Cancer Bio Exam 3 (Final)

Is EMT reversible? If so, how?

•"Reactive" stromal cells at site of primary tumor contain inflammatory cells •These cells release EMT-inducing signals •Upon metastasis, the new stromal cells do not contain inflammatory cells •Thus, the EMT reverses

What are some of the major categories of chemotherapies?

•DNA-Damaging Agents (DNA alkylators, cross linkers, topoisomerase inhibitors) •Antimetabolites (base analogs, dNTPs, synthesis inhibitors) •Microtubule disrupters •Signal transduction agents /targeted agents •Hormonal antagonist

In NSCLC, which domain of the EGFR is frequently mutated to become constitutively active? Why might one want to treat NSCLC patients who express oncogenic EGFR in their cancer cells with both a first generation EGFR-targeting small molecule drug (such as Iressa) and a second generation small molecule drug (such as Osimertinib) at the same time?

- In NSCLC, which domain of the EGFR is frequently mutated to become constitutively active? In the majority of NSCLC cases, all of the somatic activating EGFR mutations were found to be occurring in the ATP-binding pocket inside the tyrosine kinase (TK) domain of the EGF receptor. As a result of these mutations, the EGFR becomes constitutively active and keeps sending a signal to the downstream effector molecules which ultimately leads to uncontrolled cell proliferation, growth, differentiation, migration, and inhibition of apoptosis. All the activating mutations of EGFR were found in the first four exons -18 to 21 of the TK domain. These mutations have been categorised into three classes which are as follows :- Class I mutations are in-frame deletions that occur in exon 19. These deletions mostly happen in amino-acid residues from leucine-747 to glutamic acid-749 and are responsible for around 44% of all EGFR TK mutations. Class II mutations are single-nucleotide substitutions which lead to an amino-acid change. The major single-point mutation is in exon 21, in which an arginine is substituted with a leucine at codon 858 (L858R). L858R has been found to be the highest occurring single-point activating mutation in EGFR TK and comprises approximately 41% of all EGFR TK activating mutations. Other class II activating mutations involve a glycine-719 change to serine, alanine or cysteine. Class III mutations are those that involve in-frame duplications and/or insertions in exon 20. - Why might one want to treat NSCLC patients who express oncogenic EGFR in their cancer cells with both a first generation EGFR-targeting small molecule drug (such as Iressa) and a second generation small molecule drug (such as Osimertinib) at the same time? Iressa is the trade name of Gefitinib and is the most commonly used first line of drug in case of NSCLC. Gefitinib is an EGFR inhibitor, which disrupts signalling through the EGFR in target cells by binding to the ATP binding site of the enzyme. However, it was seen that patients who were treated with a first generation EGFR targeting small molecule drug like Iressa, often develop resistance due to T790M mutations. As a result Iressa can no longer effectively disrupt the EGFR signalling leading to relapse of cancer. Hence to avoid drug resistance a second generation small molecule EGFR inhibitor known as Osimertinib was introduced. Osimertinib was found to be capable of effectively targeting T790M mutations thereby preventing drug resistance and increasing chances of survival. Therefore it is a better treatment option to use both Iressa and Osimertinib at the same time.

What are some ways in which cancer therapeutics can take advantage of the properties of cancer cells in order to kill them?

1)Proliferate regardless of cell cycle checkpoints- Damage DNA so catastrophic event occurs during replication 2)Cancer cells develop oncogenic addiction through mutation for proliferation-target the mutation within the kinase/cascade with a drug 3)Cancer cells evade immune system- turn immune system back on 4)Cancer cells have altered DNA repair mechanism (ex: BRCA mutations)- target repair pathways they depend on 5) cancer cells have altered metabolism- target need for excess metabolites

Describe the different steps of the invasion-metastasis cascade. What does each step entail, and what must be overcome for the cancer cells to make it through each step? Which step is the hardest?

1. primary tumor formation 2. localized invasion- basement membrane breached 3. intravasation- move into lymph or blood vessels 4. transport through circulation 5. arrest in microvessels of organs 6. extravasation 7. form micrometastasis 8. colonization- form macrometastasis colonization is most inefficient- depends on rate of adaptation of tumor cells to new environment localized invasion- breach basement membrane, breach means more probability for metastasis - invasion needs release of secreted proteases which remodel ECM and allow cancer cells to move through, stromal cells lead the way in remodeling ECM, Tumor cells recruit macrophages (green) which secrete cathepsin proteases (red), allowing tumor cells to invade into the adjacent exocrine pancreas intravasation- move into lymphatic or blood vessels, involves macrophage assistance as they release EGF to allow intravasation cancer cell transport thru circulation- life of circulating tumor cell dangerous: when they lose stromal support they may die due to loss of growth factors, cells may tear apart due to the hydrodynamic forces in circulation, the cancer cells are often too big to pass through the small capillaries so they get trapped Many CTCs become lodged in capillary system of the lungs Many cancers to metastasize to the lungs simply due to being trapped in the capillaries there •Some cancer cells then leave lungs to metastasize to other parts of the body- how they do so is unclear o Some CTCs may pinch off parts of their cytoplasm to reduce size? o CTCs may bypass capillaries by traveling through arterial-venous shunts (fistulas)? extravasation- two routes: cells push themselves through the blood/lymph vessel wall, or cells proliferate in the lumen first before pushing the endothelial cells out of the way 1. ctc trapped 2. platelets attach to CTC, forming microthrombus 3. endothelial cells pushed aside 4. platelets dissolve microthrombus 5. ctc proliferates 6. cancer breaks through basement membrane formation of micrometastasis colonization- cancer cells must be able to live in foreign environment

Compare and contrast the advantages of small molecule vs. monoclonal antibody therapeutics.

Advantages of small molecules such as Tarceva: •Can penetrate more easily into tumor tissue due to small size •Less expensive to produce •Can take in tablet form (instead of IV for antibodies) •Will still work on a receptor mutated to lack the extracellular domain Advantages of monoclonal antibodies such as Herceptin: •Longer half-life (Herceptin is ~28 days vs. 1.5 days for Tarceva) •Can link toxic drugs or other molecules to the antibody for uptake into the cancer cell EGF-R: Tarceva HER2:Herceptin

How does breast cancer, metastasized to the bone, establish a "vicious cycle"? What are some strategies to therapeutically block this cycle?

A breast cancer cell releases parathyroid-hormone related peptide (PTHrP) •This increases the RANKL:OPG ratio •Causes bone degradation by osteoclasts •This releases many growth factors from the bone ECM •Enables cancer cell proliferation - cancer cells then produce more PTHrP Therapeutic blockage of the "vicious cycle" of osteolytic metastasis A monoclonal antibody- Denosumab- binds to and neutralizes RANKL •This inhibits breakdown of bone ECM, stopping the vicious cycle

What is a confounding issue with surgery for breast cancer (and other cancers)?

A mouse study found a link between the healing process after breast cancer tumor removal and the spread of cancer cells •One in four women who have a lumpectomy or mastectomy will experience a cancer relapse •Anti-inflammatory medications were found to inhibit relapse

Which pathway does cyclopamine inhibit? Is this compound useful for human cancer therapy? Why or why not?

A sheep teratogen may be useful as an anti-cancer drug •The "corn lily" plant grows in the American West •Pregnant sheep that graze on this plant often give birth to stillborn lambs- some with cyclopia - a single central eye •Active compounds identified and named cyclopamine and jervine (a close chemical structure made in same plant) •Natural products are a good source for the identification of biologically active small molecules- evolution has generated biological activity in these molecules as many are used to defend against predators or eliminate competitors •Mechanism linked to blocked Patched-Smoothened pathway that is important in development Treatment of chicken embryo with jervine causes cyclopia •This deformity resembles an affliction seen in human fetuses •Cyclopia associated with 1 in 250 aborted human fetuses •23 distinct mutations in the Hedgehog gene and 3 distinct mutations in Patched are associated with this defect in humans •These findings gave the first clue that jervine and cyclopamine perturbes the Hedgehog signaling pathway Cyclopamine prevents Smoothened activity to prevent Hedgehog signaling •Cancers with constitutively activated Smoothened, inactivated Patched, or excessive levels of Hedgehog lead to Gli-induced activation of genes promoting cancer progression •These cancers include sporadic basal cell carcinomas of the skin, pancreatic carcinomas, glioblastomas, medulloblastomas, breast and esophageal carcinomas, rhabdomyosarcomas, cholangiosarcoma (bile duct tumors) Effect of cyclopamine on human cholangiosarcoma xenografts •Human cholangiosarcoma cells (with activated Patched-Smoothened pathway) were propagated in culture and then grown as xenografts in mice •Half of mice treated with cyclopamine, the other half with vehicle control •Tumor cells did not grow in the cyclopamine-treated mice •Unfortunately, cyclopamine was too toxic for use in humans. Alternative small molecules developed that target the GPCR-like structure of Smoothened for use in humans Alternative inhibitors in development that lack toxicity, including HhAntag Mouse model of medulloblastoma responds to Smoothened inhibitor HhAntag •Mice are Patched +/- and p53 null •These mice will develop LOH at Patched locus and develop medulloblastoma by 3 months of age •Treatment with 20 mg/kg HhAntag causes partial tumor regression •Treatment with 100 mg/kg HhAntag causes complete tumor regression

Oncogenic Addiction & Resistance

Cancer cells can exhibit dependence on a single oncogenic protein or signaling pathway for sustaining proliferation and survival, despite the wide burden of genetic lesions characterizing their genomic background. •A promising Achilles' heel of cancer.

Which types of cells display antigens on MHC I molecules? How are the antigens loaded onto the MHC I molecules and what type of antigens are displayed?

All cells display antigens from internal proteins on MHC I molecules •Up to 1/3 of all proteins made by a cell are diverted to specialized proteasomes to form 8-11 aa peptides •These are attached to MHC Class I molecules and displayed on the cell surface •Includes all proteins expressed by cell, including viral and bacterial proteins if the cell is infected, and a high number of mutant proteins if the cell is a cancer cell Display of intracellular antigens by the MHC class I molecules •Almost all cell types, including cancer cells, present a portion of all recently synthesized proteins as antigens on MHC class I molecules •Proteins degraded into 8-11 aa peptides by specialized proteasome •Peptides enter lumen of ER through TAP channels and are loaded onto MHC I molecules •Then taken via specialized vesicles from the ER to the plasma membrane •The display of "non-self" antigens in this way allows cells to be targeted for death by cytotoxic T cells Cells present diverse peptides through diversity in their MHC class I molecules -Each cell has multiple variant forms of MHC class I molecules, each with a different antigen-binding domain •This allows each cell to present a diverse set of peptides Structure of antigen-presenting domain of MHC class I bound to oligopeptide is similar to that of MHC class II Class 1: 8-10 aa peptide Class 2: 15-24 aa peptide Structure of TCR bound to MHC class I molecule presenting antigen •TCR: a and b chains •Oligopeptide (yellow) •MHC class I complex •b2 microglobulin (dark green): accessory protein This specific interaction then leads to killing of the aberrant cell by cytotoxic T cells

How do cancer cells evade cytotoxic T cell killing by the extrinsic apoptotic pathway?

