TIN Week 6

Give 2 examples of alterations in photo-oncogenes or tumour suppressor genes currently targeted by specific drug therapy...

. Herceptin - involved in Breast cancer - HER 2 receptor - • Lots work on growth factor receptors Proto-oncogenes - HER2/neu is a proto-oncogene in normal cells that helps them grow. It becomes an oncogene when a cell has too many copies of this gene. When this happens, the cells make too much HER2/neu protein and the cancer is said to be HER2 positive. Patients with breast cancer with cells that are HER2 positive do not respond as well to certain chemotherapy drugs. But newer drugs such as trastuzumab (Herceptin®), lapatinib (Tykerb®), and several others, have been designed to specifically attack cells that are HER2 positive. These drugs can slow cancer cell growth and improve outcomes in patients with HER2 positive cancers. Breast cancers are now routinely tested to see if they are or the HER2 positive to identify which patients will benefit from these drugs. Other cancers also can be HER2 positive. Anti-HER2 therapy has also helped people with stomach cancer that is HER2-positive. - In chronic myeloid leukemia (CML), the cancer cells have a gene change called BCR-ABL that makes a type of protein called a tyrosine kinase. Drugs that target the BCR-ABL protein, such as imatinib (Gleevec®), are often very effective against CML. They lead to remission of the leukemia in most patients treated in the early stages of their disease. Tumour Suppressor Genes - RB's tumour suppressor function is largely mediated through the transcriptional repression of RB/E2F, which can be alleviated by phosphorylation on RB via cyclin/CDK complexes. Cyclin-CDK complexes themselves are subjects of multiple signalling pathways (Ras, β-catenin and NF-κB). Inhibitors for these oncogenic kinases have been shown to indirectly activate RB and restore transcriptional repressions. Such RB-reactivating agents include MEK inhibitor (Trametinib), PPARα agonist (Fenofibrate), and PI3K inhibitor (LY294002). - Development of p53 mimicking peptide inhibitors as therapeutic agents to disrupt p53/MDM2 interaction in tumour cells. A number of more effective small molecule inhibitors have been identified through high-throughput screening of large chemical libraries, including Nutlins and MI-219, which structurally mimic the p53 peptide and bind to MDM2 pocket. On the other hand, small molecules mimicking MDM2, such as RITA, have also been identified to bind p53, although the cellular activities for RITA seemed to be p53-independent. In addition, a recent study using computational methods has revealed a novel pocket in the p53 core domain, mutation of which can abolish the reactivation of p53 by known reactivating drugs. This pocket can be a potential target for direct pharmaceutical reactivation of p53

What is the role of the TSG APC and what does its mutation result in?

APC • encodes factor that negatively regulates WNT pathway in colonic epithelium by forming of a complex that degrades β-catenin • mutated in familial adenomatous polyposis (FAP) - disorder associated with development of 1000s of colonic polyps and early onset colon carcinoma - at a young age, they are not a particular problem, but they will develop into cancer in a few decades time • also mutated in 70% of sporadic colon carcinomas

How is tumour-promoting inflammation an enabling characteristic of cancer?

Chronic inflammation of cancer can benefit the tumour by : • release of growth factors / proteases that promote proliferation • inhibition of growth suppressors (eg adhesion molecules) • enhancing resistance to cell death • inducing angiogenesis • facilitating invasion and metastasis (eg by degrading ECM or by inducing tumor cell mobility) • Contributing to immune evasion (eg activating M2 macrophages) Not only does cancer cause inflammation, but inflammation can lead to tumour growth

What is the role of the TSG PTEN and what does its mutation result in?

PTEN • negative regulator of PI3K/AKT signalling • loss-of-function mutations associated with both familial and diverse sporadic cancers

What is the TSG Rb the governor of? Where is it located and what are its functions? What can mutations in it lead to?

Rb - Governor of Proliferation • normal retinoblastoma gene product (Rb protein) controls the cell cycle at the checkpoint transition from G1 to S phase • key cell cycle regulator required for normal cell cycle regulation in every cell • RB1 gene located on the Q arm of chromosome 13 • Rb protein binds regulatory transcription factor E2F which is required for synthesis of DNA replication enzymes • when Rb is bound to E2F, transcription/replication is blocked. • growth factors (via Ras pathway) activate CDK4/6 which in turn phosphorylates and inhibits Rb, removing block of E2F, and transition to S phase occurs • Disruption/deletion of the Rb gene therefore leads to uncontrolled cell proliferation • directly or indirectly inactivated in most human cancers

What is the role of the TSG TP53 and what is it the guardian of? What are its roles and what are the implications of its mutation?

