockout mice, tumor growth and progression were impaired, as evidenced by the appearance of comparatively small, benign tumors showing high rates of apoptosis (Christofori et al. 1994). In these cells, the absence of IGF-2 did not affect cell proliferation rates, clearly identifying it as an antiapoptotic survival factor. Collectively, these observations argue that altering components of the apoptotic machinery can dramatically affect the dynamics of tumor progression, providing a rationale for the inactivation of this machinery during tumor development.Resistance to apoptosis can be acquired by cancer cells through a variety of strategies. Surely, the most commonly occurring loss of a proapoptotic regulator through mutation involves the p53 tumor suppressor gene. The resulting functional inactivation of its product, the p53 protein, is seen in greater than 50% of human cancers and results in the removal of a key component of the DNA damage sensor that can induce the apoptotic effector cascade (Harris 1996). Signals evoked by other abnormalities, including hypoxia and oncogene hyperexpression, are also funneled in part via p53 to the apoptotic machinery; these too are impaired at eliciting apoptosis when p53 function is lost (Levine 1997). Additionally, the PI3 kinase-AKT/PKB pathway, which transmits antiapoptotic survival signals, is likely involved in mitigating apoptosis in a substantial fraction of human tumors. This survival signaling circuit can be activated by extracellular factors such as IGF-1/2 or IL-3 (Evan and Littlewood 1998), by intracellular signals emanating from Ras (Downward 1998), or by loss of the pTEN tumor suppressor, a phospholipid phosphatase that normally attenuates the AKT survival signal (Cantley and Neel 1999). Recently, a mechanism for abrogating the FAS death signal has been revealed in a high fraction of lung and colon carcinoma cell lines: a nonsignaling decoy receptor for FAS ligand is upregulated, titrating the death-inducing signal away from the FAS death receptor (Pitti et al. 1998). We expect that virtually all cancer cells harbor alterations that enable evasion of apoptosis.It is now possible to lay out a provisional apoptotic signaling circuitry (Figure 2); while incomplete, it is evident that most regulatory and effector components are present in redundant form. This redundancy holds important implications for the development of novel types of antitumor therapy, since tumor cells that have lost proapoptotic components are likely to retain other similar ones. We anticipate that new technologies will be able to display the apoptotic pathways still operative in specific types of cancer cells and that new drugs will enable cross-talk between the still intact components of parallel apoptotic signaling pathways in tumor cells, resulting in restoration of the apoptotic defense mechanism, with substantial therapeutic benefit.

angiogenesis

Angiogenesis is involved in both normal and pathological processes.

Sustained angiogenesis

Stimulate angiogenesis to form their own blood supply- delivers O2 and nutrients The oxygen and nutrients supplied by the vasculature are crucial for cell function and survival, obligating virtually all cells in a tissue to reside within 100 μm of a capillary blood vessel. During organogenesis, this closeness is ensured by coordinated growth of vessels and parenchyma. Once a tissue is formed, the growth of new blood vessels—the process of angiogenesis—is transitory and carefully regulated. Because of this dependence on nearby capillaries, it would seem plausible that proliferating cells within a tissue would have an intrinsic ability to encourage blood vessel growth. But the evidence is otherwise. The cells within aberrant proliferative lesions initially lack angiogenic ability, curtailing their capability for expansion. In order to progress to a larger size, incipient neoplasias must develop angiogenic ability ( 8, 41, 32).Counterbalancing positive and negative signals encourage or block angiogenesis. One class of these signals is conveyed by soluble factors and their receptors, the latter displayed on the surface of endothelial cells; integrins and adhesion molecules mediating cell-matrix and cell-cell association also play critical roles. The angiogenesis-initiating signals are exemplified by vascular endothelial growth factor (VEGF) and acidic and basic fibroblast growth factors (FGF1/2). Each binds to transmembrane tyrosine kinase receptors displayed by endothelial cells (29, 93). A prototypical angiogenesis inhibitor is thrombospondin-1, which binds to CD36, a transmembrane receptor on endothelial cells coupled to intracellular Src-like tyrosine kinases (Bull et al. 1994). There are currently more than two dozen angiogenic inducer factors known and a similar number of endogenous inhibitor proteins.Integrin signaling also contributes to this regulatory balance. Quiescent vessels express one class of integrins, whereas sprouting capillaries express another. Interference with signaling from the latter class of integrins can inhibit angiogenesis (90, 36), underscoring the important contribution of cell adhesion to the angiogenic program (Hynes and Wagner 1996). Extracellular proteases are physically and functionally connected with proangiogenic integrins, and both help dictate the invasive capability of angiogenic endothelial cells (Stetler-Stevenson 1999).Experimental evidence for the importance of inducing and sustaining angiogenesis in tumors is both extensive and compelling (8, 41, 32). The story begins almost 30 years ago with Folkman and colleagues, who used in vivo bioassays to demonstrate the necessity of angiogenesis for explosive growth of tumor explants (reviewed in Folkman 1997). Molecular proof of principle came, for example, when anti-VEGF antibodies proved able to impair neovascularization and growth of subcutaneous tumors in mice (Kim et al. 1993), as did a dominant-interfering version of the VEGF receptor 2 (flk-1) (Millauer et al. 1994); both results have motivated the development of specific VEGF/VEGF-R inhibitors now in late stage clinical trials.The essential role of angiogenesis is further supported by the ability of an increasing catalog of antiangiogenic substances to impair the growth of tumor cells inoculated subcutaneously in mice (Folkman 1997). Tumors arising in cancer-prone transgenic mice are similarly susceptible to angiogenic inhibitors (Bergers et al. 1999).The ability to induce and sustain angiogenesis seems to be acquired in a discrete step (or steps) during tumor development, via an "angiogenic switch" from vascular quiescence. When three transgenic mouse models were analyzed throughout multistep tumorigenesis, in each case angiogenesis was found to be activated in midstage lesions, prior to the appearance of full-blown tumors. Similarly, angiogenesis can be discerned in premalignant lesions of the human cervix, breast, and skin (melanocytes) (Hanahan and Folkman 1996); we expect that induction of angiogenesis will prove to be an early to midstage event in many human cancers. These observations, taken together with the effects of angiogenesis inhibitors, indicate that neovascularization is a prerequisite to the rapid clonal expansion associated with the formation of macroscopic tumors.Tumors appear to activate the angiogenic switch by changing the balance of angiogenesis inducers and countervailing inhibitors (Hanahan and Folkman 1996). One common strategy for shifting the balance involves altered gene transcription. Many tumors evidence increased expression of VEGF and/or FGFs compared to their normal tissue counterparts. In others, expression of endogenous inhibitors such as thrombospondin-1 or β-interferon is downregulated. Moreover, both transitions may occur, and indeed be linked, in some tumors (82, 94).The mechanisms underlying shifts in the balances between angiogenic regulators remain incompletely understood. In one well-documented example, the inhibitor thrombospondin-1 has been found to positively regulated by the p53 tumor suppressor protein in some cell types. Consequently, loss of p53 function, which occurs in most human tumors, can cause thrombospondin-1 levels to fall, liberating endothelial cells from its inhibitory effects (Dameron et al. 1994). The VEGF gene is also under complex transcriptional control. For example, activation of the ras oncogene or loss of the VHL tumor suppressor gene in certain cell types causes upregulation of VEGF expression (75, 66).Another dimension of regulation is emerging in the form of proteases, which can control the bioavailability of angiogenic activators and inhibitors. Thus, a variety of proteases can release bFGF stored in the ECM (Whitelock et al. 1996), whereas plasmin, a proangiogenic component of the clotting system, can cleave itself into an angiogenesis inhibitor form called angiostatin (Gately et al. 1997). The coordinated expression of pro- and antiangiogenic signaling molecules, and their modulation by proteolysis, appear to reflect the complex homeostatic regulation of normal tissue angiogenesis and of vascular integrity.As is already apparent, tumor angiogenesis offers a uniquely attractive therapeutic target, indeed one that is shared in common by most and perhaps all types of human tumors. The next decade will produce a catalog of the angiogenic regulatory molecules expressed by different types of tumors, and in many cases, by their progenitor stages. Use of increasingly sophisticated mouse models will make it possible to assign specific roles to each of these regulators and to discern the molecular mechanisms that govern their production and activity. Already available evidence indicates that different types of tumor cells use distinct molecular strategies to activate the angiogenic switch. This raises the question of whether a single antiangiogenic therapeutic will suffice to treat all tumor types, or whether an ensemble of such therapeutics will need to be developed, each responding to a distinct program of angiogenesis that has been developed by a specific class of human tumors.

Invasion & Metastasis

1. Cancer cells invade surrounding tissues and blood vessels 2. Cancer cells are transported by the circulatory system to distant sites 3. Cancer cells reinvade and grow at new location Two Components 1. Changes in the physical coupling of cells. 2. Activation of extracellular proteases.

Herceptin's Dual-Kill Mechanism

1. Antibody Dependent Cellular Toxicity (ADCC) - Attached herceptin signals immune destruction 2. Prevents Intracellular Cell Signaling - Induces Apoptosis- Prevents Proliferation

Essential Contributors to Metastasis

1. Attachmenttodifferenttissues Cadherins, Desmosomes 2. Degradation of extracellular matrix Matrix Metalloproteinases 3. Angiogenesis VEGF Lymphangiogenesis

Primary Tumour Growth

1. Breaking free from the tumour mass. Requires: Motility, Altered adhesion & Degradation of ECM. 2. Intravasation. Requires: Binding to epithelium. 3. Survival in circulation. Requires: Lack of adhesion. 4. Extravasation. Requires: Binding to epithelium, motility. 5. Growth at secondary site. Requires: Reformed cellular adhesion, loss of motility

Cell junctions can fail as a result of mutations that alter adhesion molecules.

A loss of function allele of a cadherin for example, can lead a cell to lose its adhesion to proper neighbours. The cell can start to wander.

E-Cadherin Mutations alter Cell Adhesion

A major function of the cell-to-cell adhesion molecule E-cadherin is the maintenance of cell adhesion and tissue integrity. E-cadherin deficiency in tumours leads to changes in cell morphology and motility, so that E-cadherin is considered to be a suppressor of invasion. In this study we investigated the functional consequences of three tumour-associated gene mutations that affect the extracellular portion of E-cadherin: in-frame deletions of exons 8 or 9 and a point mutation in exon 8, as they were found in human gastric carcinomas. Human MDA-MB-435S breast carcinoma cells and mouse L fibroblasts were stably transfected with the wild-type and mutant cDNAs, and the resulting changes in localization of E-cadherin, cell morphology, strength of calcium-dependent aggregation as well as cell motility and actin cytoskeleton organization were studied. We found that cells transfected with wild-type E-cadherin showed an epitheloid morphology, while all cell lines expressing mutant E-cadherin exhibited more irregular cell shapes. Cells expressing E-cadherin mutated in exon 8 showed the most scattered appearance, whereas cells with deletion of exon 9 had an intermediate state. Mutant E-cadherins were localized to the lateral regions of cell-to-cell contact sites. Additionally, both exon 8-mutated E-cadherins showed apical and perinuclear localization, and actin filaments were drastically reduced. MDA-MB-435S cells with initial calcium-dependent cell aggregation exhibited decreased aggregation and, remarkably, increased cell motility, when mutant E-cadherin was expressed. Therefore, we conclude that these E-cadherin mutations may not simply affect cell adhesion but may act in a trans-dominant-active manner, i.e. lead to increased cell motility. Our study suggests that E-cadherin mutations affecting exons 8 or 9 are the cause of multiple morphological and functional disorders and could induce the scattered morphology and the invasive behaviour of diffuse type-gastric carcinomas.

Anti-angiogenesis:

A process or agent that prevents new blood vessel growth. •Thrombospondin-1 •Angiostatin •Endostatin

Polyp Formation

APC is expressed in colorectal epithelial cells • There is no increase in the rate of cell proliferation in the early stage of polyp formation • Polyps represent and increase in the size of the proliferating crypt compartment

Epigenetics and Cancer

Abnormal DNA methylation is associate with many types of cancers Epigenetic changes to DNA can alter gene expression and contribute to cancer

Are there any unusual cases where e.g. prostate tumours have ended up somewhere other than bone?

About 80 percent of the time prostate cancer cells metastasize, they will spread to bones.

Epigenetics

Age is the leading risk factor for cancer, normally associated with accumulation of mutations. The new suggestion is that it might be predominantly due to epigenetic changes which leads to increased mutations. One of the biggest challenges in this field: Designing the experiments that allow us to look at/ measure/ quantify these alterations in vivo.

Factors Associated with an Increased Risk of Breast Cancer

Age of menarche, first child, onset menopause Diet, level of exercise, obesity, alcohol consumption Presence of benign breast disease (DCIS) Exposure to radiation Exogenous hormones • Family history and genetics (estimated 5% of total cases can be contributed to genetic factors)

hypoxia

Among the first responses at the onset of hypoxia is an increase in the protein levels of Hypoxia Inducible Factor-1 (HIF-1). The oxygen and nutrients display a gradient away from the necrotic core.

The Debate of Organ Specificity II

Anatomical 1920's: James Ewing challenged that it is the anatomic circulatory patterns between the tumour and the metastatic site that determines metastatic spread.

There can be large benign tumours like a lipoma. What makes a malignant primary tumour different from a benign tumour? (from a benign tumour to grow it must also induce angiogenesis).

Angiogenesis in benign tumours is very slow compared to malignant tumours. Benign tumour cells still have stong adherence to their neighbours.