Cancer cells can overexpress FLIP or IAPs to resist apoptotic cell death from cytotoxic T cells by the FASL-induced extrinsic apoptotic pathway- inhibit caspases

What are the structural features of antibodies? Where does the antigen bind? What happens to the antibody when it is digested with papain?

Antibodies •Soluble molecules that can bind to antigens •There are five subclasses of antibodies- IgA, IgD, IgE, IgG and IgM •Antibodies are heterotetramers of 2 heavy and 2 light chains •Constant regions (C) do not change for each subclass •Variable regions (V) bind distinct antigens- one per each antigen binding domain An antibody digested by the protease papain yields three fragments, two Fab fragments and one Fc fragment

What are 4 different ways discussed in class by which antibodies can eliminate damage by pathogens?

Antibodies function to neutralize or eliminate pathogens 1.Can prevent pathogen absorption to epithelial cells 2.Can induce phagocytosis/elimination of the pathogen by phagocytosis 3.Can induce killing of infected cells in the body by NK cells 4.Can induce bacterial or infected cell killing by complement Neutralization: prevention of pathogen absorption to epithelial cells by antibodies A: Virus particles (red) are coated by antibodies (blue), which recognize viral antigen molecules (red spikes). Antibody coating blocks adsorption of viruses onto host cells. B: A bacterium expressing surface antigens (red) can also be bound by antibody and prevented from adhering to host cell. Opsonization: coating of pathogen by antibodies to allow elimination by phagocytosis Macrophages use Fc receptors (green) to recognize and bind to constant regions of the antibody, resulting in phagocytosis and destruction of the pathogen or infected cell in lysosomes inside the macrophage Killing of infected mammalian cells coated with antibodies by natural killer (NK) cells Fc receptors on NK cells bind to mammalian cells coated with antibodies •NK cell becomes activated •NK cell destroys target cell by releasing cytotoxic granules containing perforins and granzyme B •Perforin makes holes in cell membrane allowing granzyme B to enter and induce apoptosis (extrinsic apoptotic pathway) Antibodies coating a bacteria or infected cell can also lead to killing by plasma proteins called complement •Bacterial or mammalian cell expressing antigen is coated by antibody •Instead of binding to Fc receptors, the antibody antigen complexes bind to complement factors (soluble factors in plasma) •Formation of complement complexes induces the formation of channels in the cell membrane of the bacterial cell or the infected mammalian cell •Channels destroy osmotic integrity of the cell, leading to death

How are antigens displayed on dendritic cells? What size antigenic peptides are displayed on the MHC II molecules? How do dendritic cells cooperate with helper T cells to induce antibody production in plasma cells?

Antigen presentation by antigen-presenting cells (APCs) is the first step in antibody production •Professional antigen-presenting cells include dendritic cells and macrophages •Dendritic cells phagocytose diverse pathogens •B cells can also phagocytose specific pathogens through interactions with IgM receptors •The pathogens are degraded into peptides, which are then loaded onto MHC II molecules •While dendritic cells can present antigens from multiple pathogens, B cells only present antigens from pathogens that can bind to their antigen-specific IgM molecules Antigen presentation by MHC Class II molecules •Structure shows antigen-presenting groove of MHC Class II molecule bound to antigen •Hydrogen bonds shown in blue •Bound peptides are ~15-24 residues •The antigen presenting cells displaying MHC class II/peptides then interact with specific helper T cells (TH cells) APCs cooperate with TH cells to induce antibody production in B cells •Dendritic cell displays MHC II-antigen complexes •Only TH cells with compatible TCRs will bind Dendritic cells cooperate with TH cells to induce antibody production in B cells -TH cells with compatible TCRs bind to dendritic cell •TH cell then becomes activated and leaves dendritic cell •TH cell searches for B cell displaying same MHC II-antigen complex •B cell becomes activated and differentiates into a plasma cell •Plasma cell secretes antibodies specific for the antigen

What are some differences between apoptosis and necrosis?

Apoptosis •Tumorigenesis is associated with acquired mutations including dysregulation of apoptosis •Membrane blebbing (no loss of integrity) / Condensation of nucleus) •Shrinking cytoplasm •Energy Dependent •No inflammatory response •Initiator caspases: 2, 8, 9, 10,& 12 •Effector caspases: 3,6, and 7 Necrosis •Loss of membrane integrity •Swelling of cytoplasm/mitochondria •Groups of cells rather than single cells •Random digestion of DNA (smear on gel electrophoresis) •Ends with cell lysis •Inflammatory response

What are some of the challenges that lie ahead for cancer therapeutic research?

As cancer is a disease of aging, increased lifespans create increased incidence of cancer Cancer is not one, but many diseases •With bioinformatics to subcategorize, cancer may turn into many hundreds of distinct diseases •Diagnoses of the future may be based more on bioinformatic classification than pathological classification Multidrug treatments may be more effective than sequential treatments Some obstacles with multi-drug therapies •Harder for patients to tolerate •Hard to know which drugs to combine •Some drugs that may only work synergistically with others could have already been passed by •Drug companies do not have a financial interest in combining their drugs with drugs developed from another company Expression of the P-glycoprotein allows acquisition of multi-drug resistance (MDR) •The MDR1 gene encodes P-glycoprotein (P-gp) •Expression is likely the cause of multi-drug resistance •P-gp often elevated in cancer cells that have become resistant to chemotherapies •ATP-dependent transmembrane pump- can pump a wide variety of small molecules out of cells •Drug binds in drug-binding pocket •ATP binding then causes conformational change •Drug released outside cell Cancer stem cells create treatment obstacles •Cancer stem cells are resistant to therapy •Ideal therapies should strike both cancer stem cells, as well as transit amplifying cells (as these may revert back to cancer stem cells) •Cancer stem cell field is still in its infancy Biological models create issues for initial stages of drug development Mouse models are currently limited in their ability to predict clinical responses to drug treatments "For the moment, truly useful animal tumor models are little more than a fond hope." (Weinberg) Tumor stromal cells should also be considered in therapies Questions for the future Weinberg: •Will "personalized" medicine for various cancers, based on their unique differences, be realized, and if so, at an affordable cost? •Should the goal be to simply keep cancer under control and to manage it as a chronic disease?

Which secreted factor is important in inducing EMT of cancer cells?

Aside from TGF-β, other factors also contribute to wound healing process (with effects also on the inflammatory process, EMT, and angiogenesis) These factors are released from the cancer cells, the cancer-associated stromal cells, or both Some of these factors include: •Epidermal growth factor (EGF) •Fibroblast growth factor (FGF) •Vascular endothelial growth factor (VEGF) •Granulocyte macrophage colony stimulating factor (GM-CSF) •Platelet-derived growth factor (PDGF) •Interleukin (IL) family •Tumor necrosis factor-α (TNFα)

How to PD-1 and PD-L1 work in the immune response, and how can these proteins be targeted for cancer therapy?

Blockade of CTLA-4 together with PD-1 and PD-L1 to induce antitumor responses •Anti-CTLA-4 allows for enhanced T cell activation by dendritic cells in the lymph node upon recognition of a specific tumor antigen by eliminating this "checkpoint" •The activated T cell then circulates throughout the body •When it encounters a cancer cell with the correct MHC-I/antigen, it can bind to this cell and kill it •PD-1 binding to PD-L1 is another "checkpoint" of the immune response, inhibiting the killing of cancer cells by cytotoxic T cells •Antibodies against these proteins can then further enhance the immune response against cancer cells

How does Nutlin work, and which type of cancer patient would see the most benefit from this drug?

Blocking protein-protein interactions Interaction surfaces between two proteins generally not druggable, due to an extended interaction surface that is hard to block with a small molecule. However, there have been some recent successes including: •Mdm2-p53 (Nutlin) •b-catenin-CBP (ICG-001) Nutlin-2 fits in the MDM2 binding pocket for p53 and blocks interaction •Blocking interaction prevents p53 ubiquitination by Mdm2 •Stabilizes p53 How can specificity in kinase inhibition be achieved when kinases are structurally very similar? •Kinase domains of several kinases shown as ribbon diagrams •Catalytic clefts sandwiched between the two major lobes of the proteins in all cases •Achieving specificity is difficult, if not impossible

Why might the inhibition of miR-200 cause an epithelial cancer cell line to switch to a mesenchymal state? What does miR-200 target?

Cancer cell lines grown in vitro reside stably in the epithelial, mesenchymal, or an undefined state. This is controlled by miR-200, an EMT-TF regulator. NCI panel of 60 distinct cancer cell lines assayed for levels of E-cadherin (epithelial marker) vs. vimentin (mesenchymal marker). Graphed as ratio. •Cells appear to be in the epithelial, the mesenchymal, or an undefined state •If miR-200 is inhibited experimentally in one cell line (degrades the EMT-TFs ZEB1 and ZEB2), cells can change from the epithelial to mesenchymal state Mutually antagonistic interactions between EMT- TF ZEB1/2 and miR-200 operate a bistable switch NCI panel of 60 distinct cancer cell lines assayed for levels of ZEB1 mRNA (an EMT-TF) and miR-200c (degrades ZEB1 mRNA). •ZEB1 mRNA 3'UTR has multiple binding sites for miR-200 family members

Why are combination therapies useful? How are they designed?

Combination therapies often are used to combat drug resistance •Cocktails work best if each drug has a different mechanisms of action •Likelihood of cancer cells developing resistance is therefore severely decreased •Multi-drug resistance can develop if cancer cells begin to express plasma membrane pumps that export all drugs

What are 2 ways that cytotoxic T cells may kill target cells?

Cytotoxic T cells: cellular adaptive immunity Cellular adaptive immunity causes activation of cytotoxic T cells that kill infected cells and cancer cells T cell receptors (TCRs) bring cytotoxic T cells to specific infected cells to kill them -Each cytotoxic T cell displays a particular antigen-recognizing TCR Cytotoxic T cell binds to target cell displaying a specific antigen bound to MHC I, and then it kills the cell- extrinsic pathway T cell receptors (TCRs) bring cytotoxic T cells to specific infected cells to kill them Each cytotoxic T cell displays a particular antigen-recognizing TCR -Two methods of cell death: granzyme B and Fas L/Fas R Cell death of infected/cancer cells through release of granzyme B by cytotoxic T cell -Cytotoxic T cell binding to target cell over time -Cytotoxic granules (red spots) migrate to point of contact -Contents of granules (including granzymes) are then released Cell death of infected/cancer cells through binding of Fas ligand on cytotoxic T cell to Fas receptor on target cell

Do cancer cells and stromal cells co-evolve with each other?

it is realized that cancer cells and the corresponding stroma co-evolve with time. Cancer cells recruit and transform the stromal cells, which in turn remodel the extracellular matrix of the stroma.