TP53 - Guardian of the Genome • tumor suppressor gene that regulates cell cycle progression, DNA repair, cellular senescence and apoptosis • most frequently mutated gene in human cancers • loss of function mutations in more than 50% of cancers • TP53 mutations occur in virtually every type of cancer • inheritance of a mutated copy of TP53 predisposes individuals to malignant tumours - Li-Fraumeni syndrome - 25 X greater chance of developing a malignant tumor by age 50 - they have one hit already - only need 1 more hit to develop cancer - high risk • p53 loss has important therapeutic implications - radiation therapy and conventional chemotherapy work by inducing DNA damage and subsequent apoptosis - tumours may not undergo apoptosis because there are mutations in the apoptosis pathway - cells with wild type TP53 alleles are more likely to be killed - thus some lung cancers and colorectal cancers, which frequently carry TP53 mutations, are relatively resistant

Examples of TSG/DNA Repair Genes a) target type b) gene c) functional alteration d) cancers involved e) targeted therapy

• can argue that tumour suppressor genes are DNA repair genes • there are no targeted therapy drugs for these yet - harder to switch things on where they have been switched off - gene therapy is on the horizon

Name the 10 different hallmarks of cancer...

• Characteristic cellular genetic alternations and processes that cancers need to have in order to be cancer cells • Sustained proliferative signalling - oncogenes • Evading growth suppressors - tumour suppressor genes - The above 2 are the most important characteristics and targets of cancer drugs • Resisting cell death - apoptosis • Enabling replicative immortality - cancer cells keep replicating • Inducing angiogenesis - to enable quick growth - tumours need blood vessels • Activating invasion and metastasis - have mechanisms to leave the primary site and go to other tissues (normal cells can't do this) ABOVE ARE THE ORIGINAL 6 HALLMARKS NEW HALLMARKS: • Deregulating cellular energetics - reprogramming metabolism • Avoiding immune destruction - important in terms of drug and immunotherapies ENABLING CHARACTERISTICS - DON'T REALLY NEED TO KNOW THESE • Processes that make the situation worse - Genome instability and mutation - Tumour-promoting inflammation - Inflammation can lead to increases in the other factors

How is genome instability and mutation an enabling characteristic of cancer?

• DNA repair genes are not directly oncogenic; however, defective proteins permit mutations to occur in other genes • solid tumours show high degree of genetic abnormalities, such as aneuploidy, chromosome translocations etc. • likely due to the lack of active p53, and the ability of cancer cells to avoid cell death through apoptosis. • example - BRCA1 or BRCA2 mutations in breast cancer • DNA repair and tumour suppressor genes belong in the same camp Need problems in the DNA repair genes for everything to go crazy in cancer in the first place

What are some examples of targeted therapy for the hallmarks of cancer?

• Drugs may be being used clinically now, or in clinical trials

What drug therapy could be used to target this aberrant protein product? How does it work?

• Inhibitors of the BCR-ABL tyrosine kinase (e.g. imatinib and niiltinib) induce complete remission in a high fraction of patients with little associated ctoxicity • Treatment with tyrosine kinase inhibitors (particularly in patients with early disease) induces sustained remissions - may prevent the appearance of blast crisis by suppressing the proliferative dirve that leads to the acquisition of additional mutations - When patients on tyrosine kinase inhibitors relapse, their tumors frequently are found to have acquired mutations in the kinase domain of BCR-ABL that prevent the drugs from binding.. The selective outgrowth of these cells is explained by the powerful antitumor effects of BCR-ABL inhibitors, and indicates that many resistant tumors are still "addicted" to the pro-growth signals created by BCR-ABL. - For those with resistant disease, hematopoietic stem cell transplantation is curative in 70% of patients, but carries with it substantial risks, particularly in the aged.

What are some mechanisms that cancer cells use to evade the host immune response?

• Loss or reduced expression of histocompatibility molecules - Tumor cells may fail to express normal levels of human leukocyte antigen (HLA) class I, escaping attack by CTLs. Such cells, however, may trigger NK cells. • Immunosuppression. - Many oncogenic agents (e.g., chemicals, ionizing radiation) suppress host immune responses. - Tumors or tumor products also may be immunosuppressive. - For example, TGF-β, secreted in large quantities by many tumors, is a potent immunosuppressant. - In some cases, the immune response induced by the tumor may inhibit tumor immunity. Mechanisms: ϖ May lead to engagement of the T cell inhibitory receptor, CTLA-4, or activation of regulatory T cells that suppress immune responses. ϖ Expressions of FasL on tumours, which can engage Fas on immune cell surfaces and induce the immune cell to enter apoptosis! • Antigen masking. - Many tumor cells produce a thicker coat of external glycocalyx molecules, such as sialic acid-containing mucopolysaccharides, than normal cells. - This thick coat may block access of immune cells to antigen-presenting molecules, thereby preventing antigen recognition and cell killing. • Downregulation of co-stimulatory molecules. - Costimulatory molecules are required to initiate strong T cell responses. - Many tumors reduce expression of these costimulatory molecules.

What is CML and what are the symptoms?

• Principally affects adults between 25 and 60 y/o • Chronic myeloproliferative disorder - is marked by the hyperproliferation of neoplastic myeloid progenitors that retain the capacity for terminal differentiation; as a result, there is an increase in one or more formed elements of the peripheral blood.

What is the role of the TSG TGF-beta and what does its mutation result in?