Therapeutic Approaches

Anti-angiogenesis strategies: • Inhibit endogenous pro-angiogenic factors, such as VEGF. Inhibit degradative enzymes (Like MMPs). Inhibit endothelial cell proliferation. Inhibit endothelial cell migration. Inhibit the activation and differentiation of endothelial cells. There is an urgent need for a non-invasive method to accurately determine the different stages of tumour angiogenesis.

HER2-positive clinical impact

Associated with poor outcomes: - ↑ Distant metastases- ↑ Nodes + disease- ↑ Highest risk of recurrence ↓Overall Survival- High grade tumours- Endocrine therapy resistance

biogenesis

As many as 40% of miRNA genes may lie in the introns or even exons of other genes.[42] These are usually, though not exclusively, found in a sense orientation,[43][44] and thus usually are regulated together with their host genes. The DNA template is not the final word on mature miRNA production: 6% of human miRNAs show RNA editing (IsomiRs), the site-specific modification of RNA sequences to yield products different from those encoded by their DNA. This increases the diversity and scope of miRNA action beyond that implicated from the genome alone. transcription : miRNA genes are usually transcribed by RNA polymerase II (Pol II).[47][48] The polymerase often binds to a promoter found near the DNA sequence, encoding what will become the hairpin loop of the pre-miRNA. The resulting transcript is capped with a specially modified nucleotide at the 5' end, polyadenylated with multiple adenosines (a poly(A) tail),[47][43] and spliced. Animal miRNAs are initially transcribed as part of one arm of an ∼80 nucleotide RNA stem-loop that in turn forms part of a several hundred nucleotide-long miRNA precursor termed a primary miRNA (pri-miRNA).[47][43] When a stem-loop precursor is found in the 3' UTR, a transcript may serve as a pri-miRNA and a mRNA.[43] RNA polymerase III (Pol III) transcribes some miRNAs, especially those with upstream Alu sequences, transfer RNAs (tRNAs), and mammalian wide interspersed repeat (MWIR) promoter units Nuclear processing : Nuclear processing[edit] A crystal structure of the human Drosha protein in complex with the C-terminal helices of two DGCR8molecules (green). Drosha consists of two ribonuclease III domains (blue and orange); a double-stranded RNA binding domain (yellow); and a connector/platform domain (gray) containing two bound zinc ion (spheres). From PDB: 5B16​. A single pri-miRNA may contain from one to six miRNA precursors. These hairpin loop structures are composed of about 70 nucleotides each. Each hairpin is flanked by sequences necessary for efficient processing. The double-stranded RNA (dsRNA) structure of the hairpins in a pri-miRNA is recognized by a nuclear protein known as DiGeorge Syndrome Critical Region 8 (DGCR8 or "Pasha" in invertebrates), named for its association with DiGeorge Syndrome. DGCR8 associates with the enzyme Drosha, a protein that cuts RNA, to form the Microprocessor complex.[50][51] In this complex, DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs by cleaving RNA about eleven nucleotides from the hairpin base (one helical dsRNA turn into the stem).[52][53] The product resulting has a two-nucleotide overhang at its 3' end; it has 3' hydroxyl and 5' phosphate groups. It is often termed as a pre-miRNA (precursor-miRNA). Sequence motifs downstream of the pre-miRNA that are important for efficient processing have been identified.[54][55][56] Pre-miRNAs that are spliced directly out of introns, bypassing the Microprocessor complex, are known as "Mirtrons." Originally thought to exist only in Drosophila and C. elegans, mirtrons have now been found in mammals.[57] As many as 16% of pre-miRNAs may be altered through nuclear RNA editing.[58][59][60] Most commonly, enzymes known as adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine (A to I) transitions. RNA editing can halt nuclear processing (for example, of pri-miR-142, leading to degradation by the ribonuclease Tudor-SN) and alter downstream processes including cytoplasmic miRNA processing and target specificity (e.g., by changing the seed region of miR-376 in the central nervous system

Intergenerational Impact

BACKGROUND: The involvement of epigenetic mechanisms in intergenerational transmission of stress effects has been demonstrated in animals but not in humans. METHODS: Cytosine methylation within the gene encoding for FK506 binding protein 5 (FKBP5) was measured in Holocaust survivors (n = 32), their adult offspring (n = 22), and demographically comparable parent (n = 8) and offspring (n = 9) control subjects, respectively. Cytosine-phosphate-guanine sites for analysis were chosen based on their spatial proximity to the intron 7 glucocorticoid response elements. RESULTS: Holocaust exposure had an effect on FKBP5 methylation that was observed in exposed parents as well in their offspring. These effects were observed at bin 3/site 6. Interestingly, in Holocaust survivors, methylation at this site was higher in comparison with control subjects, whereas in Holocaust offspring, methylation was lower. Methylation levels for exposed parents and their offspring were significantly correlated. In contrast to the findings at bin 3/site 6, offspring methylation at bin 2/sites 3 to 5 was associated with childhood physical and sexual abuse in interaction with an FKBP5 risk allele previously associated with vulnerability to psychological consequences of childhood adversity. The findings suggest the possibility of site specificity to environmental influences, as sites in bins 3 and 2 were differentially associated with parental trauma and the offspring's own childhood trauma, respectively. FKBP5 methylation averaged across the three bins examined was associated with wake-up cortisol levels, indicating functional relevance of the methylation measures. CONCLUSIONS: This is the first demonstration of an association of preconception parental trauma with epigenetic alterations that is evident in both exposed parent and offspring, providing potential insight into how severe psychophysiological trauma can have intergenerational effects Methylation levels of gene encoding for FK506-binding-protein-5 was measured in holocaust survivors and their offspring. 3 generations looked at. Methylation levels were highest in F0 but present in F1. Why is this interesting? Parental trauma exposure is associated with greater risk of PTSD, mood and anxiety disorders in their offspring. FKBP5 a regulator of glucocorticoid receptor (GR) - it decreases glucocorticoid binding to GR. This has an effect on developmental processes. The trauma impact is being passed on through methylation of specific genes.

metastasis

Cancer 5 Metastasis: By the end of this lecture students should be able to: Appreciate the impact metastatic spread has on tumour therapy and survivability rates. Discuss the genetic and physical changes required for a tumour cell to become metastatic. Discuss patterns of metastatic spread observed in solid tumours.

Avastin ( Anti-VEGF antibody )

Bevacizumab, sold under the brand name Avastin, is a humanized monoclonal antibody that binds VEGF and blocks its binding to its signaling receptor, VEGFreceptor 2, and is used to treat patients with a variety of cancers

Cadherin-mediated cell-cell adhesion

Cadherins bind the MF cytoskeleton of one cell to that of its neighbours, forming a mechanical unit. This coupling contributes to the mechanical integrity of a tissue

E-cadherin Summary

Calcium dependent function Expression drives formation of cell sheets (responsible for intermediate junctions) Associates with actin cytoskeleton via catenins Down regulation in tumour progression

aims

Cancer 6 Epigenomics: By the end of this lecture students should be able to: • Define epigenetics and relate its influence within the context of cancer genetics. Describe the mechanisms behind the 4 main types of epigenetic inheritance. Discuss the current level of understanding of epigenetic inheritance.

Why is cancer regarded as a disease of the elderly?

Cancer can be considered an age-related disease because the incidence of most cancers increases with age,2 rising more rapidly beginning in midlife. ... Some of the same biologic mechanisms that regulate aging also may be involved in the pathogenesis of age-related diseases such as cancer

Oestrogen-Induced Proliferation of Existing Mutant Cells

Cancer is caused by DNA damage (i.e., mutations) in genes that regulate cell growth and division. Some mutations are inherited, while others are caused by exposure to radiation or to mutation-inducing chemicals such as those found in cigarette smoke. Mutations also can occur spontaneously as a result of mistakes that are made when a cell duplicates its DNA molecules prior to cell division. When cells acquire mutations in specific genes that control proliferation, such as proto-oncogenes or tumor suppressor genes, these changes are copied with each new generation of cells. Later, more mutations in these altered cells can lead to uncontrolled proliferation and the onset of cancer. (For more information on how gene mutations cause cancer, see Understanding Cancer.) Although estrogen does not appear to directly cause the DNA mutations that trigger the development of human cancer, estrogen does stimulate cell proliferation. Therefore, if one or more breast cells already possesses a DNA mutation that increases the risk of developing cancer, these cells will proliferate (along with normal breast cells) in response to estrogen stimulation. The result will be an increase in the total number of mutant cells, any of which might thereafter acquire the additional mutations that lead to uncontrolled proliferation and the onset of cancer. In other words, estrogen-induced cell production leads to an increase in the total number of mutant cells that exist. These cells are at increased risk of becoming cancerous, so the chances that cancer may actually develop are increased.

Group 3: Cell Proliferation/ Viability

Cell Proliferation/ Viability Insulin-like growth factor IGF binding protein p35srj

What happens when cell junctions are broken or weakened?

Characterisation of cadherins is important for our understanding of metatstasis Thus, we can either compare properties of metastatic cells or set up experiments that knock out known components of cell adhesion and see how their loss compares with the onset of metastasis.

DNA Methylation and Cancer

DNA methylation is probably the best understood and was first investigated in cancers by Feinberg and Vogelstein in 1980s. Investigating how changes to a tumours microenvironment impacted on the frequency of mutations in tumour cells. They were the first to show that sites where normal tissue showed methylation were unmethylated in all the tumour types they tested. i.e. tumour cells had lost the gene control that methylation allows. Hypomethylation.

Epigenetics

DNAMethylation: Occurs almost exclusively at CpG nucleotides. Linked directly to the regulation of gene expression and the silencing of repeat elements in the genome. Genomic Imprinting : The silencing of one parental allele compared with the other parental allele. 3. HistoneModifications : Important in transcriptional regulation and are normally rigidly maintained and regulated during cell division. Noncoding RNAs: MicroRNAs (miRNAs) playing an important role in gene regulation and control.

Transformation

Definition: cultured cells that become able to divide indefinitely and undergo various other changes characteristic of a potential cancer cell NIH 3T3 Transformed • Much higher cell density• Cells often poorly spread • Colonies form in soft agar • Grow in very low serum Typically highly tumourigenic - e.g. less than 10 cells may cause tumour formation in animals.

risk factors for colorectal cancer

Diabetes, alcohol use, obesity, smoking, high-fat diet

Oestrogen and Breast Cancer

During each menstrual cycle, estrogen normally triggers the proliferation of cells that form the inner lining of the milk glands in the breast. If pregnancy does not occur, estrogen levels fall dramatically at the end of each monthly menstrual cycle. In the absence of high estrogen levels, those milk gland cells that have proliferated in any given month will deteriorate and die, followed by a similar cycle of cell proliferation and cell death the following month. For the average woman, this means hundreds of cycles of breast cell division and cell death repeated over a span of roughly 40 years, from puberty to menopause. But how do these estrogen-induced cycles of breast cell proliferation increase the risk of developing cancer?

What is the difference between E Cadherin and Cadherin?

E-cadherin is one of the most important molecules in cell-cell adhesion in epithelial tissues. Cadherin-1 is the same as E- Cadherin.

Endogenous Angiogenesis Inhibitors experiment 1

Endostatin injections into mice with multiple tumours. Primary tumours decreased in size and stopped growing. Tumours did not develop resistance to endostatin. Conclusion: Addition of angiogenesis inhibitors halts primary tumour growth.

What does HIF-1 do?

Enhances the ability to make ATP anaerobically and stimulates new blood vessel growth. Helps normal tissues as well as tumours to survive under hypoxic conditions. HIF-1 is a transcription factor that turns on genes that are needed for survival under hypoxic conditions. So far, more than 40 target genes have been found to be regulated by HIF-1. These genes can be classified into 3 main groups.

group : Glucose/Energy metabolism

EnolaseGlucose Transporter 1Glyceraldehyde phosphate dehydrogenase HexokinaseLactate dehydrogenase PhosphofructokinasePhosphoglycerate kinasePyruvate kinase

The Debate of Organ Specificity I

Environmental 1889: Stephen Paget's Seed and Soil Hypothesis. The "seed" (cancer cell) will grow only when it falls on congenial "soil" (factors in the organs environment). Certain organs are better for certain cancer cells.

Epigenetics a

Epigenetics and developmental biology are closely related. You should be aware that:Someone's phenotype arises due to the sequential activation and deactivation of genetic information within the genome. This is highly influenced by the environmental stimuli present. E.g. Genes within skeletal muscle can be shown to respond quickly after the start of exercise. Therefore: The study of epigenetic inheritance accounts for the relationship between an individuals genetic background, their environment and other aspects like aging and disease.

Oestrogen Receptors

Estrogens act on target tissues by binding to parts of cells called estrogen receptors. An estrogen receptor is a protein molecule found inside those cells that are targets for estrogen action. Estrogen receptors contain a specific site to which only estrogens (or closely related molecules) can bind. The target tissues affected by estrogen molecules all contain estrogen receptors; other organs and tissues in the body do not. Therefore, when estrogen molecules circulate in the bloodstream and move throughout the body, they exert effects only on cells that contain estrogen receptors. Estrogen receptors normally reside in the cell's nucleus, along with DNA molecules. In the absence of estrogen molecules, these estrogen receptors are inactive and have no influence on DNA (which contains the cell's genes). But when an estrogen molecule enters a cell and passes into the nucleus, the estrogen binds to its receptor, thereby causing the shape of the receptor to change. This estrogen-receptor complex then binds to specific DNA sites, called estrogen response elements, which are located near genes that are controlled by estrogen. After it has become attached to estrogen response elements in DNA, this estrogen-receptor complex binds to coactivator proteins and more nearby genes become active. The active genes produce molecules of messenger RNA, which guide the synthesis of specific proteins. These proteins can then influence cell behavior in different ways, depending on the cell type involved.

Why does Prostate Cancer Metastasize to Bone?