What is meant by "oncogene addiction", and why is this important when considering targeted cancer therapies?

Despite the diverse array of genetic lesions typical of cancer - some tumors rely on one single dominant oncogene for growth and survival, so that inhibition of this specific oncogene is sufficient to halt the neoplastic phenotype •Therefore, targeting these oncogenes may be a good strategy

Discuss the differences in cancer risk between immunocompromised individuals and the general population.

Do immunocompromised people have a higher incidence of cancer? Groups available to test via epidemiological data: •Organ transplant patients receiving immunosuppressive drugs •HIV-infected people Immunocompromised people have a higher incidence of cancer due to infectious agents •SIR: standard incidence ratio oNumber of cancer cases in the immunocompromised population/ number of cases in age-matched general population oIf no effect: SIR = 1 •"Box and whisker" plots: box shows median of lower half of values up to median of higher half of values, whiskers show 95% confidence intervals Possible explanations for why immunocompromised patients have more virally-induced cancer 1.The immune system protects us against viral infections. Without the immune response, these infections are around longer in the body, allowing for higher chance for cancer-promoting viruses to cause cancer. 2.The immune system specifically recognizes and eliminates virally-transformed cancer cells Does the immune response also hinder non-virally associated cancers? Yes- transplant patients have higher incidences of many cancer types not associated with viral infection

Why are the draining lymph nodes of a cancerous tissue a good "surrogate marker" of metastasis? Are these lymph nodes generally thought to aid in metastasis themselves?

Draining lymph nodes of the mammary gland as "surrogate markers" of metastasis •Lymph ducts (red) •Lymph nodes (swellings along ducts) •Carcinoma cells carried to the lymph ducts by the flow of lymph •Presence of cancer cells in lymph nodes near tumor increases chance that cancer has metastasized •When lymph nodes are removed surgically, no effect on survival •It may be that cancer cells become trapped in lymph nodes, but do not spread from lymph nodes

Discuss the stages that Gleevec likely had to pass through in order to go from "bench to bedside" (i.e. from laboratory bench to use in patients) after its initial discovery as a small molecule hit in a high-throughput screen, giving details of each stage.

Drug candidates are generally tested in cell culture prior to testing in mice and finally in people Testing Gleevec for activity in cell culture •Pre-B lymphocytes depend on IL-3 for survival •Gleevec not intrinsically toxic to these cells •Bcr-Abl introduction eliminates IL-3 requirement, as it would in a cancer cell •Gleevec kills these cells •These studies also show the drug is able to cross the cell membrane Next step for the drug candidate that works in vitro: assay in vivo •Usually human cancer cells are tested in immunocompromised mouse hosts as a next step in drug testing •Presumption: Human tumor cells in immunocompromised mice will behave similarly to tumor cells in human patients •Caveats: o Interactions with immune response not taken into account o If cancer is not orthotopic (at original site), may not accurately represent what would happen in a cancer patient o Also, cancer cells grown in culture are often not like most cancers encountered in people Cancer cells propagated in culture are not representative of cancer cells from most patients •12 year study of esophageal carcinoma cell lines •Cell lines were more likely to be established from the minority of patients with the poorest outcomes •Suggests only the most malignant cancer cells can be propagated in vitro Pharmacokinetics and pharmacodynamics of Gleevec as assessed in the mouse •Pharmacokinetics: kinetics of Gleevec accumulation and disappearance from the plasma. •Pharmacodynamics: kinetics of Gleevec activity. (Kit kinase is a "surrogate marker" of Bcr-Abl function, as it is also targeted by Gleevec.) If initial animal experiments are successful, further in vivo drug assessments are made •Are there any toxic side effects in the liver, kidneys, GI tract, hematopoietic system, heart, or other places? •Does it have a high therapeutic index? (destroying cancer cells, but leaving other cells relatively unharmed?)

What are the desired properties of a kinase inhibitor to be useful as a cancer therapy?

Druggable" targets often have catalytic clefts vulnerable to specific attack by small molecules •Kinases have catalytic clefts and are thus generally "druggable" o 518 kinases in genome, of which 90 are tyrosine kinases (major players in cancer) •Transcription factors generally not considered druggable, due to not having catalytic clefts Rational drug design can be harnessed for cancer therapeutics •Drugs should be targeted to proteins contributing to the disease state (such as in oncogene addiction) •"Druggable" targets most frequently have a binding cleft, as found in kinases •Detailed structures of target proteins should inform design of chemical structure synthesis •Libraries of synthetic or natural compounds can also be screened, and hits can be further modified for more spec

What is secreted from stromal cells that can induce EMT?

EGF, TNF-a, TNF-B

Some NSCLC (non small cell lung cancers) that are "addicted" to signaling through the EGFR can shrink dramatically when treated with a targeted therapy against the kinase domain of the EGFR. Is the cancer remission likely to last? Why or why not?

EGFR mutants induce dramatic initial responses, but resistance emerges within the first 1-2 years so, remission is not likely to last because of resistance to treatment

What is EMT, and which biological processes does it occur in? What happens, physically, to the cell during EMT?

EMT is the epithelial mesenchymal transition involved in the biological processes of wound healing and development, the EMT allows cancer cells to break through the basement membrane and push epithelial cells aside toa become invasive cells undergoing EMT lose cytokeratin, tight junctions and others involving E-cadherin expression, and the polarity and gene expression they had as epithelial cells and gain motility, invasiveness, resistance to apoptosis, protease expression etc

How was it shown that EMT-TFs can induce the stem cell state?

EMT-TFs are induced upon wound healing Slug induction (dark brown) upon wound healing Scratch assay- wound made with pipette tip in confluent monolayer of cells Keratinocytes grown in vitro Wounded by scraping away a swath of cells EMT-TFs confer stem-cell properties on epithelial cells HMLE: hTERT-immortalized human mammary epithelial cells •Expression of Snail or Twist shifts cells to stem cell morphology and biochemical characteristics (CD44 high CD24 low)

List some examples of EMT-TFs. In general, where were these factors first identified?

EMTs are programmed by transcription factors that orchestrate embryogenesis Snail- Amphioxis, Small translucent fish Slug- Xenopus Twist-Drosophila MT-TF regulatory properties •Often expressed in combination •More than one is required for EMT (example: Twist depends on Slug to induce EMT) •They can regulate each other (example: Twist upregulates itself, Snail, Slug, and Zeb1) •Some have redundant functions (example: Snail, Slug Zeb1 and Zeb2 can all repress E-cadherin)

What is lymphangiogenesis?

Endothelial cells construct lymph ducts in response to VEGF-C and -D •Lymph ducts drain fluid between cells which empty into veins •Bring antigens from tissues to the lymph nodes, where immune responses are often initiated •Also created from endothelial cells •Lack the surrounding pericytes that capillaries have

What is CAR-T therapy?

Engineering T cell "assassins" to recognize and kill cancer cells -taking t cells from a patient and re-engineering them to fight tumor cells The Cancer Immunotherapy Revolution •The past few years have seen unprecedented clinical responses, rapid drug development, and first-in-kind approvals from the U.S. Food and Drug Administration. •Reports of terminal cancer patients defying the odds and achieving complete remissions are accumulating •Why do only a subset of patients respond? •Hundreds of clinical trials underway Science March 23, 2018. Many checkpoint inhibitor trials underway In a trial at MD Anderson Cancer Center in Houston, Texas, bladder cancer patient David Wight received two immunotherapy drugs known as checkpoint inhibitors •Currently more than 1000 studies combining checkpoint inhibitor drugs, which unleash suppressed immune cells, with other treatments. •That's up from about 100 trials testing these combos through 2014

What is a reason, as discussed in class, that makes GPCRs difficult targets for cancer drugs?

GPCRs regulate the majority of signal transduction pathways that are relevant in cancer cells like EGFR/Ras (proliferation) , ATF4(cell stress), chemokine (metastasis) ,p53 (apoptosis) signalling. due to this pathways GPCRs frequently hijacked by malignant cell. There are three reason which makes GPCRs difficult targets for cancer drugs :- the high degree of sequence homology in ligand binding sites with in GPCRs. the difficulty of protein isolation and crystallization majority part of GPCRs buried within the membrane and ligand binding sites do not lie in the extracellular domain

Cancer cells are constantly finding ways to become resistant to cancer drugs. What is the difference between "vertical resistance" and "horizontal resistance?

Genetic resistance that is effective at preventing successful attack only by certain races of a pathogen is called specific (or vertical) resistance, whereas resistance that is effective at preventing successful attack by all races of a pathogen is called general (or horizontal) resistance.

What does Gleevec target? How can the clinical response to Gleevec be measured and how might patients develop resistance to Gleevec?

Gleevec targets the Bcr-Abl catalytic cleft to prevent the kinase from interacting. The clinical response is measured by cytological analysis of patient blood which shows disappearance of leukemia cells with Gleevec treatment. Resistance to Gleevec can sometimes develop in CML patients •Gleevec nestles tightly into the catalytic cleft of Bcr-Abl •In one mutation leading to Gleevec drug resistance, threonine 315 of Bcr-Abl is replaced by isoleucine, interfering with Gleevec binding Cancer stem cells limit the utility of Gleevec •Gleevec successfully inhibits the growth of the actively cycling leukemia cells (transit amplifying cells) •Gleevec does not kill the cancer stem cells as well- perhaps due to the elevated expression of efflux pumps that confer multi-drug resistance in these cells •This explains why Gleevec treatment must be chronic Why has Gleevec been so successful? •Most CML cases share Bcr-Abl as an oncogene to which they are "addicted" (an oncogene that is driving the cancer) •Leukemia cells in the chronic stage of the disease have been likened to carcinoma in situ, and not full-fledged malignant cancer- making it more responsive to treatment -The success of Gleevec gave unrealistically high expectations for other RTK inhibitors that were subsequently developed to target other cancers

Trastuzumab targets HER2. What kind of a molecule is this drug? What type of breast cancer patient would most likely benefit from this drug? What are some ways in which the drug is thought to work? Does this drug "cure" metastatic breast cancer patients?- discuss.

HER2 (HER2/neu or ErbB-2) a 185-kD receptor first described 3 decades ago -30% of invasive breast carcinomas •Breast cancer cells over express this receptor or have a high copy number of its gene have a worse Overall Survival (OS) before targeted therapy (Transtuzumab)

How can cancers of a particular type (such as breast cancer, lymphomas, etc.) be stratified? What additional information does this provide and why might it be helpful?