• TGF-β (via TGF-β receptors ) is potent inhibitor of proliferation (different to other growth factors) • loss-of-function mutations in TGF-β receptors or downstream signal pathways are involved in numerous cancers

How is cancer related to inflammation?

• cancer can be viewed as a form of tissue injury as tissues/organs affected fail to function correctly • hypoxia leads to necrosis and inflammatory response - it is not corrected - is like chronic inflammation - get recruitment of WBCs, etc. in a lot of cancers • surrounding stromal tissues respond by releasing chemokines/cytokines leading to a chronic inflammatory response • chronic inflammation of cancer contributes to systemic signs and symptoms (eg anemia, fatigue, cachexia - weight loss and loss of muscle)

What are some examples of mutations that can occur that affect signal transduction pathways in cancer? What are the consequences?

• RAS family genes (HRAS, KRAS, NRAS) - most common oncogene in human tumors - 20% of all tumours, 90% of pancreatic adenocarcinomas - activated by growth factors binding to RTKs - RAS proteins are small G proteins that switch between inactivated (GTP-bound) and activated (GDP-bound) states ϖ GTP gets hydrolysed (so it doesn't stay activated - automatically switch itself off) - active RAS stimulates both MAPK and PI3K/AKT pathways, which phosphorylate and activate effectors which promote growth - RAS activation terminated due to intrinsic GTPase activity o accelerated by GTPase-activating proteins (GAPs), which bind to active RAS and greatly enhance GTPase activity - numerous RAS point mutations have been identified which can: a) reduce GTPase activity of the RAS protein, locking RAS in the activated state b) mutations of GAPS - eg NF-1 a tumor suppressor gene - these usually accelerate the 'switching-off' - without these, the receptor will be constituently active • Other mutated downstream factors in the RTK (tyrosine kinase receptor) pathway - BRAF - gain of function mutations in BRAF stimulate downstream kinases and activate transcription factors (60% of melanomas) - PI3K family - stimulated by RTK activation o activates multiple kinase cascades (eg AKT, mTOR, BAD) which increase growth and cell survival - PTEN - tumor suppressor gene which normally inhibits PI3K o loss of function mutation in endometrial carcinomas • Nonreceptor tyrosine kinases - mutations create fusion genes that encode constitutively active cytoplasmic tyrosine kinases - e.g. BCR-ABL tyrosine kinase (a fusion product where 2 genes combine) in chronic myelogenous leukemia (CML) - ABL gene translocated and fuses with BCR gene - successful drugs that inhibit BCR-ABL (eg imatinib) o rare CML stem cells with BCR-ABL fusion gene persist o resistant to therapy and can repopulate if treatment stopped - other mutations include loss of function point mutations in inhibitory regulators of tyrosine kinases o eg JAK2 (JAK/STAT signaling pathway) all of these steps can be potentially mutated in cancer -

What is the role of the TSG CDKN2A and what does its mutation result in?

• functions as a negative regulators of cell cycle entry / progression • encodes tumor suppressor proteins including p16/INK4a (CDK inhibitor) and ARF (stabilizes p53) • loss-of-function mutations in diverse familial and sporadic cancers

How are growth factor receptors involved in sustaining proliferative signalling as a hallmark of cancer? What are some targeted therapies for this?

• many oncogenes encode growth factor receptors • drive malignant transformation by constitutive activation i.e. activation in the absence of ligand binding • most important are the receptor tyrosine kinase (RTK) family - may be altered genetically to be constituently active or be more sensitive to a growth factor • Examples - point mutations in ERBB1 in lung adenocarcinomas o overexpression leads to constitutive activation of RTKs or receptors become more sensitive to growth factors o constitutive activation - receptor is active without a ligand - doesn't have to respond to any normal signals - doesn't need growth factors to stimulate it anymore - amplification of ERBB2 which causes overexpression and constitutive activation of HER2 tyrosine kinase receptors in breast cancers - fusion of tyrosine kinase ALK with another gene EML4 in some lung adenocarcinomas which has constitutive tyrosine kinase activity ϖ genes are combined to produce something with constitutive activity 3 different types of mutations • targetted therapies - antibodies or specific small molecule inhibitors of constitutively active RTKs - eg drugs that block HER2 activity in breast cancers with ERBB2 amplification and overexpression of HER2 • many tumours develop alternative activating mutations in other signalling molecules (eg another TK) - resulting in resistance - due to subclones within the genetically heterogeneous tumour cell population

What are the roles of the proteins that tumour suppressor genes encode and how can they be involved in evading growth suppressors as a hallmark of cancer?

1. Evading Growth Suppressors - TSGs • cancer cells may not respond to molecules that inhibit proliferation of normal cells, usually because of inactivation of tumour suppressor genes Tumour suppressor genes encode proteins that are: • receptors for growth factors that inhibit cell proliferation • negative regulators of cell cycle entry or progression • negative regulators of growth signalling pathways • checkpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal proteins that promote apoptosis • regulators of cell adhesion • DNA repair enzymes

How is activating invasion and metastasis a hallmark of cancer? What is it caused by? To invade surrounding tissue, what must tumour cells do?