Ewing (Anatomic) Vs Paget (seed & soil) Current data convincingly shows that prostate cancer cells disseminate widely throughout the body. • However, these disseminated cells seem to form metastases only in the bone environment. • The "seeds" are scattered everywhere, but prefer to grow in the bone environment ("soil").

cancer cell cultures

Finite number of division cycles • Must spread on surface to survive and/or grow • Require hormone-like growth factors to survive/grow

How does HIF-1 do the Job?

HIF-1 expression increasesExponentially when O2 Concentration decreases. The curve shows a point of Inflection around 4-5% O2, Which is the O2 concentration In normal human tissues.

Self-sufficiency in growth signals

Hallmark: ability to promote cell growth in absence of normal growth-promoting signals Normal cells require mitogenic growth signals (GS) before they can move from a quiescent state into an active proliferative state. These signals are transmitted into the cell by transmembrane receptors that bind distinctive classes of signaling molecules: diffusible growth factors, extracellular matrix components, and cell-to-cell adhesion/interaction molecules. To our knowledge, no type of normal cell can proliferate in the absence of such stimulatory signals. Many of the oncogenes in the cancer catalog act by mimicking normal growth signaling in one way or another. Dependence on growth signaling is apparent when propagating normal cells in culture, which typically proliferate only when supplied with appropriate diffusible mitogenic factors and a proper substratum for their integrins. Such behavior contrasts strongly with that of tumor cells, which invariably show a greatly reduced dependence on exogenous growth stimulation. The conclusion is that tumor cells generate many of their own growth signals, thereby reducing their dependence on stimulation from their normal tissue microenvironment. This liberation from dependence on exogenously derived signals disrupts a critically important homeostatic mechanism that normally operates to ensure a proper behavior of the various cell types within a tissue. Acquired GS autonomy was the first of the six capabilities to be clearly defined by cancer researchers, in large part because of the prevalence of dominant oncogenes that have been found to modulate it. Three common molecular strategies for achieving autonomy are evident, involving alteration of extracellular growth signals, of transcellular transducers of those signals, or of intracellular circuits that translate those signals into action. While most soluble mitogenic growth factors (GFs) are made by one cell type in order to stimulate proliferation of another—the process of heterotypic signaling—many cancer cells acquire the ability to synthesize GFs to which they are responsive, creating a positive feedback signaling loop often termed autocrine stimulation (Fedi et al. 1997). Clearly, the manufacture of a GF by a cancer cell obviates dependence on GFs from other cells within the tissue. The production of PDGF (platelet-derived growth factor) and TGFα (tumor growth factor α) by glioblastomas and sarcomas, respectively, are two illustrative examples (Fedi et al. 1997).The cell surface receptors that transduce growth-stimulatory signals into the cell interior are themselves targets of deregulation during tumor pathogenesis. GF receptors, often carrying tyrosine kinase activities in their cytoplasmic domains, are overexpressed in many cancers. Receptor overexpression may enable the cancer cell to become hyperresponsive to ambient levels of GF that normally would not trigger proliferation (Fedi et al. 1997). For example, the epidermal GF receptor (EGF-R/erbB) is upregulated in stomach, brain, and breast tumors, while the HER2/neu receptor is overexpressed in stomach and mammary carcinomas (84, 100). Additionally, gross overexpression of GF receptors can elicit ligand-independent signaling (DiFiore et al. 1987). Ligand-independent signaling can also be achieved through structural alteration of receptors; for example, truncated versions of the EGF receptor lacking much of its cytoplasmic domain fire constitutively (Fedi et al. 1997).Cancer cells can also switch the types of extracellular matrix receptors (integrins) they express, favoring ones that transmit progrowth signals (64, 36). These bifunctional, heterodimeric cell surface receptors physically link cells to extracellular superstructures known as the extracellular matrix (ECM). Successful binding to specific moieties of the ECM enables the integrin receptors to transduce signals into the cytoplasm that influence cell behavior, ranging from quiescence in normal tissue to motility, resistance to apoptosis, and entrance into the active cell cycle. Conversely, the failure of integrins to forge these extracellular links can impair cell motility, induce apoptosis, or cause cell cycle arrest (Giancotti and Ruoslahti 1999). Both ligand-activated GF receptors and progrowth integrins engaged to extracellular matrix components can activate the SOS-Ras-Raf-MAP kinase pathway (1, 36).The most complex mechanisms of acquired GS autonomy derive from alterations in components of the downstream cytoplasmic circuitry that receives and processes the signals emitted by ligand-activated GF receptors and integrins. The SOS-Ras-Raf-MAPK cascade plays a central role here. In about 25% of human tumors, Ras proteins are present in structurally altered forms that enable them to release a flux of mitogenic signals into cells, without ongoing stimulation by their normal upstream regulators (Medema and Bos 1993).We suspect that growth signaling pathways suffer deregulation in all human tumors. Although this point is hard to prove rigorously at present, the clues are abundant (Hunter 1997). For example, in the best studied of tumors—human colon carcinomas—about half of the tumors bear mutant ras oncogenes (Kinzler and Vogelstein 1996). We suggest that the remaining colonic tumors carry defects in other components of the growth signaling pathways that phenocopy ras oncogene activation. The nature of these alternative, growth-stimulating mechanisms remains elusive.Under intensive study for two decades, the wiring diagram of the growth signaling circuitry of the mammalian cell is coming into focus (Figure 2). New downstream effector pathways that radiate from the central SOS-Ras-Raf-MAP kinase mitogenic cascade are being discovered with some regularity (48, 77). This cascade is also linked via a variety of cross-talking connections with other pathways; these cross connections enable extracellular signals to elicit multiple cell biological effects. For example, the direct interaction of the Ras protein with the survival-promoting PI3 kinase enables growth signals to concurrently evoke survival signals within the cell

Cediranib (RECENTINTM)

Highly potential inhibitor of VEGFR tyrosine kinase activity and inhibits VEGF-dependent signaling and angiogenesis. Reduces tumour blood flow and overall size of the tumour.

Histone Modifications and Cancer

Histones are proteins that associate with DNA and help condense it into chromatin. In the majority of cancers aberrant patterns of histone modifications are being found. Tumour types can now also be categorised on the basis of the expression patterns of their histone- modifying enzymes. These modifications lead to activation or inactivation of histone which plays a vital role in gene regulation. There is the suggestion that patterns of these modifications can be linked to specific cancer types. There is also some recent findings that suggest these patterns may act as indicators for risk of tumour reaccurance.

DNA Methylation and Cancer

Hypomethylation has now been demonstrated in cancers of the: stomach kidney colon pancreas liver uterus lung cervix Hypomethylation has also recently been linked to chromosomal instability and may be one of the primary mechanisms of carcinogenic compounds.

DNA Methylation and Cancer

Hypomethylation is also being linked to how cancer cells respond to drugs, toxins etc. E.g. Hypomethylation of the multidrug-resistance gene (MDR1) leads to increased expression of the gene and increased resistance to drug treatments in acute myelogenous leukaemia. There is a lot of research continuing to investigate if this methylation can be artificially restored to allow for cell cycle to be regained Hypermethylation of gene promoters has also been linked to the inactivation of tumour suppressor genes. Drugs are available that inactivate methyltransferases. These are being used in some chemotherapy regimes, mainly for leukaemias. There is some evidence that these drugs help to reactivate growth suppressors (e.g. P15). However these options are still in their infancy and are generally only justified in patients where all other anti-cancer treatments have failed, or are expected to fail.

embryonic development ( following vasculogenesis)

In addition to its role in tumors, angiogenesis occurs normally in the human body at specific times in development and growth. For example, a developing child in a mother's womb must create the vast network of arteries, veins, and capillaries that are found in the human body. A process called vasculogenesis creates the primary network of vascular endothelial cells that will become major blood vessels. Later on, angiogenesis remodels this network into the small new blood vessels or capillaries that complete the child's circulatory system.

Must spread on surface to survive and/or grow

In vivo loss of this characteristic may contribute to metastatic capability of tumour cells. normal cells = anchorage dependent, require a surface for division density dependent inhibition= cells forma single layer, they divide to fill a gap and then stop

VEGF - Functions

Induces proliferation of vascular epithelial cells Stimulates endothelial cells to release matrix metalloproteinases. Increases blood vessel permeability (aids in degradation of the basal membrane. Cancer cells secrete VEGF or bFGF these bind to receptors on endothelial cells that induce relay proteins to activate genes in the nucleus and secretion of new endothelial cell growth is stimulated

Therapeutic Approaches

Inhibit signaling factors involved in stimulating angiogenesis. Anti-VEGF antibodies bind to the receptor tyrosine kinase,preventing the binding and downstream action of VEGF A second group of angiogenesis inhibitors being tested in human clinical trials are molecules that interfere with steps in the angiogenesis signaling cascade. Included in this category are anti-VEGF antibodies that block the VEGF receptor from binding growth factor. Bevacizumab (Avastin), a monoclonal antibody, is the first of these anti-VEGF antibodies to be FDA-approved. This new drug has been proven to delay tumor growth and more importantly, to extend the lives of patients. Another agent, interferon-alpha, is a naturally occurring protein that inhibits the production of bFGF and VEGF, preventing these growth factors from starting the signaling cascade. Also, several synthetic drugs capable of interfering with endothelial cell receptors are being tested in cancer patients.

Epigenetics

It is becoming more and more apparent that alteration to the epigenetic inheritance in a cell is key to our understanding of the differences between: Senescent & Immortal cells Normal & tumour cells Differentiated & Non-differentiated Ageing & Diseased cells The identification of these differences offers novel approaches for treatment.

The let-7 family controlscell proliferation and differentiation

Let-7 first miRNA discovered to regulate differentiation in Ceanorhabditis elegans. Highly conserved during evolution and ubiquitous. Mammalian Let-7 family has 12 members: let-7-a1, a2, a3, b, c, d, e, f1, f2, g, I, miR-98). Probably targets overlapping sets of mRNAs. e.g. CDC25A, CDK6 (cell cycle regulators), RAS, c-myc (oncogenes), HMGA2, Mlin-41, IMP-1(embryonic development).

Moving through

Locomotion is essential for metastasis, cells achieve this with the use of arms (pseudopedia).

Tumour Angiogenesis

Long-standing research shows that tumours cannot grow past about 1-2mm3 without their own blood vessel supply. These findings have instigated research of angiogenesis inhibitors to treat cancer. Traditional therapies target proliferating cells.

Genomic Imprinting and Cancer

Loss of Imprinting is defined as "the loss of parental allele-specific monoallelic expression of genes due to aberrant hypomethylation profiles at one of the two parental alleles." E.g. IGF2 - loss of imprinting has been shown to give an increased risk of cancer.

Knudson's 2-hit hypothesis

Like all genes, tumor suppressor genes may undergo a variety of mutations; however, most loss-of-function mutations that occur in tumor suppressor genes are recessive in nature. Thus, in order for a particular cell to become cancerous, both of the cell's tumor suppressor genes must be mutated. This idea is known as the "two-hit" hypothesis, and it was first proposed by geneticist Alfred Knudson in 1971. Today, this hypothesis serves as the basis for researchers' understanding of how mutations in tumor suppressor genes drive cancer. The two-hit hypothesis arose of out Knudson's interest in the genetic mechanisms underlying retinoblastoma, a childhood form of retinal cancer. Under normal circumstances, a population of cells in the developing eye, called retinoblasts, stops growing and dividing during embryogenesis and differentiates into retinal photoreceptor (light-capturing) cells and nerve cells. Typically, these differentiated cells do not divide very often, if ever. In the case of retinoblastoma, however, the retinoblasts fail to differentiate; thus, these cells continue to divide, forming tumors in the retina. If left untreated, the retinal tumor cells will eventually metastasize (spread) to other parts of the body.At the time of Knudson's study, researchers believed that retinoblastoma could be caused by either somatic or germ-line mutations. Knudson's combined data itself showed that retinoblastoma was caused by a germ-line mutation in approximately 40% of U.S. cases. However, Knudson was puzzled by the observation that some children with an affected parent were disease-free, but these unaffected individuals later bore children with retinoblastoma; this finding suggested that an individual could inherit a germ-line mutation but not have the disease. Knudson also noted that while the majority of children with an affected parent had bilateral tumors (25%-30%), some had only unilateral tumors (10%-15%). Furthermore, he determined that approximately 60% of retinoblastoma cases in the U.S. were unilateral and were not associated with a family history of the disease. These findings are summarized in Table 1; in the table, "hereditary" refers to those individuals with a family history of retinoblastoma.Knudson knew that if retinoblastoma were caused by a recessive mutation in a single gene, both copies of the gene would need to be mutated in order for retinoblastoma to occur, and the mutation ratewould be the same for each allele of the gene. Therefore, individuals who inherited a mutation in one allele of the gene would only need to accumulate a single mutation in the remaining normal allele of any retinoblast in order for cancer to occur. However, without an inherited mutation, the same cell would need to accumulate two mutations—one in each allele of the gene—and this process would be much slower.Based on the predicted mutation rate, Knudson expected that many individuals in the general population would acquire a single somatic mutation in the RB1 gene over their lifetime, and that the retinas of most people would therefore likely contain small groups of retinoblasts that had received one "hit" in the RB1 gene. In order to become cancerous, each retinoblast with one mutant copy of the RB1gene would need to acquire a mutation in the remaining wild-type copy of the gene. Most individuals who had one hit did not develop retinoblastoma, however, because most of their mutated cells had already differentiated and quit dividing before they could receive a second hit (Knudson, 2001).

Invasion & Metastasis: Extracellular Proteases

Matrix metalloproteinases secreted by cancer cells degrade the extracellular matrix allowing cells to move.

Why is Metastasis Bad?