High resolution noninvasive imaging techniques that can be used for cancers such as breast cancer can create issues in determining which cancers to treat •Allows detection of more cancers at a small and early stage •Allows detection of treatment progress •For small tumors, which proportion of these will grow to be life-threatening? •Which deserve aggressive treatment, and which can be safely ignored? Breast cancer incidence vs. mortality in the US •Incidence has risen •Is this due primarily to more sensitive detection procedures? Post-mortem cancer detection •66% of women, age 80, have breast carcinoma •80% of men, age 80, have prostate carcinoma How should limited resources for therapy be used with ever-increasing diagnoses? Weinberg's opinion: Therapy decisions should depend on accurate disease risk diagnosis . 1. Tumors with low invasive and metastatic potential that will likely remain this way for lifetime of patient- no treatment. (Surgery could even provoke some tumors to progress or cause higher rate of death due to surgery itself) 2. Highly aggressive tumors with a propensity to metastasize, and that have likely already metastasized at time of diagnosis- no treatment. Unless treatment may ameliorate symptoms or extend lifespan 3. Intermediate grade tumors that can be excised or treated before metastasis occurs- most deserving of treatment How can we distinguish breast cancers that are likely to become metastatic from those that will remain benign and not spread (and therefore do not need aggressive treatment)? -Functional genomics can stratify breast cancers into those that will become metastatic vs. those that will remain benign •Gene expression of 70 "prognostic" genes analyzed by microarray for 295 breast tumors •Threshold set for those with a "good signature" vs. a "poor signature" •Kaplan-Meier plot shows that patients separated by gene expression in panel A take dramatically different clinical courses. good signature= more survival How can we separate tumor types to allow targeted therapy? Distinct alterations in subtypes of diffuse large B-cell lymphomas (DLBCLs) PMBL: primary mediastinal B-cell lymphoma GCB: germinal center B-cell-like lymphoma ABC: activated B-cell-like lymphoma Distinct alterations in subtypes of diffuse large B-cell lymphomas could allow targeted therapy •High levels of NF-kB activation present in the ABC and PMBL subtypes •NF-k B is driving proliferation of these cells, and protecting them from apoptosis •These two subtypes are thus susceptible to killing by an IKK inhibitor

Testing Gleevec for specificity and activity on recombinant kinases

High specificity desired •Low IC50 desired (IC50: amount of drug required to inhibit 50% of the activity) oIC50 in the nanomolar range is best

What is immune tolerance, and what is the difference between central tolerance and peripheral tolerance?

Immune tolerance vs. autoimmunity T cells and B cells recognizing self antigens are normally eliminated by apoptosis "Central tolerance":T cells that recognize self-antigens in the thymus are eliminated (~50% of proteins encoded in the genome are presented in the thymus to allow for this) "Peripheral tolerance": T cells that escape central tolerance are inactivated in lymph nodes When tolerance fails, autoimmunity results Autoimmunity: Destruction of b cells in Islets of Langerhans in Type I Diabetes •Autoimmune attack on islet cells •Stage 1: lymphocytes congregate near still-intact islet •Stage 2 and 3: progressive elimination of b cells •Stage 4 total elimination of b cells

What is the tumor surveillance theory? How was it shown that mice can be immunized against cancer? Discuss how experiments in Rag2 null mice support the hypothesis that "strongly immunogenic" cancer cells are normally eliminated by the immune response.

Immunization of mice by exposure to killed cancer cells supports tumor immunosurveillance theory •3-methylcholanthrene (3-MC) induced tumor cells extracted, irradiated to prevent proliferation, and injected into syngeneic (genetically identical) mouse •These tumor cells do not grow •Subsequent injection of the same tumor cells that have not been irradiated also do not grow, as the mouse has been immunized •If the mice are injected with a second, independently induced tumor, there is no immune protection •The cells in the second tumor, although from a genetically identical mouse, had acquired distinct random mutations as a result of the 3-MC treatment Immunodeficient mice more susceptible to cancer induced by 3-MC -INF-g is a cytokine that regulates the immune system •Mice lacking this cytokine are 10-20X more susceptible to 3-MC-induced cancer •Perforin used by cytotoxic immune cells to make pores to allow granzyme entry -Null mice more susceptible to 3-MC-induced cancer -Rag-1 and Rag-2 allow antibody and TCR gene rearrangement •Null mice more susceptible to 3-MC-induced cancer Rag2 null mouse experiments support the hypothesis that strongly immunogenic cancer cells are normally eliminated

Do natural killer cells promote or inhibit cancer progression?

Important mediators of the innate immune response are natural killer (NK) cells •99% of organisms only have an innate immune response- well conserved •In the innate response, foreign particles and aberrant cells are attacked without needing a prior "education" •The recognition of specific antibodies is not required NK cells kill cancer cells •Cancer cells often lose expression of MHC I to evade immune response •But not so fast! NK cells kill cells that have lost MHC I expression! (more details in next lecture) •NK cells are thus "pre-programmed" to recognize and kill cancer cells with cytotoxic granule content release •After recognizing and killing cancer cells, NK cells also secrete cytokines including IFN-g •This causes inflammation and recruits other immune cells to the site to allow a more effective response to kill any remaining cancer cells

Why is the best therapeutic option not to just simply block a growth or survival signaling pathway at the "end" of the pathway?

In the context of cancer, the therapeutic strategy should be not focus in just one point of the cell signaling pathway for two reasons: Tumor cells are highly mutagenic: This means that tumor cells are always proliferating, and in some point they can develop a mechanism of resistance against a determined drug our therapeutic antibody. So, if we just considerate the end of signaling pathway, the cell could produce newly tumor cells with a mutation on that specific protein that we are attacking, avoiding the action of our drug or developing secondary signaling pathway Growth signaling pathways are more than one: In most of the cases, the signaling pathway that occurs in a cell when it is in contact with a growth factor can vary. This means that there are more than one pathways that could lead to promote survival and proliferation. For example, in HER 2+ breast cancer, the signaling pathway has two alternative pathways. It can be activated the PI3K/akt pathway or the ras/raf pathway.

How are breast cancer cells and their tumor-associated macrophages engaged in reciprocal stimulation?

Macrophages are recruited to some tumors by CSF-1 released by tumor cells Transgenic mouse mammary tumor model created in Csf-1 +/- and -/- animals o MMTV promoter- Polyoma Virus Middle T oncogene. o MMTV promoter active in mouse mammary epithelium. •Without CSF-1 expression, macrophages are not recruited to tumor o Macrophages stained in red- correspond with invasive tumor CSF-1 required for breast cancer cells to metastasize to lungs in mouse model -Transgenic mammary tumor model: MMTV promoter- Polyoma Virus Middle T oncogene •Presence of CSF-1 has no effect on primary tumor size (A), but does affect invasive phenotype (B) •Metastasis occurs in the lungs in presence of CSF-1 expression o evidence: PyMT RNA expression in lung tissue Reciprocal stimulation of breast cancer cells and macrophages •Carcinoma cells secrete CSF-1 •Macrophages respond to CSF-1 by proliferating and releasing EGF •EGF allows carcinoma cells to gain invasive phenotype Macrophages allow breast cancer cells to gain invasive phenotype A mixture of carcinoma cells (green) plus macrophages (red) allows carcinoma cells to invade into collagen gel layer

In heterotypic signaling, how is ligand and ligand receptor expression distributed between cells, and how can this be determined?

Localization of ligand and cognate receptors for heterotypic signaling in normal tissues is usually confined to distinct cell types ligand= parenchyma receptor= stroma can be determined by markers

Which factors regulate motility in cancer, and what types of proteins are these?

Locomotion of cells on solid substrates •Actin fibers extend lamellipodium at leading edge •Stress fibers, also made of actin, contract the lagging edge, breaking focal contacts •Making and breaking of focal contacts depends on localized modulation of affinity of integrins for ECM A. Lamellipodia (ruffles) at leading edge. These are broad, flat, sheetlike structures. B.Filopodia(stained for filamentous actin in red) are protruding from lamellipodia. Filopodia are spikelike structures that allow cell to explore and initiate focal adhesions with integrins. Lamellipodia formation stimulated by "motogens": Growth factors that allow motility Breast cancer cell, upon addition of heregulin, induces formation of lamellipodium (actin in green, nucleus in blue) Formation of stress fibers and lamellipodium by Cdc42, Rac, and Rho monomeric G proteins

The product of the MGMT gene repairs alkylated DNA. Would an alkylating chemotherapy drug be successful for a patient in which the MGMT gene promoter is highly methylated in the cancer cells? Discuss.

MGMT (O6-methylguanine-DNA methyltransferase) & Alkylating Agents •DNA repair protein that removes alkyl groups from O6 position of guanine •This site is a target site of DNA methylation •MGMT repairs this damage but is depleted, If not repleted, chemotherapy induced DNA lesions leads to cell death/apoptosis •Resistance may be associated with high levels of MGMT an alkylating drug would be successful for a patient in which the MGMT gene promoter is methylated a lot because the site where it attacks is prone to methylation

How do stromal cells contribute to invasion? What is some experimental evidence for this?

Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. Stromal changes at the invasion front include the appearance of myofibroblasts, cells sharing characteristics with fibroblasts and smooth muscle cells. The main precursors of myofibroblasts are fibroblasts. The transdifferentiation of fibroblasts into myofibroblasts is modulated by cancer cell-derived cytokines, such as transforming growth factor-β (TGF-β). TGF-β causes cancer progression through paracrine and autocrine effects. Paracrine effects of TGF-β implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts. Autocrine effects of TGF-β incancer cells with a functional TGF-β receptor complex may be caused by a convergence between TGF-β signalling and β-catenin or activating Ras mutations. Experimental and clinical observations indicate that myofibroblasts produce pro-invasive signals. Such signals may also be implicated in cancer pain. N-Cadherin and its soluble form act as invasion-promoters. N-Cadherin is expressed in invasive cancer cells and in host cells such as myofibroblasts, neurons, smooth muscle cells, and endothelial cells. N-Cadherin-dependent heterotypic contacts may promote matrix invasion, perineural invasion, muscular invasion,and transendothelial migration; the extracellular, the juxtamembrane and the β-catenin binding domain of N-cadherin are implicated in positive invasion signalling pathways. A better understanding of stromal contributions to cancer progression will likely increase our awareness of the importance of the combinatorial signals that support and promote growth, dedifferentiation, invasion, and ectopic survival and eventually result in the identification of new therapeutics targeting the stroma

How could you perform an experiment to show that an EMT-TF is involved in cancer cell motility?

Metastasis to lung in a mouse breast cancer model depends on Twist •Mouse mammary carcinoma cells (4T1) implanted in orthotopic site (mammary gland) •When treated with siRNA control retroviral vector, ~105 metastases occur to lung per mouse •When treated with siRNA against Twist, ~14 metastases occur to lung per mouse

What are some current "hot" areas of cancer research?