1. Activating Invasion and Metastasis • tumour invasion and metastases cause majority of cancer deaths • about a cell getting across ECM and basement membrane and into the blood • occurs through interplay of processes that are intrinsic to tumor cells and signals that are initiated by the stroma • to invade surrounding tissue, tumour cells must : a) detach from adjacent cells (loss of integrin, cadhedrins these molecules are involved in keeping cells connected) b) degrade the ECM (produce protesases eg MMP) c) attach to and migrate through ECM (express adhesion molecules) d) migrate (increased locomotion) • tumour cells form aggregates in the bloodstream or adhering to circulating leukocytes and platelets - this may confer some protection from host antitumour effector mechanisms • Exactly where tumor cell emboli eventually lodge and begin growing is influenced by : - vascular and lymphatic drainage from the primary tumour - interaction with specific receptors eg some tumour cells express CD44 adhesion molecules that bind to venules in lymph nodes - the microenvironment of the organ or site (eg tissue rich in protease inhibitors might be resistant to penetration by tumor cells)

What are the roles of the TSGs BRCA1 and BRCA2 and what do their mutations result in?

BRCA1 and BRCA2 • involved in the repair of double-stranded DNA breaks by homologous recombination • mutations in BRCA1 and BRCA2 account for 25% of cases of familial breast cancer • BRCA1 mutations - also higher risk of ovarian cancers in women, higher risk of prostate cancer in men • BRCA2 mutations - higher risk of breast cancer in both men and women as well as many other cancers • cells that express mutated BRCA genes develop chromosomal breaks and severe aneuploidy. • rarely inactivated in sporadic cancers - different from other tumor suppressor genes, (eg APC, p53) which are inactivated in both familial and sporadic cancers - more involved in familial breast cancer - what people are screened for to see their potential to develop cancer or prognosis

How is inducing angiogenesis a hallmark of cancer? What is it caused by? How does it promote cancer progression?

1. Inducing Angiogenesis • tumour cells need vascular supply to provide adequate nutrition and waste removal; thus must induce angiogenesis • angiogenic switch involves production of angiogenesis factors or the loss of inhibitors eg thrombospondin-1, angiostatin, endostatin • released from tumour & stroma, inflammatory cells • hypoxia is a major stimulus for angiogenesis, via HIF1α (normally activated in hypoxic environments such as a high altitude)• important endothelial growth factors include VEGF, bFGF • new tumor vessels differ from normal vasculature by being dilated and leaky with slow abnormal flow • angiogenesis also promotes cancer progression indirectly by: - stimulating tumor growth via endothelial cell production of growth factors - providing route for metastasis - (they can get into circulation)

What are the 5 categories of ways in which tumour cells can sustain proliferative signalling?

1. Sustaining Proliferative Signalling • cancer cells have the capacity to proliferate without external stimuli • consequence of oncogene activation • most fundamental trait of cancer cells • proto-oncogenes, oncogenes and oncoproteins • 5 categories - growth factors - growth factor receptors - signal transduction pathways - nuclear transcription factors - cell cycle regulators

How can cancer cells avoid immune destruction (as a hallmark of cancer)?

8. Avoiding immune destruction • innate and adaptive immune system can recognize and eliminate cells displaying abnormal antigens (eg a mutated oncoprotein) - because of change to oncogenes, tumour cells display molecules that aren't present on normal cells - allow the immune system to detect and kill them • immunosuppressed patients have an increased risk for cancer - not all cancers - predominantly cancers related to viruses • cancer cells show alterations that allow them to evade host immune response • eg selective growth of antigen-negative variants • eg loss or reduced expression of histocompatibility antigens eg increased expression of immunosuppressive factors • tumour cells that don't express the tumour antigens will not be detected and will be favoured as they are able to proliferate • Mutations in MHC genes or genes needed for antigen processing • Production of immunosuppressive proteins or expression of inhibitory cell surface proteins

Clinical features of CML...

Clinical Features: • Onset is often insidious - intial symptoms usually non-specific • Splenomegaly - have a dragging sensation in the abdomen • Usually slow progression • Without treatment, median survival is 3 years • After a variable (and unpredictable) period , approximately half of CML cases enter an accelerated phase marked by increasing anemia and new thrombocytopenia, the appearance of additional cytogenetic abnormalities, and then into a picture resembling acute leukemia (blast crisis) • In the remaining 50% of cases, blast crisis occurs abruptly, without an accelerated phase. - In 30% of cases the blast crisis resembles precursor-B cell ALL, further attesting to the origin of CML from hematopoietic stem cells. - Remaining 70% of cases, the blast crisis resembles AML. - CML progresses to a phase of extensive bone marrow fibro- sis resembling primary myelofibrosis. • receptor tyrosine kinases have a TK built into their structure • ABL is a non-receptor tyrosine kinases - tyrosine kinase enzymes that aren't part of the receptor on the membrane - Tyrosine kinases phosphorylate the tyrosine residue

A 24-year-old woman with a family history of colon cancer undergoes a routine colonoscopy to check for neoplasia. The test reveals hundreds of polyps in her colon. What is the likely diagnosis? What gene mutations would likely be responsible for this? How does the genetics of this disorder compare to hereditary nonpolyposis colon cancer syndrome (Lynch syndrome)? What is the prognosis for this patient?