Metastasis is responsible for 90% of cancer deaths. Once a tumour metastasizes it is very difficult to cure by surgery or radiation. Metastatic cancer cells forming tumours in other organs can impede the function of that organ. Growing metastases may destroy the organ they inhabit. • E.g. Prostate bone metastases: Changes bone structure and alters bone marrow function. This causes pain, blood production issues, immune system issues and skeletal problems

Global MicroRNAs and Cancer

MiRNAs are small non coding RNAs that play a specific role in regulating gene expression. Studies have now highlighted the differences in miRNA expression between normal and tumourous tissue (and between different tumour types). Some specifics: Epigenetically silenced miRNAs are being associated with greater risks of metastasis. Evidence of downregulation in tumour suppressors and overexpression in certain oncogenes.

Invasion & Metastasis: Cell Adhesion Molecules

Modulation in membrane adhesion sites allow cells to adapt to a new environment.

BRCA1 and Breast Cancer

Most cases caused by a BRCA1 or BRCA2 mutation

- Local invasion - metastasis

Most reliable and most important features that distinguish a tumor as malignant (2)

Is it only primary tumours that metastasis?

No, secondary deposits can also spread further. It is rare to see this as by this stage there would be a lot of symptoms from the secondary tumour growth.

Complex interplay of let-7 and miR200 in cancer formation

Other examples of miRs involved in metastasis are miR-7, 10b and 21, Often involves complex feed-back loops of transcriptional and post-transcriptional regulation. Among all tumor suppressor microRNAs, reduced let-7 expression occurs most frequently in cancer and typically correlates with poor prognosis. Activation of either LIN28A or LIN28B, two highly related RNA binding proteins (RBPs) and proto-oncogenes, is responsible for the global post-transcriptional downregulation of the let-7 microRNA family observed in many cancers

Why do Tumours Produce Pro- Angiogenic Factors?

Pro-angiogenic factors can be induced by: • Hypoxia • Glucose deprivation • Formation of Reactive Oxygen Species (ROS) • Cellular acidosis • Iron deficiency • Activation of some oncogenes • Loss of function of some tumour suppressor genes

Ames Test

Problem for somatic mutation hypothesis : Many carcinogens are unreactive with DNA. Therefore they are NOT mutagens Examples: X-rays, Ethidium bromide, benzene E.g. polycyclic aromatic hydrocarbons (PAH) carcinogens are chemically unreactive with DNA making them non-mutagenic in bacteria. However - in mammals they are substrates for de-toxifying enzymes (liver microsomal P450 oxidase). Enzymatic oxidation converts PAHs to electrophilic reagents. These form adducts with DNA which in turn generate mutations after replication.

Why do Tumours Produce VEGF?

Production is part response to low oxygen - Hypoxia • Most human solid tumours have pO2 values lower than their normal tissues of origin. • Tumour cells are usually proliferating faster than normal cells. • Therefore, the ability of tumour cells to sense and adapt to low oxygen (hypoxia) is essential for tumour growth.

menstrual cycle and pregnancy wound healing - Normal Angiogenesis in Adults

Proliferation of new blood vessels also takes place in adults, although it is a relatively infrequent event. In women, angiogenesis is active a few days each month as new blood vessels form in the lining of the uterus during the menstrual cycle. Also, angiogenesis is necessary for the repair or regeneration of tissue during wound healing.

Modeling Prostate Cancer Bone Metastasis in an Animal

Purpose: 1. To understand what prostate cancer cells do in the bone environment. 1. What can be done to stop them from growing and affecting bone function.

HER2 and Breast Cancer

Receptor Activation via two dimer formations. HER2 is the preferred dimer partner

Cadherin = Ca adherin

Refers to absolute dependence on Ca2+ • Unique external Cadherin domains • Family includes E(epithelial)-cadherin which is determining molecule of epithelial sheets. • Attach to F-actin filaments via catenins.

Micro RNA-mediated gene regulation

Regulate translation of >60% of protein-coding genes. Regulate cell growth, differentiation, development and apoptosis. Some miRNAs regulate individual genes Some are master regulators of multiple genes Some can coordinately regulate a set of genes Can be very complex

Tissue invasion and metastasis

Sooner or later during the development of most types of human cancer, primary tumor masses spawn pioneer cells that move out, invade adjacent tissues, and thence travel to distant sites where they may succeed in founding new colonies. These distant settlements of tumor cells—metastases—are the cause of 90% of human cancer deaths ( Sporn 1996). The capability for invasion and metastasis enables cancer cells to escape the primary tumor mass and colonize new terrain in the body where, at least initially, nutrients and space are not limiting. The newly formed metastases arise as amalgams of cancer cells and normal supporting cells conscripted from the host tissue. Like the formation of the primary tumor mass, successful invasion and metastasis depend upon all of the other five acquired hallmark capabilities. But what additional cellular changes enable the acquisition of these final capabilities during tumorigenesis? Invasion and metastasis are exceedingly complex processes, and their genetic and biochemical determinants remain incompletely understood. At the mechanistic level, they are closely allied processes, which justifies their association with one another as one general capability of cancer cells. Both utilize similar operational strategies, involving changes in the physical coupling of cells to their microenvironment and activation of extracellular proteases. Several classes of proteins involved in the tethering of cells to their surroundings in a tissue are altered in cells possessing invasive or metastatic capabilities. The affected proteins include cell-cell adhesion molecules (CAMs)—notably members of the immunoglobulin and calcium-dependent cadherin families, both of which mediate cell-to-cell interactions—and integrins, which link cells to extracellular matrix substrates. Notably, all of these "adherence" interactions convey regulatory signals to the cell (Aplin et al. 1998). The most widely observed alteration in cell-to-environment interactions in cancer involves E-cadherin, a homotypic cell-to-cell interaction molecule ubiquitously expressed on epithelial cells. Coupling between adjacent cells by E-cadherin bridges results in the transmission of antigrowth and other signals via cytoplasmic contacts with β-catenin to intracellular signaling circuits that include the Lef/Tcf transcription factor (Christofori and Semb 1999). E-cadherin function is apparently lost in a majority of epithelial cancers, by mechanisms that include mutational inactivation of the E-cadherin or β-catenin genes, transcriptional repression, or proteolysis of the extracellular cadherin domain (Christofori and Semb 1999). Forced expression of E-cadherin in cultured cancer cells and in a transgenic mouse model of carcinogenesis impairs invasive and metastatic phenotypes, whereas interference with E-cadherin function enhances both capabilities (Christofori and Semb 1999). Thus, E-cadherin serves as a widely acting suppressor of invasion and metastasis by epithelial cancers, and its functional elimination represents a key step in the acquisition of this capability.Changes in expression of CAMs in the immunoglobulin superfamily also appear to play critical roles in the processes of invasion and metastasis (Johnson 1991). The clearest case involves N-CAM, which undergoes a switch in expression from a highly adhesive isoform to poorly adhesive (or even repulsive) forms in Wilms' tumor, neuroblastoma, and small cell lung cancer (52, 54) and reduction in overall expression level in invasive pancreatic and colorectal cancers (Fogar et al. 1997). Experiments in transgenic mice support a functional role for the normal adhesive form of N-CAM in suppressing metastasis (Perl et al. 1999).Changes in integrin expression are also evident in invasive and metastatic cells. Invading and metastasizing cancer cells experience changing tissue microenvironments during their journeys, which can present novel matrix components. Accordingly, successful colonization of these new sites (both local and distant) demands adaptation, which is achieved through shifts in the spectrum of integrin α or β subunits displayed by the migrating cells. These novel permutations result in different integrin subtypes (of which there are greater than 22) having distinct substrate preferences. Thus, carcinoma cells facilitate invasion by shifting their expression of integrins from those that favor the ECM present in normal epithelium to other integrins (e.g., α3β1 and αVβ3) that preferentially bind the degraded stromal components produced by extracellular proteases (90, 64). Forced expression of integrin subunits in cultured cells can induce or inhibit invasive and metastatic behavior, consistent with a role of these receptors in acting as central determinants of these processes (Varner and Cheresh 1996).Attempts at explaining the cell biological effects of integrins in terms of a small number of mechanistic rules have been confounded by the large number of distinct integrin genes, by the even larger number of heterodimeric receptors resulting from combinatorial expression of various α and β receptor subunits, and by the increasing evidence of complex signals emitted by the cytoplasmic domains of these receptors (1, 36). Still, there is little doubt that these receptors play central roles in the capability for tissue invasion and metastasis.The second general parameter of the invasive and metastatic capability involves extracellular proteases (21, 14). Protease genes are upregulated, protease inhibitor genes are downregulated, and inactive zymogen forms of proteases are converted into active enzymes. Matrix-degrading proteases are characteristically associated with the cell surface, by synthesis with a transmembrane domain, binding to specific protease receptors, or association with integrins (96, 86). One imagines that docking of active proteases on the cell surface can facilitate invasion by cancer cells into nearby stroma, across blood vessel walls, and through normal epithelial cell layers. That notion notwithstanding, it is difficult to unambiguously ascribe the functions of particular proteases solely to this capability, given their evident roles in other hallmark capabilities, including angiogenesis (Stetler-Stevenson 1999) and growth signaling (96, 3), which in turn contribute directly or indirectly to the invasive/metastatic capability.A further dimension of complexity derives from the multiple cell types involved in protease expression and display. In many types of carcinomas, matrix-degrading proteases are produced not by the epithelial cancer cells but rather by conscripted stromal and inflammatory cells (Werb 1997); once released by these cells, they may be wielded by the carcinoma cells. For example, certain cancer cells induce urokinase (uPA) expression in cocultured stromal cells, which then binds to the urokinase receptor (uPAR) expressed on the cancer cells (Johnsen et al. 1998).The activation of extracellular proteases and the altered binding specificities of cadherins, CAMs, and integrins are clearly central to the acquisition of invasiveness and metastatic ability. But the regulatory circuits and molecular mechanisms that govern these shifts remain elusive and, at present, seem to differ from one tissue environment to another. The acquired capability for invasion and metastasis represents the last great frontier for exploratory cancer research. We envision that evolving analytic techniques will soon make it possible to construct comprehensive profiles of the expression and functional activities of proteases, integrins, and CAMs in a wide variety of cancer types, both before and after they acquire invasive and metastatic abilities. The challenge will then be to apply the new molecular insights about tissue invasiveness and metastasis to the development of effective therapeutic strategies.

Do cancer cells have a preference for specific bones/ bones in specific parts of the body?

Spine, pelvis, proximal femur, and skull are the most common sites of bone metastases.

History

Tamoxifen was first developed in 1960s as a morning-after birth control pill • Tamoxifen is the best-known hormonal treatment and the most prescribed anti-cancer drug in the world. • Used for over 20 years to treat women with advanced breast cancer, tamoxifen also is commonly prescribed to prevent recurrences among women with early breast cancer. • Selective estrogen receptor modulator (SERM). Since estrogen can promote the development of cancer in the breast and uterus, it seems logical to postulate that substances that block the action of estrogen might be helpful in preventing or treating these two types of cancer. This rationale has led scientists to work on the development of "antiestrogen" drugs that can block the action of estrogens and thereby interfere with, or even prevent, the proliferation of breast and uterine cancer cells. Antiestrogens work by binding to estrogen receptors, blocking estrogen from binding to these receptors. This also blocks estrogen from activating genes for specific growth-promoting proteins. In working on the development of antiestrogens, scientists have made a somewhat surprising discovery. Some drugs that block the action of estrogen in certain tissues actually can mimic the action of estrogen in other tissues. Such selectivity is made possible by the fact that the estrogen receptors of different target tissues vary in chemical structure. These differences allow estrogen-like drugs to interact in different ways with the estrogen receptors of different tissues. Such drugs are called selective estrogen receptor modulators, or SERMs, because they selectively stimulate or inhibit the estrogen receptors of different target tissues. For example, a SERM might inhibit the estrogen receptor found in breast cells but activate the estrogen receptor present in uterine endometrial cells. A SERM of this type would inhibit cell proliferation in breast cells, but stimulate the proliferation of uterine endometrial cells.

Herceptin: Humanized Anti-HER2 Antibody

Targets HER2 oncoprotein • High affinity (Kd = 0.1 nM) and specificity • 95% human, 5% murine - Decrease potential for immunogenicity - Increase potential for recruiting immune-effector mechanisms

An Enabling Characteristic: Genome Instability

The acquisition of the enumerated six capabilities during the course of tumor progression creates a dilemma. The available evidence suggests that most are acquired, directly or indirectly, through changes in the genomes of cancer cells. But mutation of specific genes is an inefficient process, reflecting the unceasing, fastidious maintenance of genomic integrity by a complex array of DNA monitoring and repair enzymes. These genome maintenance teams strive to ensure that DNA sequence information remains pristine. Karyotypic order is guaranteed by yet other watchmen, manning so-called checkpoints, that operate at critical times in the cell's life, notably mitosis. Together, these systems ensure that mutations are rare events, indeed so rare that the multiple mutations known to be present in tumor cell genomes are highly unlikely to occur within a human life span. Yet cancers do appear at substantial frequency in the human population, causing some to argue that the genomes of tumor cells must acquire increased mutability in order for the process of tumor progression to reach completion in several decades time (Loeb 1991). Malfunction of specific components of these genomic "caretaker" systems has been invoked to explain this increased mutability (Lengauer et al. 1998). The most prominent member of these systems is the p53 tumor suppressor protein, which, in response to DNA damage, elicits either cell cycle arrest to allow DNA repair to take place or apoptosis if the damage is excessive. Indeed, it is now clear that the functioning of the p53 DNA damage signaling pathway is lost in most, if not all, human cancers (Levine 1997). Moreover, a growing number of other genes involved in sensing and repairing DNA damage, or in assuring correct chromosomal segregation during mitosis, is found to be lost in different cancers, labeling these caretakers as tumor suppressors (Lengauer et al. 1998). Their loss of function is envisioned to allow genome instability and variability and the generation of consequently mutant cells with selective advantages. Interestingly, recent evidence suggests that apoptosis may also be a vehicle of genomic instability, in that DNA within apoptotic cell bodies can be incorporated into neighboring cells following phagoctytosis (Holmgren et al. 1999), in principle genetically diversifying any of the constituent cell types of a tumor. We place this acquired characteristic of genomic instability apart from the six acquired capabilities associated with tumor cell phenotype and tumor physiology: it represents the means that enables evolving populations of premalignant cells to reach these six biological endpoints.

process

The angiogenesis process begins with the degradation of the basement membrane by proteases secreted by activated endothelial cells that will migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space. Then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane.