Metastasis: Slipping Control" Metastatic colonization is the rate-limiting step •What are the tumor-specific genes required? oOral squamous cell carcinoma: CD36, a cell surface fatty acid receptor, is required for metastasis to the lymph nodes • What are the microenvironment-specific genes required? o Mouse metastasis assay screen identified 23 genes o Strongest hit: Spns2- a phospholipid transporter linked to lymphocyte trafficking Lipids could be highly relevant to metastasis Hot areas of cancer research: 1.Genomics: What is the genetic complexity of cancer and how can individualized therapies be promoted with this knowledge? 2.Metabolism: How do cancer cells use the nutrients that they guzzle down, and how can this be targeted? 3.Microenvironment: How do cancer cells coevolve with stromal cells? How can immune checkpoint blockade therapeutics be best utilized? Combining emerging technologies to fight cancer: 1.CRISPR-based genome editing combined with immunotherapies- use to create specific T cell-based therapies 2.High throughput sequencing combined with high resolution imaging: could use to improve early detection 3.Tumor antigen detection combined with the identification of infiltrating immune cell types: could be used to create personalized immunotherapies Social issues 1.How can we have affordable drugs and therapies? 2.How can we improve the inclusion of diverse ethnicities in clinical trials? 3.How can we balance patient privacy vs. data sharing to enhance research?

What type of cell death is radiation most likely to induce?

Mitotic Catastrophe •Failure to undergo mitosis (mitotic failure) after DNA damage (coupled to defective checkpoints)- tetraploidy/endopolyploidy -Cell death occurs during mitosis as a result of DNA damage or deranged spindle formation + debilitation of different checkpoint mechanisms (Cdk1/cyclin B) (progression to M phase) •After radiotherapy most cells die by mitotic death (mitotic catastrophe) •Sometimes occurs after several cell divisions

How is Herceptin thought to fight breast cancer?

Monoclonal antibodies against HER2/Neu can be used to kill breast cancer cells Herceptin (mAb against HER2/Neu) •Generated first in mice, using a specific HER2/Neu epitope as an antigen •Then "humanized" the antibody, by replacing all but the complementarity- determining regions with human sequences to prevent immune response in patients against the mouse constant region •This also allows Fc receptor recognition of antibody for attack by NK cells Herceptin thought to work by recruiting NK cells, not simply by blocking signaling through HER2/Neu Herceptin (trastuzumab), together with paclitaxel, prevents breast cancer metastasis to distant sites two years after cancer removal

What is the concept behind immunotoxins?

Monoclonal antibodies such as Herceptin can be linked to cytotoxic substances, which will then be guided like "smart bombs" to tumor cells Various toxic biological substances can be attached: immunotoxins •Chemotherapeutic drugs such as Adriamycin (topoisomerase inhibitor- prevents DNA replication) •Radioactive molecules- kill cells by radioactive decay CTLA-4 interaction with B7 inhibits Th cell activation by APCs •CD28/B7 interactions at first phase of Th/APC interaction stimulate T cell response •CTLA-4 is then expressed on the cell surface of the Th cell •CTLA-4 interaction with B7 inhibits Th cell activation (negative feedback to turn off response) How can the natural response of a Helper T cell to an APC be enhanced? •Anti-CTLA-4 antibody (ipilimumab) injection into a patient can enhance the immune response by blocking the CTLA-4/B7 interaction •Also increases risk for developing autoimmunity Anti-CTLA-4 shows efficacy in tumor-bearing mice Anti-CTLA-4 called a "checkpoint inhibitor" •CTLA-4 keeps the immune response in check- it's thus an immune response "checkpoint" •Anti-CTLA-4 is thus called a "checkpoint inhibitor" •Checkpoint inhibitors currently all target either CTLA-4 or PD-1 on immune cells, orPD-L1 (ligand for PD-1 receptor) on cancer cells Occasionally, anti-CTLA-4 shows dramatic effects in cancer patients •Metastatic melanoma patient with metastasis to the lung •Exposed to repeated injections of dendritic cells with MHC II receptors loaded ex vivo with the MART-1 melanoma antigen •Treatment followed with anti-CTLA-4 •This should create lots of MART-1-specific antibody to fight the cancer cells MART-1 melanoma antigen

Which type of cancer has risen the most strongly within the last century and why?

Mortality from some cancers has declined since 1930 •Stomach cancer:changes in food storage techniques o Greater use of refrigeration for food storage rather than preserving foods by salting, pickling, and smoking. •Cervical and colorectal cancer: Pap smears (catch early), hpv vaccine (preventative), colonoscopies (catch early) Mortality from other cancers has remained constant or risen Lung and bronchus: rise in smoking (peak in early 1960s)

How does Gleevec work as a targeted cancer therapy? What does it inhibit?

Multiple contacts form between Gleevec and the Bcr-Abl catalytic cleft Gleevec fits into the catalytic cleft of Bcr-Abl •Drug optimized to have precise structure allowing for hydrogen bonds (red) and van der Waals interactions within the cleft

What is a pathway by which proteasome inhibitors may be effective for certain cancer patients?

NF-kB pathway, blocks the formation of the proteasome so that misfolded proteins and those no longer being used cannot be degraded, and build up in the cell which leads to apoptosis

How can NK cells "fight back" against immunoevasive cancer cells?

NK cells kill cancer cells by: 1.Killing cells that have lost expression of MHC I 2.Killing cells that express the stress-induced family of MICA proteins NK cells are activated to kill cells that have lost MHC I expression •NK cells express killer inhibitory receptor (KIR) •KIR binds to MHC I and produces inhibitory signals to prevent attack by NK cell •If NK cell encounters target cell without MHC I, it will attack and kill! This may explain why the loss of MHC-I sometimes correlates with a better clinical outcome, as these cells are killed by NK cells NK cells are activated to kill cells that have gained expression of stress-induced proteins -These stress-induced transmembrane proteins are induced by physiological stress, genetic damage, viral infection, transformation, etc. on many types of human cells. Binding of NKG2D to MICA proteins activates NK cell's cytotoxic response

induction of angiogenic switch needed for tumor expansion

Not all developing tumors will acquire a blood supply •Tumors that do not develop a blood supply cannot grow •Thus, the acquisition of a blood supply is a rate-limiting step in tumor progression

What are usually the best targets for small molecules in targeted therapy?

Oncoproteins and downstream signaling pathways as targets for inhibition

How do osteoblasts and osteoclasts regulate bone creation and resorption?

Osteotropic metastasis •10% of bone mass is replaced per year •Bone is remodeled in response to weight-bearing signals by osteoclasts and osteoblasts •Osteolytic metastasis (breast cancer): activates osteoclasts, which breaks down mineralized bone •Osteoblastic metastasis (prostate cancer): activates osteoblasts, which reconstructs bone Balance between bone creation and resorption is controlled by RANKL and OPG levels •Osteoblasts display RANKL on their cell surface •RANKL binds to RANKL-R on osteoclast precursors and causes them to become osteoclasts, degrading bone •Osteoblasts also secrete osteoprotegerin (OPG- soluble) which acts as a decoy receptor for RANKL to block its function •Balance of OPG and RANKL levels determine bone fate

What are the differences between stage I, II and III clinical trials?

Phase I trials in humans is the next step •Begin at ~1/10th of concentration that caused toxicity in animals •In a series of patients, increase the dose incrementally until drug levels are reached that begin to have unacceptable toxicities: this is the "maximum tolerated dose". •Make pharmacokinetic measurements •Occasionally patients in the trials (these patients have generally failed other therapies) may see a positive result, but these results are not statistically significant at this stage due to low patient numbers •Purpose of Phase 1 is to discover unanticipated toxicities and tolerable levels of drugs in patients Phase II trials in humans •Drug efficacy tested in Phase II trials •Larger groups of patients •How are patients selected? o Sometimes obvious (anti-HER2 drug should be tested on HER2-positive breast cancer patients), but sometimes patients chosen by chance o Have some effective drugs been missed due to poor choice of cancer patient selection? •If Phase II trials show efficacy, move on to Phase III trials Phase III trials in humans •Larger patient populations •Show whether responses seen are statistically significant •Compare response of test drug to a drug that is the current standard of care for that cancer •Is the benefit greater than the current drug? •Patients in Phase III trials usually have gone through many rounds of treatment and have tumors that are refractory to current treatment

Why were proteasome inhibitors first considered for cancer patients?

Proteasome inhibitors yield unexpected therapeutic benefit •Developed to stop cachexia, a muscle wasting that occurs late in tumor progression and that accounts for ~20% of cancer deaths •The thought was that they would prevent protein degradation that occurs in muscle cells during cachexia •It turns out that many signaling processes important in cancer depend on proteasomal degradation (NF-kB, for example) •Proteasomal inhibitors therefore turned out to be useful for a different reason than the one they were created for Velcade/bortezomib

What type of cancer would Tarceva benefit?

Rationally designed drugs that inhibit the EGF-R kinase domain •EGF-R is an RTK that is overexpressed in ~1/3 of carcinomas. •Iressa and Tarceva can be effective chemotherapeutic agents for these cancers by binding the ATP-binding cavity to prevent signaling NSCLC patients that express oncogenic EGF-R can respond well to EGF-R inhibitors •Red arrows point to tumor mass in right lung •After a 6-week treatment with Iressa, tumor has shrunk -Initial clinical trials of this drug did not separate out NSCLC patients with and without oncogenic EGF-R, and thus the results were not striking -This is a reminder of the importance of subclassification of tumor type, as only tumors expressing oncogenic EGF-R will respond

studying cancer cells in mice at orthotopic vs subcutaneous sites

Subcutaneous site: •Advantages o Easier to inject cells o Easier to monitor tumor growth •Disadvantages o Not in normal cancer cell microenvironment with correct stromal cells Orthotopic site: •Advantage- o this is the site where the cancer would normally grow, so the stromal cells located at this site are most likely to have similar properties to those found in the human cancer •Disadvantages- o may have to perform surgery to get cells to this site o Harder to measure size of growing tumor

What are some natural biological programs that cancer cells co-opt to allow metastasis?

wound healing, EMT, angiogenesis

In immunotherapy, what are two common targets, and why is inhibiting these targets effective in fighting some cancers?

Regulation of T Cell Responses Via Multiple Co- Stimulatory and Inhibitory Interactions Drugs that target PD-1 or PD-L1 PD-1 is a checkpoint protein on immune cells called T cells. It normally acts as a type of "off switch" that helps keep the T cells from attacking other cells in the body. It does this when it attaches to PD-L1, a protein on some normal (and cancer) cells. When PD-1 binds to PD-L1, it basically tells the T cell to leave the other cell alone. Some cancer cells have large amounts of PD-L1, which helps them evade immune attack. Monoclonal antibodies that target either PD-1 or PD-L1 can block this binding and boost the immune response against cancer cells. These drugs have shown a great deal of promise in treating certain cancers. Drugs that target CTLA-4 CTLA-4 is another protein on some T cells that acts as a type of "off switch" to keep the immune system in check. Ipilimumab (Yervoy) is a monoclonal antibody that attaches to CTLA-4 and stops it from working. This can boost the body's immune response against cancer cells. This drug is used to treat melanoma of the skin and some other cancers.