Familial Adenomatous Polyps - due to the family history of autosomal dominant disorder, appearance and early onset - A count of at least 100 polyps is necessary for a diagnosis of classic FAP Colorectal adenocarcinomas develop in all patients with familial adenomatous polyps Adenomatous polyposis coli (APC) gene HNPCC is caused by inherited germline mutations in genes that encode proteins responsible for the detection, excision, and repair of errors that occur during DNA replication. At least five such mismatch repair genes have been recognized, but a majority of HNPCC cases involve either MSH2 or MLH1. Patients with HNPCC inherit one mutated DNA repair gene and one normal allele. When the second copy is lost through mutation or epigenetic silencing, defects in mismatch repair lead to the accumulation of mutations at rates up to 1000 times higher than normal, mostly in regions containing short repeating DNA sequences referred to as microsatellite DNA. If left untreated she will undoubtedly develop colorectal adenocarcinomas Hence, standard treatment for FAP is prophylactic colectomy for persons carrying APC mutations.

What is Lynch Syndrome and how does it relate to the hallmarks of cancer?

Hereditary Nonpolyposis Colon Cancer Syndrome (Lynch Syndrome) • inherit one defective copy of DNA repair genes involved in mismatch repair (e.g., MSH2 and MLH1) • second hit occurs in a colonic epithelial cell • gradual accumulation of errors in multiple genes including proto-oncogenes and tumor suppressor genes • mismatch repair mutations show microsatellite instability - regions of repetitive DNA sequences prone to shortening or extension when mismatch repair enzymes are defective - genetic analysis of these regions can be used to identify such defects - MICROSATELLITE INSTABILITY OCCURS WHEN THERE ARE MUTATIONS IN THE MISMATCH REPAIR GENES - Get repeats of the sequences - can detect these • treated with surgery • only make up a small portion of all colon cancers

How are transcription activators/cell cycle regulators involved in sustaining proliferative signalling as a hallmark of cancer? What are some targeted therapies for this?

MYC • most commonly involved in human tumours • master transcriptional regulator of cell growth • activates expression of genes involved in cell growth eg D cyclins • MYC can be directly altered, or affected by mutations involving upstream pathways which elevate MYC levels • Responsible for most cases of Burkitt's Lymphoma Cyclins, CDKs and CDK inhibitors • regulate cell cycle progression via checkpoints • eg. gain of function mutations in D cyclin / CDK4 which promote G1/S progression • eg loss of function mutations in tumor suppressor genes that inhibit G1/S progression (eg p16, RB, TP53) - leads to progression through the cell cycle

What is the TSG Rb the governor of? Where is it located and what are its functions? What can mutations in it lead to?

Rb - Governor of Proliferation • normal retinoblastoma gene product (Rb protein) controls the cell cycle at the checkpoint transition from G1 to S phase • key cell cycle regulator required for normal cell cycle regulation in every cell • RB1 gene located on the Q arm of chromosome 13 • Rb protein binds regulatory transcription factor E2F which is required for synthesis of DNA replication enzymes • when Rb is bound to E2F, transcription/replication is blocked. • growth factors (via Ras pathway) activate CDK4/6 which in turn phosphorylates and inhibits Rb, removing block of E2F, and transition to S phase occurs • Disruption/deletion of the Rb gene therefore leads to uncontrolled cell proliferation • directly or indirectly inactivated in most human cancers • Looking at gene therapy for treatment, but Rb is involved in every cell - haven't had much success with treatment • Hypophosphorylated Rb in complex with the E2F transcription factors binds to DNA, recruits chromatin remodeling factors (histone deacetylases and histone methyltransferases), and inhibits transcription of genes whose products are required for the S phase of the cell cycle. • When Rb is phosphorylated by the cyclin D-CDK4, cyclin D-CDK6, and cyclin E-CDK2 complexes, it releases E2F. The latter then activates transcription of S-phase genes. • The phosphorylation of Rb is inhibited by CDKIs, because they inactivate cyclin-CDK complexes. • Virtually all cancer cells show dysregulation of the G1-S checkpoint as a result of mutation in one of four genes that regulate the phosphorylation of Rb; these genes are RB, CDK4, cyclin D, and CDKN2A [p16]. EGF, epidermal growth factor; PDGF, platelet-derived growth factor.

What are some ligands that stimulate receptors with intrinsic tyrosine kinase activity and what are the signalling mechanisms involved?