Epigenetics

The mechanisms for genomic imprinting and histone modification remain largely unknown. These, along with methylation changes, are all inter-related and a better understanding of their links and affects can only lead to a better understanding to change associated with cancer.

Epigenetics

The mechanisms that initiate and maintain heritable patterns of gene function and regulation in a heritable manner without affecting the sequence of the genome.

miRNAs as cancer drivers

The oncogene MYC negatively regulates multiple tumour suppressor miRNAs

is there a limit to the angiogenesis process?

The only limit is the amount of growth factors being produced.

The Diseases Associated with VEGF Signaling Pathway

The over-expression of VEGF is contributive to many diseases, such as solid tumors, breast cancer, GBM, melanoma, and hypoxic diseases. When solid tumors grow beyond a limited size, if their blood supply is insufficient, they cannot continue to grow. And then, the tumors express VEGF to promote new blood vessels formation, which facilitates self-growth and metastasis.

Alternative Pathways to Cancer

The paths that cells take on their way to becoming malignant are highly variable. Within a given cancer type, mutation of particular target genes such as ras or p53 may be found in only a subset of otherwise histologically identical tumors. Further, mutations in certain oncogenes and tumor suppressor genes can occur early in some tumor progression pathways and late in others. As a consequence, the acquisition of biological capabilities such as resistance to apoptosis, sustained angiogenesis, and unlimited replicative potential can appear at different times during these various progressions. Accordingly, the particular sequence in which capabilities are acquired can vary widely, both among tumors of the same type and certainly between tumors of different types (Figure 4). Furthermore, in certain tumors, a specific genetic event may, on its own, contribute only partially to the acquisition of a single capability, while in others, this event may aid in the simultaneous acquisition of several distinct capabilities. Nonetheless, we believe that independent of how the steps in these genetic pathways are arranged, the biological endpoints that are ultimately reached—the hallmark capabilities of cancer—will prove to be shared in common by all types of tumors.

Epigenetics

The research into epigenetic inheritance has increased massively in the past 20 years. This work is beginning to bear fruit but there are still a lot of questions. It is thought that ~50% of mutational events in cancer formation are epigenetic. (Jones & Baylin, 2002, The findamental role of epigenetic events in cancer, Nature Reviews, Vol 6 (3), pp 415-428) Epigenetic drugs are currently being used in cancer therapy.

metastasis

The spread of cancer cells to locations distant from their original site. Metastasis is the process whereby a tumour cell: 1. Invades and leaves the original tumour 2. Transports to another organ 3. Divides out of control within that organ Metastases are secondary tumours that are disjointed from the original (primary) tumour.

Integrins/Cadherins mediate like-like cell adherence. Is it possible that those molecules are just more similar e.g. between prostate and bone than say prostate and another tissue to allow initial adherence?

There is no difference in the adherence molecules (that has been discovered to date). The attraction of a tumour cell from the vasculature will not be connected to the adherence. So... a pie in Jamaica is £1.50, but in Cuba they are £2.30 and in Puerto Rico they are £3!!! ...but yeah, those are the pie-rates of the Caribbean!

How do the tumour cells leave the bloodstream? If it cannot adhere to other cells how can it leave the bloodstream?

They do adhere to the epithelial cells in the vascular cells walls. They have to do this to enter the blood stream as well.

Local invasion

This occurs in normal development as well as in tumours. e.g. Epithelial-mesenchymal transition - synaptic adhesion is involved in learning and memory.

How impactful is genetic disposition into getting cancer?

This varies from cancer to cancer.

Cadherin-mediated cell-cell adhesion

To promote tissue strength, cadherins are specifically bound to microfilaments (MFs) of the cytoskeleton. The linking proteins include catenins

Mouse Skin Model

Treatment of mice with carcinogens is the basis of numerous mouse models of cancer. Skin - 7,12-dimethylbenz[α]anthracene (DMBA) Lung - Nitrosamines Liver - vinyl chloride Breast - N-Nitroso-N-methylurea (NMU) Colon - dimethylhydrazine (DMH), Nitrosamines Bladder - Aromatic Amines

tumour promoter

Tumour Promoter (e.g. Phorbol Myristate Acetate (PMA) PMA is an activator of Protein Kinase C wounding or tumour promotion = no tumours

Definitions

Tumour initiator: a carcinogen that sows the seeds of cancer (usually without giving rise to a tumour by itself). • Initiation: Irreversible DNA damage in critical gene(s). tumour promoters: agents which, when applied to susceptible organisms after exposure to an initiator, increase tumour production. promotion: expansion of initiated cells

Tumours Rarely Kill Patients

Tumours Rarely Kill Patients • Tumour cells are rarely lethal unless they form a mass in a vital organ (e.g. brain). • Tumour cells can become lethal when they invade the surrounding tissue and spread to other organs (otherwise known as malignancy). • This process is termed metastasis: where tumour cells travel to other organs and form tumours within that organ.

Cancer is typically characterised as a disease of the elderly. What makes those that appear early (e.g. Non-Hodgkins Lymphoma) different?

Typically early onset cancers means that a certain pattern of mutated oncogenes and TSG have been inherited by the child. In this large national cohort study, family history of NHL, high fetal growth, older maternal age, low birth order, and male sex were independent risk factors for NHL in early life.

Oestrogen Receptor-Negative Breast Cancer

Unlike normal breast cells, cancer cells arising in the breast do not always have receptors for estrogen. Breast cancers that DO have estrogen receptors are said to be "estrogen receptor-positive," while those breast cancers that DO NOT possess estrogen receptors are "estrogen receptor-negative." In women with estrogen receptor-positive cancers, cancer cell growth is under the control of estrogen. Therefore, such cancers are often susceptible to treatment with tamoxifen, because tamoxifen works by blocking the interaction between estrogen and the estrogen receptor. In contrast, the growth of estrogen receptor-negative cancer cells is not governed by estrogen, or treated with tamoxifen.

How to be a Successful Cancer Cell

We suggest that the vast catalog of cancer cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth (Figure 1): self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Each of these physiologic changes—novel capabilities acquired during tumor development—represents the successful breaching of an anticancer defense mechanism hardwired into cells and tissues. We propose that these six capabilities are shared in common by most and perhaps all types of human tumors. This multiplicity of defenses may explain why cancer is relatively rare during an average human lifetime. We describe each capability in turn below, illustrate with a few examples its functional importance, and indicate strategies by which it is acquired in human cancers.

Epigenomics

Whole genome analysis (Epigenomics) allows for the identification of biomarkers associated with DNA methylation. In the past 5 years an extensive map is being drawn up reporting methylation based bio-markers. This is leading to labeling of unique methylation profiles that define each type of cancer. A DNA methylation fingerprint.

Since time is the killer, can Cadherin be used as a therapy to slow down metastasis? Or are the genetic differences between the stages of metastasis overpowering?

Yes, but Cadherin is only one adhesion system and by the time you are treating someone metastasis has most likely already occurred.

Adenomatous Polyposis Coli (APC)

a tumor suppressor gene on chromosome 5. Mutations in this gene result in familial adenomatous polyposis First mutation associated with sporadic and FAP colon cancers. Sporadic forms - spontaneous/ environmental mutation. FAP - inherit a mutation in one APC allele.

Colorectal Cancer

adenocarcinoma of the colon or rectum, or both Why is colorectal cancer a good cancer model? Why is colorectal cancer a good cancer model? tumours develop from pre- existing benign tumours • all stages of tumours are available for study

Angiogenesis via cell divisions and movement of VE cells

angiogenic stimulus

Tamoxifen and Cancer

antiestrogen work by binding to oestrogen receptors blocking' oestrogen from binding to these receptors stopping cell proliferation tamoxifen blocks the action of oestrogen in breast tissue it binds to the oestrogen receptors of breast cells thereby preventing oestrogen molecules from binding to these receptors The normal situation, when estrogen binds to its receptor, the binding of tamoxifen to the receptor does not cause the receptor molecule to acquire the changed shape that allows it to bind to coactivators.As a result, the genes that stimulate cell proliferation cannot be activated.

Current Assays of HER2

immunohistochemistry FISH

therapeutics

importing-11 identified as new therapeutic target for colorectal cancer

Why is it difficult to remove a secondary tumour but the primary tumour can be?

its not more difficult to remove one secondary tumours growth. Its just that secondary growths tend to be in several sites, not just one that can be removed by one operation

cancer

look at last slide

BRCA1/2

mutations to them are implicated in breast and ovarian cnacer. they are tumor supressor genes that play a role in DNA REPAIR (particularly double stranded DNA breaks.

Epigenetics

the study of environmental influences on gene expression that occur without a DNA change I.e. modifications that affect gene expression So we are investigating any modifications to the DNA, or associated proteins, other than DNA sequence variation

The Most Common LOF Mutations for Carcinomas are in Cadherins

• E-cadherins are frequently found mutated (down- regulated) in metastatic cancers. Mutations found in exons 8 and 9 (extracellular domain) cause decrease in cell-cell adhesion and increase in motility. Snail is a transcription factor that down-regulates cadherin expression. Its hyperactivity can lead to lowered levels of cadherin

Travel in blood is dangerous for the metastatic cell

• Fast flow of blood creates sheer forces which can destroy the metastatic cells. • Correlations between tumour cells in blood and frequency of metastatic growth are not good. • This emphasizes the difficulties faced by tumour cells in accomplishing 'extravasation'.

Metastasis is Organ Specific

• It has long been observed that certain cancers prefer certain organ sites of metastasis:

Breast Cancer

• breast cancer occurs in the cells that line the lobules that manufacture milk or more commonly in the ducts that carry it to the nipple.

Disruption of miRNAs in cancer

• miRNA expression differs between normal and tumour tissue. • miRNAs can act as oncogenes or as tumour suppressors (mainly tumour suppressors). • Defects in miRNA expression lead to a global miRNA repression. The first human disease known to be associated with miRNA deregulation was chronic lymphocytic leukemia. Many other miRNAs also have links with cancer and accordingly are sometimes referred to as "oncomirs". In malignant B cells miRNAs participate in pathways fundamental to B cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches and the production and class-switching of immunoglobulins. MiRNAs influence B cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. Another role for miRNA in cancers is to use their expression level for prognosis. In NSCLC samples, low miR-324a levels may serve as an indicator of poor survival.[142] Either high miR-185 or low miR-133b levels may correlate with metastasis and poor survival in colorectal cancer.[143] Furthermore, specific miRNAs may be associated with certain histological subtypes of colorectal cancer. For instance, expression levels of miR-205 and miR-373 have been shown to be increased in mucinous colorectal cancers and mucin-producing Ulcerative Colitis-associated colon cancers, but not in sporadic colonic adenocarcinoma that lack mucinous components.[144] In-vitro studies suggested that miR-205 and miR-373 may functionally induce different features of mucinous-associated neoplastic progression in intestinal epithelial cells.[144] Hepatocellular carcinoma cell proliferation may arise from miR-21 interaction with MAP2K3, a tumor repressor gene.[145] Optimal treatment for cancer involves accurately identifying patients for risk-stratified therapy. Those with a rapid response to initial treatment may benefit from truncated treatment regimens, showing the value of accurate disease response measures. Cell-free miRNA are highly stable in blood, are overexpressed in cancer and are quantifiable within the diagnostic laboratory. In classical Hodgkin lymphoma, plasma miR-21, miR-494, and miR-1973 are promising disease response biomarkers.[146] Circulating miRNAs have the potential to assist clinical decision making and aid interpretation of positron emission tomography combined with computerized tomography. They can be performed at each consultation to assess disease response and detect relapse. MicroRNAs have the potential to be used as tools or targets for treatment of different cancers.[147] The specific microRNA, miR-506 has been found to work as a tumor antagonist in several studies. A significant number of cervical cancer samples were found to have downregulated expression of miR-506. Additionally, miR-506 works to promote apoptosis of cervical cancer cells, through its direct target hedgehog pathway transcription factor, Gli3

How is transcription factor HIF-1 induced by low oxygen?

The transcription factor complex hypoxia-inducible factor (HIF)-1 controls the expression of most genes involved in adaptation to hypoxic conditions. HIF-1 is a heterodimer composed of oxygen-labile HIF-α and constitutively expressed HIF-β subunits. The oxygen-dependent regulation of HIF-α is a multistep process that includes degradation under normoxia but stabilisation, translocation into the nucleus and activation under hypoxic conditions Ubiquitin tagging of HIF-1 is dependent on molecular oxygen The alpha subunits of HIF are hydroxylated at conserved prolineresidues by HIF prolyl-hydroxylases, allowing their recognition and ubiquitination by the VHL E3 ubiquitin ligase, which labels them for rapid degradation by the proteasome.This occurs only in normoxic conditions. In hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate E3 complex contains product of tumour suppressor gene VHL protein (von Hippel-Lindau syndrome).