How are regulatory T cells relevant to cancer?

Regulatory T cells block action of cytotoxic T cells and helper T cells •Tregs express TCRs and block action of cytotoxic T cells with TCRs that recognize the same antigen •Tregs release TGF-b and IL-10 to suppress proliferation of nearby cytotoxic T cells and T helper cells •Tregs are thus important in preventing autoimmunity •Tregs may help allow cancer cell survival through suppression of the immune response

Cetuximab targets the extracellular domain of the EGFR. While many colon cancer patients have EGFR overexpressed, Cetuximab only gives a positive result in a small number of patients. Why might this be?

Resistance to a drug by a cell can be evidenced by an absence of positive response to therapy. In this context, remember that tumor cells are highly mutagenic. This means that in some point of cell proliferation, new cell with a determined mutation that allows them to resist the action of the treatment can be formed. Among the most studied resistance mechanisms are: the expression of obstacles between the antibody and the EGFR, the overregulation of EGFR signaling pathways, the activation of alternate signaling pathways and failures to trigger cytotoxicity immune mechanisms. For example, we can find the development of a truncated form of the EGFR receptor (lack of extracellular domain) preventing the binding of the antibody to its extracellular domain or alterations of the components that participate in the PI3K/Akt/mTOR signaling pathway. On the other hand, the acquired resistance mostly occurs as a result of alterations in the receptor, resulting in a constitutively activated form of the receptor.

What is the Rip-Tag model, and how was it used to investigate the "angiogenic switch"?

Rip-Tag transgenic mouse experiment: •Transgenic mice with rat insulin promoter expressing the SV40 Large T Antigen •Beta cells of pancreas (where insulin promoter is induced) are thus transformed, and visible tumors develop in 50% of the islets. •50% of islets become hyperplastic, but only 2-4% of these eventually form tumors with a blood supply •The ones that develop a blood supply have succeeded in undergoing an "angiogenic switch"

What are two important groups of EMT effector proteins?

SNAIL factors- MMPs ZEB factors- also MMPs Matrix metallopeptidases are EMT-TF transcriptional targets: expressed by stromal cells and cancer cells to allow tumor invasion

What is one way in which cancer cells can become resistant to multiple types of chemotherapy drugs all at once?

Some of the cells that are not killed by the chemotherapy mutate (change) and become resistant to the drug. Once they multiply, there may be more resistant cells than cells that are sensitive to the chemotherapy. Gene amplification. A cancer cell may produce hundreds of copies of a particular gene. This gene triggers an overproduction of protein that renders the anticancer drug ineffective. Cancer cells may pump the drug out of the cell as fast as it is going in using a molecule called p-glycoprotein. Cancer cells may stop taking in the drugs because the protein that transports the drug across the cell wall stops working. The cancer cells may learn how to repair the DNA breaks caused by some anti-cancer drugs. Cancer cells may develop a mechanism that inactivates the drug.

Promotion of EMT by stromal signals and cancer cell changes

Stromal cells release factors that may include TGF-b, Wnts, TNFa, EGF, HGF, IGF-1 and PGE2, which all can promote EMT

What is the vitiligo syndrome? How does this syndrome affect melanoma prognosis and why?

TAAs may evoke autoimmunity -This melanoma patient was dark-skinned prior to the onset of melanoma, and has lost most of his pigmentation. •This condition is caused by the autoimmune attack incited by the melanoma cells. •Melanomas overexpress certain normal proteins (tyrosinase, transferrin, gp100, Melan-A/MART-1, gp75) •The immune system attacks these tumor associated antigens •Cancer cells are thus attacked, as well as normal melanocytes expressing low levels of these proteins •Destroys the melanocyte pigment-producing cells (both cancerous and non-cancerous) •Causes autoimmune depigmentation syndrome: vitiligo •Melanoma patients with vitiligo live longer, implicating the immune system in controlling the cancer •Can we use TAAs to immunize against cancer?

If you could only add one factor to induce EMT to a plate of cancer cells grown in tissue culture,which factor would you add?

TGF-B because TGF-B changes the cells from E-cadherin to vimentin expression which causes them to undergo EMT, removing it causes MET

What is EMT, and how is it associated with wound healing?

TGF-β induces Epithelial-Mesenchymal Transition (EMT) for wound healing and in cancer cells - loss of signals used for contact inhibition - gain of mesenchymal markers TGF-β often has increased expression by cancer cells, and affects the cancer cells themselves (autocrine signaling) as well as the cancer-associated stromal cells Aside from TGF- β, other factors also contribute to wound healing process (with effects also on the inflammatory process, EMT, and angiogenesis)

Inhibitors of TORC may have promise in preventing cell growth to inhibit cancer proliferation. What is a confounding issue with TORC inhibitors, as discussed in class, that may limit their effectiveness as cancer therapeutics?

TORC inhibitors have many side effects: hypertension, increased lipoproteins, hyperlipidemia, GI effects such as ulcers, high risk of infection especially bacterial, bone marrow toxicity, respiratory problems etc.

in the abscopal effect, it was noted that local irradiation of a tumor can also shrink tumors at distinct sites of metastasis. How, at a very big-picture level, is this thought to occur?

The abscopal effect is a hypothesis in the treatment of metastatic cancer whereby shrinkage of untreated tumors occurs concurrently with shrinkage of tumors within the scope of the localized treatment. R.H. Mole proposed the term "abscopal" ('ab' - away from, 'scopus' - target) in 1953 to refer to effects of ionizing radiation "at a distance from the irradiated volume but within the same organism A simplified depiction of the immune effects of localized irradiation is shown. Radiation can affect the immune state of a tumor in many ways. Irradiation of tumor vasculature can result in expression of adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1). Irradiated tumor cells increase the expression of major histocompatibility complex class I (MHC-I) and Fas. Tumor antigens are released from dying tumor cells, and injured and dying cells may translocate calreticulin to the cell surface. Factors that can activate dendritic cells, such as damage-associated molecular patterns (DAMPs), high-mobility group box 1 protein (HMGB1), and ATP, are also released from irradiated tumor cells. Dendritic cells present tumor-cell antigens to naive T cells, facilitating their conversion to cytotoxic effector cells, which may recognize tumor cells, a process that may also be enhanced by increased MHC-I expression. These events may be highly dependent on the radiation dose, target, and volume and may vary as a function of time after radiation exposure.

Why is the recruitment of mast cells and macrophages to a growing tumor an important part of the "angiogenic switch"?

The angiogenic switch requires recruitment of inflammatory stromal cells •The angiogenic switch is not based solely on VEGF production, as this production occurs before the time of the switch •VEGF is sequestered in the ECM and is not functional •The hyperplastic islet that will be successful in initiating angiogenesis must recruit inflammatory cells that then release the matrix metalloprotease MMP-9 •MMP-9 then digests the ECM and allows release of VEGF, thus tripping the angiogenic switch

Describe the history of how Gleevec came to be a drug (dates and names not needed).

The history of Gleevec A "bench to bedside" success story of the first targeted cancer therapy 1960: 2 cytologists in Philadelphia discovered an unusually small chromosome present in CML. Called it the "Philadelphia chromosome" (Ph) 1972:A researcher in Chicago showed that a reciprocal translocation between chromosome 22 and 9 creates the Philadelphia chromosome. Present in >95% of CML cases. 1982: Bcr-Abl fusion identified. Various breakpoints in the BCR (breakpoint cluster region) gene fuse to the ABL gene 1984: Bcr-Abl found to function as a constitutively active tyrosine kinase Origin and structure of the Bcr-Abl protein •Translocation causes a lengthened chromosome 9 and a truncated chromosome 22 The history of Gleevec, continued: 1990: Bcr-Abl fusion protein found to induce leukemia in mice. If the tyrosine kinase domain of the fusion protein is mutated, the protein no longer induces cancer. Early 1990s: Drug screens performed to identify small molecule antagonists of tyrosine kinase activity of Bcr-Abl. A positive small molecule was found and further optimized by structure/activity analysis to create Gleevec The history of Gleevec, continued: 1996: Gleevec inhibits growth of CML cells in vitro, while not affecting normal bone marrow cells 1998: Clinical trials begin and remissions found in all 31 treated CML patients 2002: 6000 patients in Gleevec clinical trials 2006: Clinical study shows that when chronic-phase CML patients are treated with Gleevec, fewer than 5% of patients die of CML within 5 years

What are the traditional cancer therapies, and how do they work in general? Include the main types of chemotherapies and what they target.

Traditional cancer therapies •Surgery often used to remove bulk of cancer cells •Radiation damages DNA o Although this is toxic to all cells, cancer cells are preferentially killed as they are usually unable to repair DNA (due to defects in DNA repair pathways) and die after several cell divisions due to severe genetic damage o Radiation can be targeted to cancer tissue •Chemotherapy often works by damaging DNA or interfering with mitosis o Cancer cells are more sensitive as they have a higher rate of proliferation than other cells and lack DNA repair capabilities Chemotherapies-"All substances are poisonous, there is none that is not a poison; the right dose differentiates a poison from a remedy."-Paracelsus, alchemist and physician, 1538 •Cancer cells preferentially killed by chemotherapies. •Must find correct dose that kills the cancer cells but not the patient. Various Chemotherapies •Alkylating agents •Nucleoside analogs •Synthetic compounds •Natural products Alkylating agents create DNA that is difficult to replicate/repair -Ultimately cause apoptosis, preferentially to cancer cells. -cisplatin Nucleoside analog chemotherapies create DNA that is difficult to replicate/repair •Fludarabine and gemcitabine are incorporated into DNA, creating residues that are difficult to replicate and/or repair. Gemcitabine is incorporated into DNA instead of dCTP Synthetic compounds have diverse modes of action -Etoposide forms a ternary complex with DNA and the topoisomerase II enzyme -prevents DNA unwinding required for DNA replication and transcription •Bortezomib disrupts proteasome function by binding to the catalytic site, disrupting multiple signaling pathways within the cell which can lead to cell death. Natural products paclitaxel and vincristine stabilize and destabilize microtubules, respectively- Both disrupt cell division process

T/F: The EGFR family includes EGFR, Her2 and Kit.

True

What are the various ways in which tumor antigens may arise? What is the difference between TSAs and TAAs?