Receptors with intrinsic tyrosine kinase activity: • ligands = EGF, VEGF, FGF, HGF • Signalling Mechanisms = Ligand binding one chain of the receptor activates tyrosine kinase on the other chain, resulting in activation of multiple downstream signalling pathways (RAS-MAP kinase, PI-3 kinase, PLC-gamma) and activation of various transcription factors - Binding of ligand to the extracellular portion of the receptor causes dimer- ization and subsequent phosphorylation of the receptor subunits. - Once phosphorylated, the receptors can bind and activate other intracellular proteins (e.g., RAS, phos- phatidylinositol 3[PI3]-kinase, phospholipase Cγ [PLC- γ]) and stimulate downstream signals that lead to cell proliferation, or induction of various transcriptional programs. IMAGE • General pathway by which growth factors work • GFR on the plasma membrane • Complicated signal transduction pathways downstream of the receptor tyrosine kinases - these are mutated in many cancers - become an important target for drugs - This identifies what the mutations are and target this - new approach to treatment (not like chemotherapy which targets highly proliferative cells - lots of side effects) • Nuclear transcription factors regulate entry into the cell cycle, etc. • Inactive RAS converted to active RAS with conversion of GDP to GTP - similar to GPCRs • Pathways to know - RAS, MAPK, PI3K

What are some examples of ligands that act via receptors with intrinsic tyrosine kinase activity and their main signalling mechanism?

Receptors with intrinsic tyrosine kinase activity: • ligands = EGF, VEGF, FGF, HGF • Signalling Mechanisms = Ligand binding one chain of the receptor activates tyrosine kinase on the other chain, resulting in activation of multiple downstream signalling pathways (RAS-MAP kinase, PI-3 kinase, PLC-gamma) and activation of various transcription factors - Binding of ligand to the extracellular portion of the receptor causes dimer- ization and subsequent phosphorylation of the receptor subunits. - Once phosphorylated, the receptors can bind and activate other intracellular proteins (e.g., RAS, phos- phatidylinositol 3[PI3]-kinase, phospholipase Cγ [PLC- γ]) and stimulate downstream signals that lead to cell proliferation, or induction of various transcriptional programs. • General pathway by which growth factors work • GFR on the plasma membrane • Complicated signal transduction pathways downstream of the receptor tyrosine kinases - these are mutated in many cancers - become an important target for drugs - This identifies what the mutations are and target this - new approach to treatment (not like chemotherapy which targets highly proliferative cells - lots of side effects) • Nuclear transcription factors regulate entry into the cell cycle, etc. • Inactive RAS converted to active RAS with conversion of GDP to GTP - similar to GPCRs • Pathways to know - RAS, MAPK, PI3K

Given that the RB protein is expressed in all cells, one why do patients with germline RB mutations only a few types of cancer, such as retinoblastoma?

• Don't know the answer to this - There may be other members of Rb gene family in tissues other than the retina that can compensate • Rb is pretty central to the cell cycle - Evidence suggests that homozygous loss of RB1 triggers apoptosis, and that unrestrained action of E2F proteins (as would occur with loss of both RB1 alleles) not only drives the cell cycle, but also triggers apoptosis. ϖ Plausible that although in most tissues, homozygous loss of RB1 induces cell death, the retinoblasts are relatively resistant to the apoptosis-inducing effect. In these cells, therefore, dysregulated E2F gives rise to tumors. - There are also likely parallel regulatory pathways in different cell types. • While some cell types, e.g. breast, lung or bladder epithelial cells, require additional loss of other tumor suppressor genes or activation of oncogenes, the proliferation of retinoblasts in early childhood may be uniquely controlled by Rb, such that few other genetic changes are required for tumor formation • May be because retinoblasts terminally differentiate by the age of six years, and thus do not need extensive safeguards against uncontrolled proliferation.

Name of chromosomal alteration in chronic myeloid leukaemia and resulting protein product...

• Gene fusion - oncogenic translocation that create encoding novel chimeric proteins • This example - Philadeplphia (Ph) chromosome in CML • BCR-ABL rearrangement is the crucial consequence of Ph translocation - has potent tyrosine kinase activity • Balanced Translocation RESULTING PRODUCT: • Balanced translocation between chromosome 9 and 22 results in Philadelphia chromosome (pH) - leads to formation of BCR/ABL fusion gene - There is no ABL/BCR protein - Final product of this genetic rearrangement is a 210 kDa cytoplasmic fusion protein - p210BCR/ABL - essential and sufficient for the malignant transformation of CML - The BCR-ABL hybrid protein maintains the tyrosine kinase domain; the BCR domain self- associates, a property that unleashes a constitutive tyrosine kinase activity. - There is cross-talk between BCR-ABL and RAS pathways, since BCR-ABL protein activates all of the signals that are downstream of RAS. - BCR-ABL fusion gene formation is an early, perhaps initiating, event that drives leukemo- genesis. Development of leukemia probably requires other collaborating mutations, but the transformed cell continues to depend on BCR-ABL for signals that mediate growth and survival. - BCR-ABL signaling can be seen as the central lodgepole around which the structure is built. If the lodgepole is removed by inhibition of the BCR-ABL kinase, the structure collapses

How can cancer cells resist cell death (as a hallmark of cancer)?