Angiogenesis

development of new blood vessels angio = blood/vessel genesis= beginning Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplying nutrients and oxygen and removing waste products. Tumor angiogenesis actually starts with cancerous tumor cells releasing molecules that send signals to surrounding normal host tissue. This signaling activates certain genes in the host tissue that, in turn, make proteins to encourage growth of new blood vessels.

Tumour-specific defects in miRNA processing machinery.

27% of human tumours have hemizygous deletion of the gene encoding DICER1. Reduction in DICER1 leads to inhibition of miRNA processing and an increase in rate of tumour formation. TRBP2 is a DICER cofactor. Erk phosphorylation of TRBP2 stimulates pre-miRNA processing of oncogenic miRs. Decreases production of tumour suppressor miRs.

angiogenic phenotype

somatic mutation - small tumour - proangiogenic factors secreted by tumour ( VEGF) - tumour growth and metastasis - angiogenic inhibitors may prevent new vascularization and induce vascular regression

order is imp

wounding or tumour promotion then initiation = no tumours if the other way round it will lead to tumours

Why is Angiogenesis Important in Cancer?

• Allows for expansion of a tumour. • Metastasis requires angiogenesis. 1) cancer cells invade surrounding tissues and vessels 2) cancer cells are transported by the circulatory system 3) cancer cells reinvade and grow at a new location Cancer researchers studying the conditions necessary for cancer metastasis have discovered that one of the critical events required is the growth of a new network of blood vessels. This process of forming new blood vessels is called angiogenesis.

short term assay:Ames test

Bruce Ames Histidine biosynthesis 1. Assumes that any substance that is mutagenic may also turn out to be a carcinogen (not always true!). 2. The test uses a strain of Salmonella that carries a mutant gene rendering it unable to synthesis Histidine. 3. This mutation may be reversed by a potential mutagen with the gene regaining its function.

Loss of Anchorage Dependence

Can be measured experimentally by investigating the ability of cells to form colonies in soft agar. A sensitive test of malignant transformation - colony formation assay hard agar keeps cells off surface and soft agar keeps cells suspended tumour cells form colonies - Lung carcinoma cell line High magnification of colony from A C. Normal lung fibroblast

Finite Number of Division Cycles

Cells from normal human tissues divide in culture for about 9 months only. • They cease replication. • Known as Senescence. Reaching the "Hayflick limit" Countdown mechanism may be Telomere shortening.

The Hayflick Phenomenon

Cellular Senescence The Hayflick limit or Hayflick phenomenon is the number of times a normal human cell population will divide before cell division stops. Hayflick demonstrated that a normal human fetalcell population will divide between 40 and 60 times in cell culture before entering a senescence phase. Each time a cell undergoes mitosis, the telomeres on the ends of each chromosome shorten slightly. Cell division will cease once telomeres shorten to a critical length. Hayflick interpreted his discovery to be aging at the cellular level. The aging of cell populations appears to correlate with the overall physical aging of an organism. limit to the number of times normal cells can divide cells reach a predefined limit (Hayflick Limit) replicative senescence causes a nondividing state inability to divide represents ageing

Let-7 and miR-200, Global Regulators of Differentiation

Specific miRNAs have been shown to regulate mammalian cellular differentiation as well as developmental patterning and morphogenesis in a tissue specific fashion.23-29 In addition to these specific regulators, there exist classes of miRNAs that have universal functions which are dependent not on the tissue, per se, but rather on the differentiation state of the tissue. Two of the largest miRNA families, let-7 and miR-200 seem to have such activities. The ubiquitously expressed let-7/miR-98 family was one of the first mammalian miRNAs to be identified.13-16,30,31 The let-7 family is comprised of 12 family members (let-7-a1, a2, a3, b, c, d, e, f1, f2, g, i and miR-98) located on 8 different chromosomes.32 These 12 family members represent 9 distinct let-7 sequences with identical seed sequences (the 5' sequence of the miRNA responsible for initating binding) and, very likely, overlapping sets of targets. Let-7 is expressed late in mammalian embryonic development and plays an evolutionarily conserved role from Caenorhabditis elegansto Drosophila to mammals.31,33-35 The let-7 targets that have been identified include cell cycle regulators such as CDC25A and CDK6,36 promoters of growth including RAS and c-myc16,37,38 and a number of early embryonic genes including HMGA2, Mlin-41 and IMP-1.39-44 The miR-200 family is comprised of 5 members (miR-200a, b, c, miR-141 and miR-429). They are located within two clusters on separate chromosomes. Interestingly the 5 members can be subdivided into two subgroups according to their seed sequences. MiR-200a and miR-141 comprise group I and miR-200b, c and miR-429 comprise group II. Although target prediction algorithms predicted little overlap in the targets of these groups, experimental approaches suggested that their sets of targets are highly overlap- ping.45 The most prominent targets of the miR-200 family are two E box binding transcription factors, ZEB1 (also known as TCF8 and δEF1) and ZEB2 (also known as ZFXH1B and SMAD interacting protein 1 (SIP1)).45-48 Both are key regulators within a complex network of transcriptional repressors that regulate the expression of E-cadherin and a number of master regulators of epithelial polarity.49-52 Consistent with their function, the miR-200 family was recently identified as both a marker and a powerful regulator of the epithelial-to-mesenchymal transition (EMT).45,48,53,54 For simplicity I will refer to these families of miRNAs as either let-7 or miR-200 unless specific activities of individual miRNA species are being discussed. Mounting evidence links two miRNA families, let-7 and miR-200, which are significantly correlated with dedifferentiation of cancer cells, to stem cells (Fig. 3). In addition to experimental evidence linking let-7 to the stem cell factors Lin28 and Lin28B, Sox2 and Klf4 are predicted to be targets of miR-200b/c/429 (TargetScan. org). Expression of both let-7 and miR-200 was correlated with more differentiated cancer using genome wide miRNA arrays or real time PCR.45,48,187 Interestingly in all of these studies additional miRNAs that were (less significantly) associated with the dedif- ferentiated phenotype were identified. These "runners up" can also be connected to stem cell regulation. In the study by Goodall and colleagues, in addition to miR-200, miR-205 was identified as a marker for mesenchymal cells, and miR-205 was shown to target the EMT regulators ZEB1 and ZEB2.48 In a recent genome wide analysis of the connections between the core transcriptional machinery and miRNAs, both Sox2 and Nanog were found to be bound to the promoter of miR-205, suggesting a direct link between the EMT regulator, miR-205 and ES cell regulators.63 In our own study in which we identified miR-200 as a regulator of EMT, the miRNA that followed the miR-200 family member in significance was miR-203.45 miR-203 was shown to negatively regulate stemness during skin development by suppressing expression of p63.188,189 Finally, in the screen to identify miRNAs that are preferentially expressed in Type II cells we identified miR-128a as more highly expressed in Type II cells.42 Interestingly, it was shown that the stem cell factors (and let-7 targets), Lin28 and Lin28B, bind not only to the loop region of let-7,but also to the loop region of miR-128,of let-7 and miR-128 is coregulated. Of note, both let-7 and miR-128 are predicted to target Lin28, and miR-128 is also predicted to target Nanog (TargetScan.org). In summary, although other miRNAs have similar functions that need to be experimentally explored, I propose that let-7 and miR-200 are major guardians against inappropriate inclinations toward stemness. One of their normal roles may be to keep differentiated cells in their differentiated state.

How do you get cancer?

Spontaneous Mutations v Induced Mutations - Excessive sunbathing = Increased UV exposure = Melanoma - Cigarette smoking = benzopyrene = lung cancer or Carcinogen v Mutagen - physical carcinogens like radiation, ultraviolet light or ionising radiation - biological carcinogens - chemical carcinogens

miRNAs can be transmitted from cell to cell in Extracellular Vesicles

MiRNA release mechanisms into extracellular space. Precursor miRNAs are processed by ribonuclease Dicer to mature double-stranded miRNAs (miRNA duplex). One strand of the miRNA duplex is selectively loaded into the RNA-induced silencing complex (RISC), which contains the Argonaute (AGO) family protein as a core component. A fraction of miRNAs are released from living cells into the extracellular environment via the following mechanisms: (1) sorting into multivesicular bodies (MVB) and secretion via exosomes, (2) incorporating into microvesicles that are formed by the outward shedding of the plasma membrane, (3) associating with RNA-binding proteins, such as AGO2 and release of the miRNA-AGO complexes, and (4) exporting and incorporating into high-density lipoprotein (HDL) particles. Extracellular vesicle miRNAs are possibly involved in cell-cell communication.

MicroRNA transcription and step-wise maturation

MicroRNAs (miRNAs) constitute a novel, phylogenetically extensive family of small RNAs (∼22 nucleotides) with potential roles in gene regulation. Apart from the finding that miRNAs are produced by Dicer from the precursors of ∼70 nucleotides (pre-miRNAs), little is known about miRNA biogenesis. Some miRNA genes have been found in close conjunction, suggesting that they are expressed as single transcriptional units. Here, we present in vivo and in vitro evidence that these clustered miRNAs are expressed polycistronically and are processed through at least two sequential steps: (i) generation of the ∼70 nucleotide pre-miRNAs from the longer transcripts (termed pri-miRNAs); and (ii) processing of pre-miRNAs into mature miRNAs. Subcellular localization studies showed that the first and second steps are compartmentalized into the nucleus and cytoplasm, respectively, and that the pre-miRNA serves as the substrate for nuclear export. Our study suggests that the regulation of miRNA expression may occur at multiple levels, including the two processing steps and the nuclear export step Most are probably transcribed separate to protein-coding genes, either individually on in multi-pri-miRNA transcripts. Some are located in pre-RNA introns Pri-miRNAs are transcribed by RNA Pol II and are capped and polyadenylated

ZEB1/miR-200

MicroRNAs as regulators of epithelial-mesenchymal transition Epithelial-mesenchymal transition (EMT) describes the molec- ular reprogramming and phenotypic changes involved in the conversion of polarised immotile epithelial cells to motile mesen- chymal cells. This process allows the remodelling of tissues during embryonic development and is implicated in the promotion of tumor invasion and metastasis. Several recent studies have identi- fied the miR-200 family and miR-205 as key regulators of EMT and enforcers of the epithelial phenotype. The miR-200 family participates in a signalling network with the E-cadherin transcrip- tional repressors ZEB1/δEF1 and ZEB2/SIP1, and TGFβ2 that is postulated to facilitate maintenance of stable epithelial or mesen- chymal states but also allow reversible switching between these states in response to EMT effectors (such as TGFβ). This review summarises these recent findings and their implications in both developmental EMT and tumor progression. Epithelial-Mesenchymal Transition (EMT) pithelial-mesenchymal transition (EMT) describes a reversible series of events during which an epithelial cell loses cell-cell contacts and acquires mesenchymal characteristics. These events involve molecular reprogramming of the cell, including loss or redistribu- tion of epithelial-specific junctional proteins such as E-cadherin and turning on of mesenchymal markers including vimentin and N-cadherin.19 The resulting mesenchymal cell has a spindle-shaped morphology and possesses an enhanced migratory ability. EMT is essential for embryonic development in higher vertebrates, facilitating the cell movements required for gastrulation,20 and subse- quently being utilised in the delamination of the neural crest,21 in heart-valve formation,22 palate fusion23 and in the development of various other tissues and organs. Although EMT is not a common event in the adult, this process has been implicated in such instances as wound healing and kidney fibrosis.24,25 The reverse of EMT, mesenchymal to epithelial transition (or MET), is also important in formation of the kidney nephron epithelium.26 An accumulating body of evidence suggests that EMT can be recapitulated during tumor progression, constituting an early step in metastasis of tumors from their primary site.19,27 Cells that have undergone EMT are proposed to be endowed with properties allowing their detachment from the primary tumor, invasion through the basement membrane into the circulation, and conversion back to an epithelial phenotype to form a metastasis at a secondary site These observations have intensified the study and elucidation of key signalling pathways involved in EMT, with players including TGFβ/ BMP family members, Wnt, Notch and growth factors such as PDGF and FGF having tissue and context-specific roles in this process.28 Many of these signalling pathways appear to converge on a small number of transcription factors capable of repressing the E-cadherin gene. E-cadherin is a central component of the adherens junction complex responsible for calcium dependent cell-cell adhesion and maintainenance of cytoskeletal organisation through its links to the actin cytoskeleton via β-catenin.29 Loss of E-cadherin expression has been identified as a causal factor in cancer progression where it is often associated with an aggressive phenotype and poor clinical prognosis.30,31 Transcriptional repression of the E-cadherin gene is emerging as an important mechanism through which E-cadherin is downregulated during tumor progression and several factors including snail,32,33 slug/snail2,34 ZEB1/δEF1,35 ZEB2/SIP1,36 and E47,37 have been shown to directly bind to the E-cadherin promoter and repress its transcription. Many of these E-cadherin repressors are induced by TGFβ pathway stimulation and are also able to repress the transcription of other cell polarity and adhesion genes suggesting they function in multiple ways to induce EMT. EMT is Regulated by the miR-200 Family and miR-205

What are proto-oncogenes and oncogenes (with an example)? What roles do these have in cells?

Proto-oncogenes are a group of genes that cause normal cells to become cancerous when they are mutated Mutations in proto-oncogenes are typically dominant in nature, and the mutated version of a proto-oncogene is called an oncogene. Often, proto-oncogenes encode proteins that function to stimulate cell division, inhibit cell differentiation, and halt cell death. All of these processes are important for normal human development and for the maintenance of tissues and organs. Oncogenes, however, typically exhibit increased production of these proteins, thus leading to increased cell division, decreased cell differentiation, and inhibition of cell death; taken together, these phenotypes define cancer cells. Thus, oncogenes are currently a major molecular target for anti-cancer drug design.