Tumor antigens include: •Mutations in oncogenes (Ras, p53, etc.) •Fusion proteins from chromosomal translocations (Bcr-Abl, etc.) •Passenger mutations •Alternative splicing that occurs in tumor cells may create new antigens •hTERT may become immunogenic when re-expressed at high levels •Overexpression of oncogenes normally expressed at low levels (such as HER2/Neu) may make them immunogenic Tumor-specific antigens (TSAs): •Mutations in oncogenes (Ras, p53, etc.) •Fusion proteins from chromosomal translocations (Bcr-Abl, etc.) •Passenger mutations •Alternative splicing that occurs in tumor cells may create new antigens Tumor-associated antigens (TAAs) •hTERT may become immunogenic when re-expressed at high levels •Overexpression of oncogenes normally expressed at low levels (such as HER2/Neu) may make them immunogenic Some human TSAs and TAAs MAGE: (TAA) regulates protein ubiquitylation. Completely silent in normal adult tissues, but is expressed in various kinds of tumors. •Tyrosinase: (TAA) required for melanin production. Normally produced in minute quantities but its levels are highly elevated in melanoma cells. •CEA: (TAA) normally produced in gastrointestinal tissue during fetal development •Caspase 8: (TSA) frequently mutated in head and neck cancer •PSA: (TAA) present in small quantities in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer Bcr-Abl is TSA and HER2/Neu is TAA

How does the presence of TILs in cancer tissue correlate with prognosis?

Tumor infiltrating lymphocytes (TILs) associate with tumors- are they targeting tumor cells for destruction? Immunostaining of oral carcinoma shows recruitment of TILs to the tumor tissue (stained in brown for anti-CD3, a marker for both cytotoxic T cells and T helper cells) Are these lymphocytes trying to eliminate the cancer cells? The presence of TILs in tumor tissue correlates with increased survival in ovarian cancer patients •Similar data obtained for melanoma, breast, bladder, colon, prostate and rectal cancers •Suggests a role for TILs in hindering tumor progression TH1 cell markers correlated with protection from cancer relapse •RNA made from tumor tissue (containing cancer cells and stromal cells) from 75 colorectal cancer patients •qRT-PCR performed for seven genes that make up a "gene signature" of TH1 cells oTH1 cells are a type of helper T cell that enhances the immune response •The higher the expression of the markers, the lower the relapse rate over a 15-year period

How may tumor cells get around anti-angiogenic therapies?

Tumors may outsmart anti-angiogenic therapies via strategy switch Example strategy switch: •Cancer treated with VEGF antibody to block VEGF signaling to endothelial cells •Surviving tumor cells may develop ability to produce FGF instead, which also promotes angiogenesis •Thus, using multiple inhibitors at once may be most effective

What do various anti-angiogenic drugs target?

VEGF-R inhibitors can block angiogenesis Tumor cells implanted subcutaneously in mice •Mice treated with vehicle control or with VEGF-R2 inhibitor (small molecule called vandetanib -ZD6474) •Mice treated with inhibitor show less vascularization of tumor vandetanib is increasing progression-free survival time for thyroid cancer patients by about 11 months

What are some reasons why particular cancers may metastasize to particular sites?

Width of arrow correlates with relative proportion of clinically apparent metastases •In some cases, due to the ease of the primary tumor cells to adapt to new microenvironment •In other cases, due to the layout of the circulatory system (for example, the colon is linked via the portal vein directly to the liver) Sites of chronic inflammation or wounding may be more hospitable to metastases •Target organs may release specific chemoattractants •Vascular "zip codes": endothelial cells of capillaries in different tissues may carry specific molecules that allow cancer cells to adhere •Some tissues may actively repel wandering cells

How do cancer-associated fibroblasts (CAFs) induce angiogenesis?

Wound healing response releases factors that allow fibroblasts to change into myofibroblasts: o Myofibroblasts can contract by using smooth muscle type actin-myosin complex. o These cells are capable of speeding wound repair by contracting the edges of the wound. Myofibroblasts secrete: oTGF-β, which induces EMT o Stroma-Derived-Factor-1 (SDF-1) which recruits endothelial precursor cells (EPCs) oVEGF, which induces angiogenesis

Why is it typical to give multiple rounds of radiation treatments to a tumor?

because each dose effects a set of chromosomes from each phase of the cell cycle

How does hypoxia stimulate the production of VEGF?

because it allows HIF1 alpha to be expressed which stimulates VEGF production lack of oxygen stabilizes HIF1a and triggers VEGF secretion which starts angiogenesis VEGF-A and -B stimulates endothelial cells to produce capillaries Two endothelial cells, responding to VEGF, have joined to form a capillary •The nucleus of one cell bulges into lumen •The nucleus of the other cell is not visible

Radiation causes multiple types of DNA damage, but double strand breaks (DSBs) are the most important lesion for cancer therapy. Why is this?

because it destroys the dna rapidly, 40 DSBs per gray of radiation= 2,500 base damages, 150 crosslinks, 1000 SSB Single Strand Breaks are generally not lethal 1 Gy = ~1000 single strand breaks Base excision repair-removes damaged bases Nucleotide excision repair-DNA helix distortions Mismatch repair-repairs defects in replication and recombination and certain types of damage D.S. breaks are lethal unless repaired-->mitotic catastrophe (majority) or apoptosis

Why are E-cadherin expression levels so important in EMT?

because when cells lose their e-cadherin expression they are able to become motile and detach from the basement membrane, eventually breaking out of the tissue they were first in to metastasize E-cadherin loss and cancer •The E-cadherin promoter is methylated in many invasive human carcinomas •Reading frame mutations occur in the E-cadherin gene in some cancers •Re-expression of E-cadherin in cancer cell lines by transfection suppresses the invasiveness of these cells

Signaling through BRAF (a member of the Raf family of kinases), involves a complex signaling network. How can cancer cells that are addicted to oncogenic BRAF become resistant to BRAF inhibitors, generally speaking?

cancer cells addicted to BRAF can overcome its inhibitors by switching RAF isoforms, activating RTKs, or activating the PI3K pathway to promote cell survival

compare the features of normal vs. tumor capillaries.

capillary networks more crowded in tumor than normal ones Pericytes and smooth muscle cells (red) tightly surround tubes of endothelial cells green) in normal venules and arterioles. B: In normal capillaries, the pericytes are sparse, but still tightly attached C: In a tumor-associated capillaries, the pericyte attachment is not tight Tumor vessels have a chaotic layout as compared to vessels in normal tissue •They are also ~3X wider •The chaotic nature of blood vessels can be mimicked in normal skin by the overexpression of VEGF Normal blood vessels: tight seals between endothelial cells (white arrows). Black arrows indicate bulges from endothelial cell nuclei. •Cancer blood vessels: Gaps of significant size occur between endothelial cells. •These gaps allow cancer cells to enter the bloodstream

What is the difference between cellular immunity and humoral immunity, generally

cellular immunity- mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and antimicrobial proteins humoral immunity- activation of phagocytes, antigen specific cytotoxic t cells or NK cells to kill infected cells

What type of cell death is lack of oxygen most likely to induce?

chronic necrosis

What we know about cancer so far

disease of cellular phenotype - have to gain mutations in oncogenes - lose tumor suppressors - become immortal this allows cancer to form

Which scientist was first to discover angiogenesis inducers, and how did he do it?

dr. judah folkman discovered angiogenesis by hypothesizing that tumors secrete a factor that induces angiogenesis, and implanting a piece of tumor tissue into a rabbit cornea, he saw that the blood vessels were growing toward the tumor, which proved that tumor cells stimulate angiogenesis Tumors secrete FGF and VEGF to induce angiogenesis Made tumor tissue extract,separated protein fractions by size, charge, and affinities and tested each fraction for activity, and discovered FGF (fibroblast growth factor) •When FGF was put in a slow-release pellet in the cornea, it induced angiogenesis •VEGF was later identified similarly by other labs

What are the conditions under which angiogenesis occurs?

embryonic development, wound healing, angiogenesis, induced by heterotypic signaling between cells Each muscle fiber cell lies adjacent to one or more capillaries (brown spots, arrows) •Allows direct access by each cell to oxygen and nutrients, and allows cells to shed metabolic waste products and carbon dioxide •During development, blood vessels form where they are needed •Stromal cells provide signals needed for angiogenesis

Describe the basic anatomy of a blood vessel. Which types of cells are present?

endothelial cells line the interior surface of blood vessels pericytes wrap around the endothelial cells

Which markers would you expect to see in epithelial cells vs. mesenchymal cells?

epithelial: e-cadherin mesenchymal: vimentin EMT-TFs and other EMT markers have altered expression between stem cells vs. non-stem cells

What is the evidence showing that EMT-TF expression correlates with poor survival?

high expression of the EM-TFs snail, slug, and twist greatly decreased patients progression free survival for many types of cancers

Discuss the analogy of cancer being like a weed in a garden. How is this analogy useful in terms of thinking about cancer therapies?

imagine your body as a garden, where the soil is your immune system. When you're healthy, the soil is rich and well tended, and the garden is green. Normally, the soil is able to prevent weeds from growing out of control. Cancer cells are like weeds in your garden. Sometimes, the soil can allow weeds to grow and spread, and soon, the entire garden suffers as your plants compete for space and nutrients. Immunotherapy is like adding weed-control fertilizer to the soil. It enriches the garden's existing soil. Now the soil can help keep the weeds under control and can maintain the garden's health. But too much fertilizer may harm your garden. If your body is like a garden, you and your cancer care team will decide how to remove the weeds (cancer cells) while doing the smallest amount of harm to the good plants (healthy cells) Surgery removes large patches of weeds and the soil around them, sometimes disturbing the good plants and leaving some weed roots behind. Chemotherapy is like spraying a general weed killer on the whole garden. This approach may not kill all the weeds, and may also harm some good plants Radiation is like increasing the power of the sun with a magnifying glass to target and dry out the weeds, but in the process, some of the good plants can also be harmed. With targeted therapy, weeds are directly sprayed with weed killer. Good plants may still be harmed. Instead of targeting the weeds, immunotherapy is like adding a weed-control fertilizer to the soil. This fertilizer enriches the soil to help control weeds, which in turn restores the health of your garden. But too much fertilizer in the soil might harm your garden. Depending on the cancer you have, your cancer care team may mention biomarkers when talking about any cancer treatment options. To understand how biomarkers may be used, let's continue with the garden analogy Every person's garden may have different types of weeds. And even if those weeds may look the same, they each have their own unique characteristics which would change the way a gardener might go about removing them. One way to help choose what to use to remove a particular weed is to take a closer look. Getting a biomarker test could be like taking a few pieces of your weeds out of your garden to take a look at them more closely. Testing for specific biomarkers may help guide treatment decisions by providing you and your cancer care team with information about: • How your body may respond to a potential treatment • How your body is responding to treatment Depending on the cancer you have, your cancer care team may mention biomarkers when talking about any cancer treatment options. To understand how biomarkers may be used, let's continue with the garden analogy. Your cancer care team may order several tests throughout your cancer treatment to better understand your cancer. Some of these tests are known as biomarker tests. The test results may help you and your cancer care team understand more about your garden and how to try controlling the weeds.