• cancer cells are resistant to apoptosis • cancer progression requires mutations in pathways that would otherwise induce apoptosis in cancer cells • example - BCL-2 overexpression in follicular B-cell lymphomas - there is more inhibition of apoptosis because of this • Genes not directly related to the BAL family can also regulate apoptosis - eg p53 which normally induces apoptosis when DNA repair is ineffective - eg reduced levels of Fas (CD95) can also render tumor cells less susceptible to apoptosis through extrinsic pathways involving Fas ligand (FasL) ϖ part of extrinsic pathway of apoptosis

Stem cell theory of carcinogenesis (as part of enabling replicative immortality as a hallmark of cancer)

• cancer stem cells (CSCs) have been identified in some tumours (eg acute myeloid leukemia • like normal SCs, CSCs divide infrequently, are capable of self- renewal, assymetric division and generation of a diversity of mature cell types - different to other cells in the cancer - impacts on treatment and why cancer can reoccur after treatment • role in cancer is uncertain • traditional chemotherapy targets rapidly dividing non- CSCs - may spare quiescent CSCs, may explain instances of cancer recurrence - because the CSCs don't divide rapidly, they will survive treatment and be able to repopulate • therapies being developed to specifically target CSCs

What is the role of the TSG E-Cadherin/Beta-Catenin and what does its mutation result in?

• cell surface protein that maintains intercellular adhesiveness • inhibit proliferation due to contact inhibition - involved in metastases if altered • binds to β-catenin • when there is loss of cell-cell contact (eg epithelium injury), interaction between E-cadherin and β-catenin are disrupted • promotes translocation of β-catenin to nucleus, where it stimulates genes that promote proliferation - normal step in repair process • cell to cell E-cadherin connections inhibit proliferation (contact inhibition) • mutation of the E-cadherin/β-catenin axis results in loss of contact inhibition in many cancers • contributes to the local invasion and metastatic ability Alpha-oxidation of very long chain fatty acids and amino acid catabolism Alpha-oxidation of very long chain fatty acids and amino acid catabolism- beta-catenin ϖ Binds to the cytoplasmic portion of E-cadherin - inhibits cell proliferation action ϖ Translocates to the nucleus to activate cell proliferation - this occurs when cell to cell contact is lost and consequently E-cadherin and β-catenin interaction is broken such as epithelial injury hence stimulates cell proliferation to replace injured cells - E-cadherin: mediates cell-cell contact in epithelial layers - Mutation of the E-cadherin/β-catenin axis results in loss of contact inhibition in many cancers and therefore cell proliferation is constitutively active. This mutation contributes to metastasis and local invasion. - Involved in adhesion and trafficking of cells and connections with ECM - Mutations in which cells can disconnect from one another and infiltrate tissues - Contact inhibition - when cells are stuck to one another, they inhibit growth because they have a neighbour on each side (don't think they need to proliferate)

Familial Adenomatous Polyposis - pathogenesis and gene involved

• disorder associated with development of thousands of colonic polyps and early onset colon carcinoma • individuals born with one mutant allele (hit) develop thousands of adenomatous polyps in their teens or 20s - get the second hit quite early in their lives • both copies of the APC gene must be lost (2 hits) • invariably, some of the polyps will undergo malignant transformation to colon cancer • additional mutations must occur for adenomas to progress to cancers • APC is a component of the WNT signaling pathway - major role in controlling cell fate, adhesion, polarity • treated with prophylactic surgery (to prevent it progressing onto colon cancer) • NSAIDs significantly decrease number of polyps • APC is an intermediate molecule in a transcription pathway • WNT is another growth factor which is involved - involved in lots of regulation of cellular proliferation, cell communication, invasion/metastases, etc. • Normal signaling pathway that is activated by GF's • APC normally forms a destruction complex with beta-catenin - leads to beta-catenin being degraded - can't go into nucleus and activate transcription

How are growth factors involved in sustaining proliferative signalling as a hallmark of cancer? What are some examples?

• growth factor genes are not usually mutated • instead, some cancer cells develop ability to synthesise the same growth factors to which they are responsive • produces an autocrine stimulation loop - normally cells that release the growth factors don't respond to them, but this changes in cancer - a cell will release a GF which acts positively back on the cell to stimulate growth • increased GF = increased replication = increases risk of acquiring mutations during increased proliferation • examples - PDGF, FGF, TGF-alpha

How are growth factor receptors involved in sustaining proliferative signalling as a hallmark of cancer? What are some targeted therapies for this?