Hallmarks of Cancer

Sustaining proliferative signaling Evading growth suppressors Activating invasion and metastasis Enabling replicative immortality Inducing angiogenesis Resisting cell death Complexity of cancer reduced to a small number of principles • Hallmarks of Cancer are eight, acquired, functional capabilities that allow cancer cells to survive, proliferate and disseminate • First proposed by Hanahan and Weinberg in 2000 Emerging Hallmarks Reprogramming metabolism Evading immune destruction Enabling characteristics Genomic instability Inflammation

HIF-1 Target Genes

group 1 : O2 delivery Erythropoeitin (EPO) Nitric Oxide synthase 2 TransferrinTransferrin receptor VEGF VEGF receptor

Problems with Traditional Cancer Therapies

Targets not only cancer cells but any cells that are normally rapidly dividing: - Stomach Lining: Causes nausea/ vomitting - Hair follicles: Results in hair loss Cancer cells may become resistant to therapies: - Survive chemo and gain additional growth advantages - Survival of the fittest

Ames test

Test in which special strains of bacteria are used to evaluate the potential of chemicals to cause cancer. mutagen-His bacteria ( medium containing histidine will grow ), normal medium it dies reverse mutation - allows his bacteria to grow on normal medium

experiment 2

The discovery that angiogenesis inhibitors such as endostatin can restrain the growth of primary tumors raises the possibility that such inhibitors might also be able to slow tumor metastasis. To test this hypothesis, researchers injected several kinds of mouse cancer cells beneath the animals' skin and allowed the cells to grow for about two weeks. The primary tumors were then removed, and the animals checked for several weeks. Typically, mice developed about 50 visible tumors from individual cancer cells that had spread to the lungs prior to removal of the primary tumor. But mice treated with angiostatin developed an average of only 2-3 tumors in their lungs. Inhibition of angiogenesis by angiostatin had reduced the rate of spread (metastasis) by about 20-fold. Induction of multiple tumours in mice. • Removal of primary tumours. • Inject half of the mice with angiostatin. • Many metastases tumours in untreated mice - few metastases in treated mice. conclusion : addition of angiogenesis inhibitors can reduce the number of metastases even well into cancer progression

Contribution of miRNAs to cancer pathways.

Tumour-suppressor miRNAs, which repress oncogenes in healthy cells, are lost in cancer cells, leading to oncogene upregulation, whereas oncogenic miRNAs inhibit tumour-suppressor genes, giving rise to cancer. b, The presence of different target genes in different cell lines can modify the function of an miRNA, both in healthy cells and cancer cells, which can lead to the development of cancer or a different outcome. c, Two miRNAs can function together to regulate one or several pathways, which reinforces those pathways and can result in the development of cancer. d, The oncogene MYC can either repress tumour-suppressor miRNAs (in blue) or activate oncogenic miRNAs (in red) and can therefore orchestrate several different pathways. MYC can repress let-7, directly, or indirectly, through LIN28 activation. Conversely, let-7 can also repress MYC, which closes the regulatory circle. e, Tumour suppressor p53 can regulate several tumour suppressor miRNAs (blue), activating different antitumoral pathways. The regulation of MDM2 by some of these miRNAs leads to interesting feedforward loops. At the same time, p53 can be negatively regulated by oncogenic miRNAs (in red). In addition, p53 is involved in the biogenesis of several tumour suppressor miRNAs.

Signalling pathways Activated by VEGF

VEGF (vascular endothelial growth factor) signaling pathway is a complex process in which VEGF stimulates new blood vessel formation. VEGF, also known as vascular permeability factor (VPF), is a dimer glycoprotein that binds to the VEGF receptor (VEGFR) on the surface of the cells, thereby activating intracellular tyrosine kinase and initiating a series of signaling cascades events involved in vasculogenesis and angiogenesis.

The Function of VEGF Signaling Pathway

VEGF signaling pathway plays a significant role in vasculogenesis and angiogenesis. Angiogenesis is important in various physiologic and pathologic diseases. VEGF signaling pathway performs a dual function that either beneficial or harmful for humans. On one hand, the process of the VEGF signaling pathway helps bone formation, hematopoiesis, wound healing and development; on the other hand, it prompts the growth of tumors. According to the tumor angiogenesis of Moses Judah Folkman, when tumor size gets bigger, it will induce the formation of new blood vessels to nourish itself and sustain its existence.

What Is Metastasis?

cancer cells leaving a tumor and invading other parts of the body When patients are diagnosed with cancer, they want to know whether their disease is local or has spread to other locations. Cancer spreads by metastasis, the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then invade and grow in normal tissues elsewhere. In large measure, it is this ability to spread to other tissues and organs that makes cancer a potentially life-threatening disease, so there is great interest in understanding what makes metastasis possible for a cancerous tumor.

initiation

low dose of DMBA , no tumours

Summary

low oxygen = no OH proline on HIF-1 = Ubiquitination stops = HIF-1 transcription factor accumulates = VGEF etc induced

The miR-200 family regulates development (EMT)

miR-200 family has 5 members (miE-200a, b, c, miR- 141, miR-429. Target overlapping sets of mRNAs. Important targets are E-box transcription factors ZEB1/2 that control the key cell polarity regulator E-cadherin. E-cadherin is involved in EMT (epithelial-to- mesenchymal transition).

Noncoding RNAs in cancer diagnosis and prognosis

miRNAprofilesreflecttumourembryonicorigin (EMT). miRNA expression profiles are linked to clinical outcome. e.g. let-7 down-regulation in small cell lung tumours is associated with poor prognosis. Next Generation Sequencing (NGS) will define miRNA profiles in individuals for assessment of prognosis and response to treatment.

miRNA binding to target

miRNAs bind to mRNA 3'UTRs 5' miRNA seed sequence has complete homology but complementarity outside this region can be less than 100%. This leads to translational repression If miRNA has complete complementarity over its entire region mRNA degradation occurs Any one mRNA may be the target of more than one miRNA.

miRNAs and p53 and tumour suppressors

p53 interacts with DROSHA complex During cell stress/DNA damage drosha complex = processing of tumour suppressor miRNAs leading to tumour suppression CDK6 - proliferation BCL2 -survival HIF1A - angiogenesis ZEB1/2 - EMT MDM2 interacts with p53 and p53 interacts with drosha complex

Methods for Detecting and Identifying Carcinogens

see slide cancer 1

VEGF (vascular endothelial growth factor)

stimulates growth of new blood vessels Vascular Endothelial Growth factor is secreted by tissues needing new vasculature. VEGF is secreted by tumour cells - very important in tumour expansion and metastasis. Very specific for vascular endothelial cells - binds to tyrosine kinase on endothelial cell surface.

The Angiogenic Switch

the ability of the tumor to promote the formation of new capillaries from pre-existing host vessels The shift by a clump of tumor cells from a state in which they are unable to induce neovascularization to one in which they exhibit this ability. Angiogenesis is tightly controlled through a balance of pro- and anti-angiogenic factors. a discrete component of multistage tumour development.A time restricted event where the balance tilts towards pro-angiogenic side

The Related Inhibitors

the process of the VEGF signaling pathway, the combination of the VEGF with VEGFR can initiate the occurrence of the enzymatic signaling cascades. Therefore, blocking their binding can effectively terminate the VEGF signaling pathway. VEGF inhibitors are some specific monoclonal antibodies and VEGFR inhibitors are some particular tyrosine kinase inhibitors. Existing drugs such as aflibercept bevacizumab ranibizumab and pegaptanib can inhibit VEGF and control or slow those diseases.

Types of Tumour Suppressor Genes

there are 2 major classes of tumour suppressor genes: 1. Growth control e.g. Retinoblastoma protein RB, cyclin-dependent kinase inhibitors (e.g. p16) normal cells = Cyclin D1/cdk4 help drive cells through G1 phase - p16 puts a break on the process cancer cells = p16 is deleted or mutated in many cancers 2. Genes that maintain stability and integrity of the genome, excision repair genes, mismatch repair genes e.g. transcription factors such as p53. Inactivation of p53 in cancer disrupts the G1/S damage checkpoint pathway p53 transactivation can activate bax - activate cytochrome C release - leads to apoptosis or p53 transactivation activates p21cip1 that activate G1/S DNA damage checkpoint to allow for DNA repair

Angiogenesis vs Vasculogenesis

vasculogenesis = building blood vessels from scratch (from stem/ progenitor cells). angiogenesis = New blood vessels form by 'sprouting' from existing vessels. Angiogenesis (also known as neovascularization) is the generation of new blood vessels from pre-existing vasculature. It is a normal process in growth and development and is required for the formation of arteries, veins, and capillaries in an embryo. By definition, angiogenesis is the development of new blood vessels. While this sounds like a relatively innocuous occurrence, it becomes problematic when the blood supply is for a cancerous tumor. When someone has cancerous cells, the last thing they want is for those cells to be fed a rich and nourishing blood supply. Microscopic cancer cells can multiply, mutate, and metastasize given the "right" conditions. Those conditions include a vasculature system. A tumor needs nutrients and oxygen to grow and spread. So, in order to stop the progression of cancer, blood supply to tumors needs to be shut off by eliminating these aberrant vessels or preventing them from developing.

porto-oncogenes and tumor suppressor genes

work together to stimulate, inhibit growth and cell division Tumor suppressor genes often function to restrain inappropriate cell growth and division, as well as to stimulate cell death to keep our cells in proper balance. In addition, some of these genes are involved in DNA repair processes, which help prevent the accumulation of mutations in cancer-related genes. In this way, tumor suppressor genes act as "brakes" to stop cells in their tracks before they can take the road to cancer. Given this situation, loss of tumor suppressor gene function can be disastrous, and it often puts once-normal cells on the fast track to disease.

Why Target Angiogenesis?

• Cancerous tissues would be targeted with few healthy tissues affected. • Controlled by extracellular signals and cell surface receptors - provides many experimental points of intervention.

miRNA networks

• Complex networks of miRNA repression and activation of other miRNAs, tumour suppressor proteins and oncoproteins • Multiple miRNAs can act on the same pathway • Most major pathways in tumourigenesis are controlled by miRNAs

Summary

• HIF-1 is a transcription factor that is composed of HIF-1α and HIF-1β subunits. • More than 40 target genes have been found to be regulated by HIF-1. • HIF-1 expression is positively correlated with tumour vascularity, indicating that HIF-1 plays a crucial role in tumour angiogenesis progression. • HIF-1α is degraded by proteasome via VHL.

Characteristics of normal tissue cells in culture which are informative about cancer.

• Require hormone-like growth factors to survive/grow Serum contains factors which allow cells to pass the G1/S restriction point and multiply. Called a CONFLUENT MONOLAYER The serum requirement can almost be abolished in tumour cell lines. Infection of cells with RNA tumour virus. • Transfection with cDNA isolated from tumours.

Mouse Models of Carcinogenesis

• Viral infection models Infection with natural or engineered viruses • Chemical induced carcinogen models Treatment with cancer-causing chemicals • Transplantation ModelsImplantation/injection with tumour tissues or cells • Engraftment Models Incorporation of tumorigenic cells into normal tissues • Transgenic or Genetic Engineering Models Randomly inserting tumour-promoting DNA into the mouse genome (transgenic) or targeted manipulation of specific sites • Somatic Engineering Models Targeted manipulation of specific sites in the mouse

experiment 2

• KO mice deficient in Id1 and Id3 genes (no ability to initiate angiogenesis). • Inject breast cancer cells. • KO mice did not develop significant primary tumours and no metastases. conclusion : Inhibition of angiogenesis prior to initiation of carcinogenesis effectively prevents or significantly slows tumour growth Additional support for the idea that interfering with the process of angiogenesis can restrain tumor growth has come from genetic studies of mice. Scientists have recently created strains of mice that lack two genes, called Id1 and Id3, whose absence hinders angiogenesis. When mouse breast cancer cells are injected into such angiogenesis-deficient mutant mice, there is a small period of tumor growth, but the tumors regress completely after a few weeks, and the mice remain healthy with no signs of cancer. In contrast, normal mice injected with the same breast cancer cells die of cancer within a few weeks. When lung cancer cells are injected into the same strain of angiogenesis-deficient mutant mice, the results are slightly different. The lung cancer cells do develop into tumors in the mutant, but the tumors grow more slowly than in normal mice and fail to spread (metastasize) to other organs. As a result, the mutant mice live much longer than normal mice injected with the same kinds of lung cancer cells.

Neovastat

•Inhibits MMPs •Induces apoptosis of EC •Increases the level of angiostatin

microRNAs (miRNAs)

A small, single-stranded RNA molecule, generated from a hairpin structure on a precursor RNA transcribed from a particular gene. The miRNA associates with one or more proteins in a complex that can degrade or prevent translation of an mRNA with a complementary sequence. •miRNAs are encoded as segments of longer transcripts. •miRNAs are 21/22 nts generated from a longer hairpin RNA called a pri-miRNA. A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression.[1][2][3]miRNAs function via base-pairing with complementary sequences within mRNA molecules.[4] As a result, these mRNA molecules are silenced, by one or more of the following processes: (1) Cleavage of the mRNA strand into two pieces, (2) Destabilization of the mRNA through shortening of its poly(A) tail, and (3) Less efficient translation of the mRNA into proteins by ribosomes.[4][5] miRNAs resemble the small interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway, except miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA.[2] The human genome may encode over 1900 miRNAs,[6] although more recent analysis indicates that the number is closer to 600.[7] miRNAs are abundant in many mammalian cell types[8][9] and appear to target about 60% of the genes of humans and other mammals.[10][11] Many miRNAs are evolutionarily conserved, which implies that they have important biological functions.[7][3]For example, 90 families of miRNAs have been conserved since at least the common ancestor of mammals and fish, and most of these conserved miRNAs have important functions, as shown by studies in which genes for one or more members of a family have been knocked out in mice.