What are some features of innate immunity?

immediate resistance against pathogens •First lines of defense to keep pathogens out include: •intact skin and epithelial barriers •high acidity of stomach •resident bacterial flora which prevents colonization by pathogenic bacteria •Second lines of defense to kill pathogens that enter include: •phagocytes that engulf and degrade the microbe •complement proteins (kill bacteria) •natural killer (NK) cells- provide non-specific defense against viral infections and tumor cells •fever and inflammation •interferon pathways (kill viruses) •antimicrobial proteins

What are some better ways to study cancer cells in the laboratory in lieu of simple 2D culturing of the cells? Why are these ways potentially better?

in 2D culturing, the cancer cell line has no associated stromal cells and as such treatments do not translate well to the clinic when studied in 2D culture and have little resemblance to the histology of the original tumor cancer cells that are translated directly to mice have histology similar to original tumor when tumors are surgically removed from patients and placed in an immunodeficient mouse they still retain the original tumor architecture and cells grow slowly another better representation is when the cancer cells are grown as spheroids in vitro because they more accurately represent in vivo conditions

Generally, what is the difference between the innate and adaptive immune response?

innate- immediate response to infection, no memory adaptive adaptive- delayed infectious response as result of antigenic stimulus

What are two main regions of RTKs that are targeted by cancer drugs? Which region of an RTK is targeted by monoclonal antibodies? Which region is targeted by small molecules?

ligand binding region- antibodies cytoplasmic kinase domain- small molecules

What are various ways you could determine whether cells are undergoing EMT?

look for biochemical markers Twist is a transcription factor that induces the EMT •γ-catenin is homologous to β-catenin •Fibronectin is an ECM component that links integrins with collagen •Vimentin is and intermediate filament in mesenchymal cells that is highly flexible E- to N-cadherin shifts and melanoma invasiveness -Intermolecular bonds formed with N-cadherin are weaker than those with E-cadherin •N-cadherin molecules favor motility, and allow interaction with endothelial cells and fibroblasts epithelial vs mesenchymal markers epithelial: e-cadherin, b-catenin on cell periphery mesenchymal: n-cadherin, nuclear b catenin, vimentin,

How does rapamycin inhibit tumor growth?

mTOR, a master regulator of cell metabolism, is an attractive target for cancer therapy mTOR functions in two distinct complexes to promote cell growth and proliferation -mTOR: a ser/thr kinase -S6K:phosphorylates 4E-BP1, which phosphorylates the S6 protein of the 40S ribosomal subunit, allowing association with the large ribosomal subunit and thus protein synthesis Rapamycin inhibits mTOR activity in a manner that requires FKBP12 Rapamycin isolated from Streptomyces hygroscopicus bacteria growing in Rapa Nui (Easter Island) and first used as an antifungal Biochemical studies found that it can bind to FKBP12 and mTOR •Rapamycin can tightly bind to FKBP12 •Once bound, the complex has a novel surface that can bind to mTOR (mammalian target of rapamycin) and inhibit mTOR function as a serine/threonine kinase -Rapamycin inhibits mTOR activity in a manner that requires FKBP12 Rapamycin directly inhibits the mTORC1 complex, blocking cell growth •Rapamycin first shuts down the mTORC1 complex signaling, shutting down cell growth •Continued rapamycin treatment leads to shutdown of mTORC2 as well (mechanism unclear), leading to shutdown of AKT signaling Rapamycin inhibits tumor growth •Mice injected with colon cancer cells develop large, vascularized tumors •If, after a week of tumor growth, mice are treated with rapamycin, the tumors are much smaller with less vascularization

Does the degree of angiogenesis correlate with cancer patient outcome?

more angiogenesis, less possibility of survival HER2-/VEGF- breast cancer patients have the best clinical outcome

Is Gleevec/Imatinib an antibody or a small molecule drug? What type of cancer patient might benefit from this drug? What is/are the target(s) of this drug?

small molecule, Imatinib blocks BCR-ABL, ARG, KIT, PDGFRA and PDGFRB tyrosine kinases The estimated 8-year survival rate has increased from 6% before 1975 up to 87% since 2001 The end of the Imatinib story? Imatinib Targets Kit

Is more surgery always better for cancer patients? Why or why not?

surgery not always best option "Generally speaking, surgery is more successful if it is done on a slow-growing cancer vs. a very rapid-growing cancer, because you can't get your hands on it so to speak," she explains. If the cancer cells might migrate and spread, surgery might not be an option. can reduce quality of life

What did the experiment published in 1961 with autologous transplantation of basal cell carcinoma show?

that when the cells were transplanted with stroma, the cancer cells would grow rapidly, but without the stroma nothing would happen stromal cells are the connective tissues of organs that support the parenchymal function of that organ stromal cells are involved in allowing cancer to progress

What happens to cells inside a growing tumor when they are too far from a blood vessel?

the centers of tumors become necrotic without a blood supply

T/F: Radiation and targeted therapy, when used together, can sometimes improve patient outcomes.

true

What are some examples of tumor-associated stromal cells, and how can these cells be identified?

tumor associated stromal cells Fibroblasts: create extracellular matrix •Myofibroblasts: contracting fibroblasts associated with wound repair •Endothelial cells: blood vessel cells •Pericytes: Aid in contraction of blood vessels •Smooth muscle cells: Involuntary non-striated muscle •Adipocytes: Fat storage cells •Macrophages: Immune system cells (phagocytosis) •Lymphocytes: Immune system cells (T cells, B cells, NK cells) •Mast cells: Wound healing tumor associated stromal cells can be identified by the use of markers such as antibodies and fluorescent tags Epithelial cancer cells: Antibody against epithelial cell marker used to identify these cells Stromal non-cancer cells: Only the nuclei are evident with the blue DAPI stain Depending on the cancer type, the non-cancer cells recruited to the tumor site can be of similar importance to the cancer cells themselves. CD = Cluster of Differentiation •Used to determine identities of a wide variety of cells •Proteins are cell-surface proteins with a wide variety of functions, including receptors and adhesion molecules

Which cells does VEGF act on to create blood vessels?

tumor cells tumor secretes VEGF VEGF increases blood vessel expression and movement to tumor tumor has increased blood supply

How do stromal cells and endothelial cells collaborate in wound healing?

tumor cells resemble wounded tissues that don't heal, do not have to make new heterotypic signaling patterns they exploit the wound healing response and which is a complex inflammatory response where cells move to fill in the wound and allow new blood vessels to be made TGF beta signaling allows wound healing and is also exploited by cancer cells through a similar mechanism TGF beta signaling normally is a tumor suppressor through the induction of genes including p21, p27 and INK4 •TGF-β also has other transcriptional targets, including transcription factors that induce EMT and angiogenesis, allowing a wound healing response. •During carcinoma progression, epithelial tumor cells commonly acquire mutations in components of the TGF- β pathway, such as TGF- β receptors or Smads, or in the RB pathway. These mutations render the tumor cells resistant to the growth-inhibitory effects of TGF-β

what does heterotypic signaling between cancer cells and stromal cells induce?

wound healing response, EMT, angiogenesis

Can chemotherapy enhance the effectiveness of radiation treatment of cancer cells?

yes because the tumor cells become more sensitive to the treatment and die faster

What are the different ways in which cancer cells can attempt immunoevasion?

• A tumor could stop displaying a tumor antigen, if the antigen is not essential to its growth o One way: methylate the promoter of the antigen oThis then allows "immune escape" by the cancer cell and its descendants •If the antigen is essential to tumor growth (oncogene), cancer cells can instead shut down expression of MHC I molecules or components of the MHC I antigen presentation pathway, so as not to display the antigen. •CTCs traveling in the blood are coated in platelets, preventing access by the NK cells Down-regulation of MHC I molecules as an immunoevasion strategy for cancer Ways for cancer cells to block MHC I antigen presentation •Block expression of MHC I •Block expression of b2-microglobulin (b2m): prevents delivery of MHC I to cell surface •Block expression of TAP1 or TAP2

Complement kills bacteria

• Complement system so named as it is complementary to the antibody response of the adaptive immune system •Complement proteins continuously made by liver and macrophages and are capable of an immediate response to invading pathogen •The complement system works by first having several proteins bind the surface of a pathogen •This binding event then begins a series of highly-specific and regulated sequences wherein successive proteins are activated by cleavage and/or structural changes of the preceding proteins •The complement system serves as a marker to indicate targets for phagocytic cells and promotes inflammation •Complement proteins can also combine to form membrane attack complexes (MAC) capable of bursting the pathogen open

Phagocytosis ingests and kills bacteria and viruses

• Pathogen enters the body •Phagocytes detect the specific pathogen-associated molecular patterns (PAMPs) on the pathogen's surface. •PAMPs are carbohydrate,polypeptide, and nucleic acid "signatures" that are expressed by viruses, bacteria, and parasites, but which differ from molecules on host cells. •The phagocytes have receptors that recognize these PAMPs, called complementary pattern recognition receptors (PRRs). •Peptides of the degraded pathogen are then also expressed on MHCII molecules of the phagocyte to activate the adaptive immune response. Phagocytes include neutrophils, macrophages and dendritic cells

Stephen Paget: the "seed and soil" hypothesis

•Hypothesis created in 1889 •Pathologist- studied breast cancer •Discovered that breast cancer metastasis was not random •Metastasizing cancer cell (the seed) was proposed to find the most compatible environment (the soil) seed: cancer cell soil: new stroma

How do MT1-MMP and MMP2 collaborate in the invasion process?

•MT1-MMP is a membrane-tethered protease made by the cancer cell •MT1-MMP initially allows cancer cells to degrade BM •Once cancer cell is in contact with stroma, MT1-MMP can activate stromal MMP-2 •MMP-2 dissolves the collagen 1 network, creating a channel that allows further invasion by the cancer cell

Macrophages release significant amounts of MMP-2 when co-cultured with cancer cells

•Macrophages alone release ~10 ng/ml MMP-2 •MCF-7 and SK-BR-3 release very small amounts of MMP-2 •Co-culture of macrophages with cancer cells increases release of MMP-2

Natural Killer cells can kill infected target cells non-specifically

•Called Natural Killer (NK) cells because they do not need to recognize a particular antigen in order to kill cells •NK cells lyse cells that do not contain intact MHCI molecules on their cell surface •NK cells protect against a wide variety of infectious microbes, as these microbes produce proteins that block MHCI display on the cell surface •NK cells also target tumor cells, as tumor cells also evolve to lose MHCI display •When NK cells detect an infected cell or tumor cell, they secrete granules that contain perforin, creating a pore in the target cell •Granzyme B passes through these pores, initiating the extrinsic apoptotic cascade •Most circulating tumor cells destroyed by NK cells within 24 hours perforin

Adaptive Immunity

•Synthesis of antibodies •Proliferation of cells to allow for immunological memory •Killing of specific target cells via cytotoxic T cells

EMT is often induced by the tumor microenvironment

•Transformed human mammary epithelial cells implanted into mouse •Cells in middle of tumor mass stain for cytokeratin (red) •Cells in contact with mouse stromal cells stain for vimentin (green), indicative of an EMT Cytokeratin is an intermediate filament that contains keratin and makes up the cytoskeleton in epithelial cells