• many oncogenes encode growth factor receptors • drive malignant transformation by constitutive activation i.e. activation in the absence of ligand binding • most important are the receptor tyrosine kinase (RTK) family • Examples - point mutations in ERBB1 in lung adenocarcinomas o overexpression leads to constitutive activation of RTKs or receptors become more sensitive to growth factors - amplification of ERBB2 which causes overexpression and constitutive activation of HER2 tyrosine kinase receptors in breast cancers - fusion of tyrosine kinase ALK with another gene EML4 in some lung adenocarcinomas which has constitutive tyrosine kinase activity • targetted therapies - antibodies or specific small molecule inhibitors of constitutively active RTKs - eg drugs that block HER2 activity in breast cancers with ERBB2 amplification and overexpression of HER2 • many tumours develop alternative activating mutations in other signalling molecules (eg another TK) - resulting in resistance - due to subclones within the genetically heterogeneous tumour cell population

How are signal transduction pathways involved in sustaining proliferative signalling as a hallmark of cancer? What are some targeted therapies for this?

• most common are downstream components of the RTK signalling pathway • RTK activation stimulates RAS and two main downstream signalling pathways - MAPK and PI3K/AKT most human cancers show defects in these pathways IMAGE • General pathway by which growth factors work • GFR on the plasma membrane • Complicated signal transduction pathways downstream of the receptor tyrosine kinases - these are mutated in many cancers - become an important target for drugs - This identifies what the mutations are and target this - new approach to treatment (not like chemotherapy which targets highly proliferative cells - lots of side effects) • Nuclear transcription factors regulate entry into the cell cycle, etc. • Inactive RAS converted to active RAS with conversion of GDP to GTP - similar to GPCRs • Pathways to know - RAS, MAPK, PI3K

CML pathogenesis and hallmark of cancer involved

• neoplasm of pluripotent hematopoietic stem cells leading to preferential proliferation of granulocytic progenitors • always associated with gene translocation resulting in a BCR- ABL fusion gene, which produces a constitutively active BCR- ABL tyrosine kinase - ABL should normally be inactive, unless activated by a growth factor, but it gets constituently activated • constitutively activates same pathways normally stimulated by GFs and involved in stimulation of myeloid progenitors • marrow is markedly hypercellular due to massively increased numbers of maturing granulocytic precursors • blood tests show marked leukocytosis • spleen often greatly enlarged due to extensive extramedullary hematopoiesis • imatinib (BCR-ABL kinase inhibitor), markedly decreases BCR-ABL-positive cells and yields sustained remission in 90% of patients (if given in early phase of disease) - tyrosine kinase inhibitor • HALLMARK OF CANCER = Sustained proliferative signaling

How is deregulating cellular energetics a hallmark of cancer? What does it form the basis of?

• reprogramming of energy metabolism • tumour cells undergo a metabolic switch to aerobic glycolysis (Warburg effect) from oxidative phosphorylation - get lots less ATP • shunts more metabolites into intermediates that can be used to support cellular synthetic pathways - tumours need to be able to produce lipids and proteins to grow their architecture more quickly • enables synthesis of the macromolecules and organelles needed for rapid cell growth • basis for PET imaging of tumours • In addition to doubling its DNA content before division, an actively dividing cell (whether normal or transformed) must also double all of its other components, including membranes, proteins, and organelles. • This task requires increased uptake of nutrients, particularly glucose and amino acids. • Studies of intermediate metabolism suggest that in rapidly growing cells glucose is the primary source of the carbons that are used for synthesis of lipids (needed for membrane assembly) as well as other metabolites needed for nucleic acid synthesis. • This pattern of glucose carbon use is achieved by shunting pyruvate toward biosynthetic pathways at the expense of the oxidative phosphorylation pathway and ATP generation. • Tumor cells that adapt this altered metabolism are able to divide more rapidly and outpace competing tumor cells that do not. • If cancer cells do this and it allows them to out-populate other cells, it must be advantageous • enables synthesis of the macromolecules and organelles needed for rapid cell growth • basis for PET imaging of tumours (week 10) • Studies of intermediate metabolism suggest that in rapidly growing cells glucose is the primary source of the carbons that are used for synthesis of lipids (needed for membrane assembly) as well as other metabolites needed for nucleic acid synthesis. • This pattern of glucose carbon use is achieved by shunting pyruvate toward biosynthetic pathways at the expense of the oxidative phosphorylation pathway and ATP generation. • Tumor cells that adapt this altered metabolism are able to divide more rapidly and outpace competing tumor cells that do not. • If cancer cells do this and it allows them to out-populate other cells, it must be advantageous

How can tumour cells enable replicative immortality as a hallmark of cancer? What does it result from?

• tumour cells have unrestricted proliferative capacity • normal mammalian somatic cells proliferate a limited number of times before undergoing senescence - chromosomal telomeres progressively shorten with each division until DNA replication can not proceed - shortened telomeres are interpreted by DNA-repair machinery as double-stranded breaks, leading to cell cycle arrest via p53 and RB and senescence ϖ the small telomeres send a signal saying they are not going to be able to replicate anymore • immortalisation is an essential step in malignant transformation • mostly due to telomerase - maintains telomeres at the ends of chromosomes - telomerase counters progressive telomere shortening - normal cells have undetectable levels of telomerase activity (but most cancers express this enzyme) - approx 90% of human tumours express active telomerase