Countering Angiogenesis

Angiogenesis has become an important target for cancer research with the recognition that it is one of the critical events necessary for cancer growth and metastasis. As a tumor develops, its size is limited by the diffusion of metabolites from existing blood vessels. Tumor angiogenesis, the growth of the new blood vessels, is essential for cancerous tumors to keep growing and spreading. As a tumor grows, cells at the center become starved of oxygen, inducing the expression of a transcription factor - hypoxia inducible factor-1 (HIF-1) - which upregulates the expression of a range of angiogenic factors. Growth factor signaling also initiates HIF-1 activity, pre-empting the need for growing cells to maintain oxygen homeostasis. As a result, HIF-1 itself has been isolated as a therapeutic target for cancer. More than a dozen different proteins, as well as several smaller molecules, have been identified as angiogenic meaning that they are released by tumors as signals for angiogenesis. Inhibitors of angiogenesis (antiangiogenics) are currently the focus of intense cancer research. The hypoxic environment of arthritic joints also encourages angiogenesis, which in turn generates new blood vessels to supply the expanded synovial tissue with nutrients

EMT/MET switches during embryonic development and cancer

During carcinoma invasion and metastatic spread, the formerly epithelial phenotype of cancer cells (left) can—via the processes of partial or full EMT—evolve to a partial-EMT (middle) or a mesenchymal (right) phenotype. Generally, the further to the right of the EMT spectrum a cell is located, the less it attaches to other cells. The trade-off for this gain in motility is a decreased proliferative potential. MET is the reverse process. It is indicated by the arrow along the bottom of the figure Cancer cells adapt to the environmental requirements of the various steps of the invasion-metastasis cascade via changes in phenotype . EMT and MET are a canonical group of—at least transiently—observed phenotypic changes that are assumed to be crucial for metastatic spread . Various combinations of so-called EMT-inducing transcription factors (EMT-TFs) together with a number of extracellular molecules in the tumour microenvironment and related pathways are thought to trigger EMT . The cell-cell adhesion between formerly epithelial-like cancer cells is typically reduced upon activation of EMT. At the same time, the cancer cells tend to express more cell-matrix adhesion enhancing molecules like cadherin . As part of this combination of changes, the characteristic polygonal cobblestone-like cell shape of epithelial cells is progressively replaced by a spindle-shaped morphology, as shown on the right of Figure 3. Also, the motility and invasiveness of the cancer cells are enhanced . As another result of EMT, the cells become increasingly potent at degrading the underlying basement membranes of organs and vessels as well as the ECM via the expression of metalloproteases (MMPs). As a trade-off, they become less proliferative. MET, can reverse the phenotypic changes induced by EMT, thus—generally speaking—causing the cells to become less motile and invasive while enhancing their proliferative potential.

hypoxia

Hypoxia induces gene expression via transcription factor HIF 1 Genes such as HIF-1, whose activation is prompted by hypoxic conditions, can interact with enzymes and other transcription factors in order to control vascularization and tissue growth. While microenvironments surrounding cancerous tumors are extremely hypoxic, proliferation of such masses often is made possible by HIF-1 activation, which leads to increased angiogenesis and, thus, an increased oxygen supply to the area HIF-1 is a heterodimeric transcription factor consisting of a constitutively expressed β-subunit and an oxygen-regulated α-subunit. The HIF-1α and HIF-1β proteins both contain basic helix-loop-helix motifs that bind DNA and cause subunit dimerization oth subunits also have a Per-ARNT-Sim (PAS) domain, with similar functions. In the α-subunit, there is an oxygen-dependent degradation (ODD) domain, which is hydroxylated by proline-hydroxylase-2 (PHD-2), rendering the α-subunit vulnerable to proteasomal degradation under normoxic cellular conditions One important HIF-1 function is to promote angiogenesis; HIF-1 directs migration of mature endothelial cells toward a hypoxic environment [2,5]. This is done via HIF-1 regulation of vascular endothelial growth factor (VEGF) transcription. VEGF is a major regulator of angiogenesis, which promotes endothelial cell migration toward a hypoxic area. During hypoxia, HIF-1 binds the regulatory region of the VEGF gene, inducing its transcription and initiating its expression [12,15,16]. Such endothelial cells ultimately help to form new blood vessels, supplying the given area with oxygenated blood alpha and beta subunits bind to Hypoxia response element ( HRE) , hypoxia induces gene expression via transcription factor HIF1, beta = stable ( constitutive) , alpha = α - Addition of ubiquitin (normal oxygen levels) .. synthesis of them will promote destruction by proteasome of alpha

MALAT1 alters transcription and splicing in lung cancer metastasis

MALAT1 binds to unmethylated chromodomain protein Pc2 Pc2+MALAT1 prefers to bind activated histone marks Growth control genes activated. MALAT1 binds SR proteins And activates their phosphorylation Leading to activated alternative splicing of growth promoting mRNA isoforms The over-expression of MALAT1, strongly and remarkably conserved through evolution, has revealed a high risk of metastatic progression in patients with early stage lung cancer (Ji et al., 2003). Loss of function of MALAT1 in mice showed that this is a non-essential gene in development and in the normal homeostasisof adult tissues (Nakagawa et al., 2012; Zhang et al., 2012). Switching off MALAT1 in mouse pulmonary carcinoma cells alters the in vitro cell mobility and slows its metastatic progress through the animal (Gutschner et al., 2013), suggesting that the over-expression of MALAT1 in cancer could lead to gain-of-function phenotypes not observed during normal development of tissues of homeostasis.

hay flicks three phases leading to senescence

To demonstrate the mortality of cell lines in culture, Hayflick combined female fibroblasts that had undergone approximately 10 population doublings with an equal number of male fibroblasts that had undergone approximately 40 population doublings (2). He provided unmixed populations of each as controls and found only female cells existing in the mixed culture when the male control cells had stopped dividing, revealing an ability of older cells to somehow remember being older, even when surrounded by the relatively younger cells (2). Hayflick proposed that an intrinsic counting mechanism of cell divisions was responsible for his results, and not chronological differences, based on two findings: interrupting growth with cryopreservation showed senescence after the same number of cell divisions as control cells, and that normal human fetal cells all showed the same specific number of population doublings (1,2). These results were later published in Experimental Cell Research, and the total finite number of population doublings a cell can produce was coined the 'Hayflick Limit' (2). The Hayflick Limit coincides with the cellular event of senescence, since cell growth and division arrest once it is reached - senescence describes the state of cells after reaching their Hayflick Limit (3). Hayflick proposed three phases from his observations; Phase I was the primary culture, Phase II was the luxurious growth and cumulative population doublings, and Phase III was senescence (Figure 8.6.1) (4). Hayflick's finding remains true in vivo, seen through several lines of evidence (1). For example, epithelial cells of elderly donors have a highly increased frequency of an unusual form of β-galactosidase, which is also seen in senesced epithelial culture cells (1). β-galactosidase is typically active at pH 4, but as cells approach senescence the percentage of active β-galactosidase at pH 6 drastically increases (11). Therefore, a commonly used marker for senescent cells is senescence associated β-galactosidase activity (11).

General scheme for miRNA disruption in cancer

microRNA biogenesis. miRNA genes are usually transcribed by RNA polymerase II to produce the large primary transcripts termed pri-miRNAs, which are cleaved by a microprocessor complex, composed of RNA-binding protein DGCR8 and type III RNase Drosha, into an ~85-nucleotide stem-loop structure called pre-miRNA. Following transportation by Ran/GTP/Exportin 5 complex from nucleus to cytoplasm, the pre-miRNAs are processed by another RNase III enzyme Dicer to a ~20-22-nucleotide miRNA/miRNA* duplex. After the duplex is unwound, the mature miRNA is incorporated into a protein complex termed RISC. A miRNA-loaded RISC mediates gene silencing via mRNA cleavage and degradation, or translational repression depending on the complementarity between the miRNA and the targeted mRNA transcript. In addition, miRNAs may function as ligands to directly binding with Toll-like receptor (TLR), triggering downstream signaling pathways. Methyltransferase-like 3 (METTL3) is recently discovered to methylate pri-miRNAs, marking them for recognition and processing by DGCR8 to yield mature miRNA * Cancer cells present global downregulation of miRNAs, loss of tumour-suppressor miRNAs and specific accumulation of oncogenic miRNAs. The alteration in miRNA expression patterns leads to the accumulation of oncogenes and downregulation of tumour-suppressor genes, which leads to the promotion of cancer development. a, The expression and function of oncogenic miRNAs is increased by genomic amplification, activating mutations, loss of epigenetic silencing and transcriptional activation. By contrast, tumour-suppressor miRNAs are lost by genomic deletion, inactivating mutations, epigenetic silencing or transcriptional repression. b, After transcription, global levels of miRNAs can be reduced by impaired miRNA biogenesis. Inactivating mutations and reduced expression have been described for almost all the members of the miRNA processing machinery. If there is a downreguation of DROSHA this can lead to a decrease in the cropping of primary miRNA (pri-miRNA) to precursor miRNA (pre-miRNA). In the case of XPO5 mutation, pre-miRNAs are prevented from being exported to the cytoplasm. Mutation of TARBP2or downregulation of DICER1 results in a decrease in mature miRNA levels. Pol II, RNA polymerase II; RISC, RNA-induced silencing complex.

The Process of VEGF Signaling Pathway

when a VEGF binds to its receptor, the receptor can transiently exert its kinase activity and form a complex with an intracellular tyrosine or serine/threonine kinase. And then the activated receptors result in the activation of other proteins in the signaling pathway and the production of various second messengers. Finally, these signals are transmitted into the nucleus and induce the expression of specific genes, thereby modulating proliferation, survival, migration, and permeability of vascular endothelial cells. The binding of VEGF to VEGFR-2 leads to form receptor dimer and then activates the PLCγ (phospholipases C γ) and PKC-Raf kinase-MEK-mitogen-activated protein kinase (MAPK) pathway, which initiates DNA synthesis to promote endothelial cell proliferation. At the same time, Src signaling is provoked to start the activation of the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway, which leads to increased endothelial-cell survival. The activation of the Src signaling pathway will also cause complete systemic destruction of the entire basement membrane and cell wall, resulting in increased vascular permeability. And CDC42 and p38 mitogen-activated protein kinase (MAPK) signaling are incited. Activation of p38 MAPK further triggers actin reorganization and ultimately migration of endothelial cells.

Micro RNA processing

•Drosha is the nuclear pri-miRNA processor •Hands over to Dicer in the cytoplasm •Dicer presents single stranded miRNAs to RISC •RISC is a multiprotein complex that contains Argonaut (Ago) •Ago presenst miRNAs to their targets and mediates downstream effects n the metazoa (such as human), miRNAs are processed from the primary transcript using a two-step sequential mechanism involving two RNase III nucleases (Fig. 1). As indicated before, miRNAs are generated either from the processing of a host intron or by transcription from their own dedicated promoters. The primary precursor (pri-miRNA) is processed into an approximately 70 nucleotide long stem-loop structure by nuclear RNase III Drosha present in the microprocessor complex, which in mammals also contains the double-stranded RNA-binding protein, DGCR8. In Drosophila and C. elegans, the DGCR8 homolog is known as Pasha (43, 44). The two RNase domains of Drosha help cleave the 5' and 3' ends of the pri-miRNA, which determines the length of pre-miRNA (44). The resultant pre-miRNA is exported to the cytoplasm by a complex of Exportin-5 and Ran-GTP (45). The final maturation of miRNA occurs with the help of Dicer, another RNase III nuclease that processes the pre-miRNA into a 22 bp double-stranded RNA. The processing is often coupled with the formation of the ribonucleoprotein complex known as RISC (miRNA-Induced Silencing Complex) (Figs. 1, ​,3,3, ​,4).4). The RISC minimally consists of one strand of the miRNA (called "guide strand") in addition to Dicer, TRBP, PACT and Argonaute (Ago) proteins. The complex engages with the target to execute silencing while the other strand of the miRNA (called "passenger strand") is generally, but not always, destroyed. It is unclear how the asymmetric strand selection and passenger strand destruction occurs (extensively reviewed in 46-48 and references therein).

miR-200 family

•Regulates epithelial to mesenchymal transition (EMT) via E- cadherin.•ZEB1 & ZEB2 repress expression of E-cadherin during EMT. • miR-200s control ZEB1&2 and vice versa. Proposed model of EMT regulation by the miR-200 family. The miR- 200 family is able to enforce the epithelial phenotype through post-transcrip- tional repression of ZEB1/ZEB2 (ZEB) and TGFβ2. This allows expression of E-cadherin and polarity factors which are integral in forming cell-cell junc- tions. Upon stimulation by TGFβ, ZEB transcription factors are upregulated in expression resulting in their binding to E-box elements and repression of E-cadherin and polarity factor genes. This leads to EMT in some cell types, characterised by a loss of cell-cell contacts and apical-basal polarity, and acquisition of a spindle shape morphology with front end-back end polarity. The mesenchymal phenotype is sustained by high expression of ZEB, which is facilitated by its interaction with the miR-200 family (double-negative feed- back loop), and through autocrine TGFβ signalling (generated by loss of the miR-200 family relieving repression on TGFβ2). This process is reversible (MET) but the triggers of MET in this context are currently unknown.

Pro-Angiogenesis Factors

•Vascular Endothelial Growth Factor (VEGF) •Basic Fibroblast Growth Factor (bFGF) •Angiogenin•Epidermal Growth Factor (EGF)