Tumour Biology MCQ - Small multiple choice questions prep PDF

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Tumour Biology: Lecture 1-2 Intro Tumours:       

A tumour is a group of cells which display abnormal growth. Cells which behave in this way are said to have undergone transformation. Tumour that continues to grow and becomes progressively more invasive= malignant. Malignant tumour= cancer Metastasis is the ability of a malignant tumour to form secondary tumours. Carcinomas: epithelial origin, 80/90% of cancers, Lung/Breast colon. Leukaemia, lymphoma, multiple myeloma (9%) are malignant tumours of hemoatopoitic cells derived from bone marrow. Sarcomas (1%) arise from mesodermal connective tissue such as bone, fat. Hallmarks of cancer:  Evading growth suppressors  Avoiding immune destruction  Enabling replicative immortality  Tumour promoting inflammation  Activating invasion & metastasis  Inducing angiogenesis (blood supply)  Genome instability & mutation  Resisting cell death.  Deregulating cellular energetics  Sustaining proliferative signalling

Therapeutic targeting -Cyclin dependant kinase inhibitors -Immune activating anti-CTLA4 mAb -Telomerase inhibitors -Selective anti-inflam. drugs -Inhibitors of HGF/c-Met -Inhibitors of VEGF signalling -PARP inhibitors -Proapoptotic BH3 mimetics -Aerobic glycolysis inhibitors -EGFR inhibitors

Oncogenes: 

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Proto-oncogenes are found in normal cells, may lead to cancer if altered to lose control of expression. Alterations may be due to viruses (Cervical cancer), Carcinogens, Genetics (BRCA). Proto-oncogenes function: generally, encode elements of signal transduction and cell cycle. Growth factors, GF receptors, signal transduction molecules, nuclear proteins. Signal transduction molecules: cytoplasmic membrane proteins; src (tyrosine-kinase) and ras (GTP binding protein) Soluble signal transduction proteins: mos (encodes protein for phosphorylating cyclin B) and raf (serine/threoinine kinase) Point mutation in ras is common ErbB2 is amplified by some breast cancers Burkitt’s lymphoma: Translocation event t(8;14)_ translocations place c-myc under the control of highly active transcriptional regulator. Oncogenes are activated versions of normal cellular genes. Generally, lead to increased cell division growth, avoid apoptosis, increased cell motility, invasion and loss of differentiation. Activated by genetic mutations, chromosomal translocations, gene amplification, gene amplification, epigenetic factors, increased protein stability. Oncogenes are usually gain of function genes, generally dominant. Tumour suppressor genes are usually loss of function mutations, generally recessive.

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One hit/ Two hit: Oncogenes: 1 hit= 1 allele mutation  cancer progression / TSG: 1 hit= no cancer. 2 hits= 2 allele mutations  cancer progression.

Lecture 3+4 Cell cycle      

Cell division cycle: important for: Maintenance/ repair/ selective advantage. Mitosis: PMAT- Interphase has G1, S and G2 phase. G1- (1st gap) growth or quiescence ?(quiescence = inactivity) S- synthesis of DNA G2- (2nd gap) is all DNA replicated with fidelity. G0- resting non-proliferative state of cells that have withdrawn from the active cell cycle. - Reduced protein synthesis. - Reduced growth - Reduced levels of CDKs - No G1 Cyclins

Cell cycle checkpoints:  

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G1/S is the cell ready for division- Can DNA synthesis begin? RB and p53 are important regulators here. Intra-S and G2/M – respond to DNA damage- prevent the replication of DNA damageprevent the replication of damaged DNA until repaired- if not possible the cell exits permanently from replication or is eliminated. Spindle checkpoint- are all chromosomes aligned to spindle correctly- if not mitotic arrest won’t proceed to anaphase. FAILURE of these checkpoints promotes genetic instability. Restriction point- Progression from G1  S depends on the presence of growth promoting signals in G1 phase up until R point. After cell has passed through R point it is no longer influenced by GFs. – Cell must decide whether to proceed. If so there are other checkpoints. Deregulation of R is common in cancers. Cyclins and Cyclin dependant kinases (CDKs): CDKs are serine/threonine kinases which depend on associated regulatory subunits, Cyclins to function. The cyclins associated with the CDKs activate the catalytic activity of their CDK partners. Active CDKs phosphorylate 100’s of target proteins to trigger downstream events. G1 stage: Cyclin Ds are activated by GFs  D- cyclins associate with CDK4+6 and move to nucleusThis is phosphorylated by CAK and then activated Cyclin D/CDK(4/6) phosphorylates Rb, also sequesters p27.  Rb is further phosphorylated by Cyclin E/CDK2.  leads to E2F release which is a key transcription for S phase genes.  progression of cell cycle. Brakes: CDK inhibitors can block activation of cyclin D/CDKs which is important during DNA damage and decision making. (Growth inhibitory factors eg. TGFB increased ecpresseion of CDKis and p15 and p21) p21 and p27 inhibit cyclin E,A,B and CDK1/2 complexes. G2/M Phase progression: during G2 CDK1 is kept in an inactive state by the kinases WEE1 and MYT1. As M phase approaches the phosphate CDC25 is activated activating CDK1/Cyclin B complex. CDC25A and B overexpression- frequent high-grade tumours and poor prognosis.

Lecture 5 c-Myc  

Akt, a serine/threonine kinase is activated downstream of GF receptors. Functions include inhibition of CDK inhibitors, promoting progression of cell cycle by blocking the actions of Glycogen synthase kinase 3b (GSK3b) and blocking pro-apoptotic proteins, Bad, and promotes cell growth.

Myc:   

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MYC is a transcription factor from chromosome 8. Expressed ubiquitously in cells during development and in growing tissues (skin/gut). Genetic alterations to MYC occur in multiple cancer types including cervix, colon, breast, lung, melanoma and a strong association with Burkitt’s lymphoma. MYC regulates 1000’s genes accounts 15-20% of all genes. Involved in many physiological functions: - Cell cycle control - Metabolism - Protein biosynthesis - MicroRNA regulation - Cell apoptosis - Senescence - DNA damage responses

Burkitt’s lymphoma: Aggressive B cell malignancy associated with EBV virus. Malaria linkage. Associated with chromosomal translocation of the MYC gene found at 8q24 cMYC translocation is not enough for malignancy. Tumors have extremely high rate of proliferation in addition to high rate of apoptosis. cMYC protein, short half-life, subject to post- translational modifications. Basic helix-loop-helix transcription factor. Full activity needs to dimerise with partner MAX at Leucine Zipper region and phosphorylation in transactivaiton domain. Transcriptional targets of MYC/MAX: E2F1, E2F2, E2F3, trascription factors cycliln D2, CDK4, CUL1. Normal MYC function: As MYC is rapidly degraded, pathway switches to a transcriptionally repressive sate when MAX dimerizes with a group of basic helix-loop-helix proteins, Mxd family are MYC antagonists. Phosphorylation at THR58 targets MYC for degradation. GSK3 is a serine/threonine kinase. Regulation of MYC: c-MYC stability-short half life c-MYC ubiquitination and degradation by proteasome. c-MYC regulates and is regulated by miRNAs Cancer-Threonine58 mutations In normal cells- cMYC expression/function tightly regualted MYC regulated by multiple signalling pathways; Ras,Wnt,Notch,EGFR pathways positively regulate TGFb and BRCA1 inhibit MYC gene expression and activiity respectively MYC promotes cell cycle progression; MYC/MAX regulates CyclinD2 and CDK4 expression. MYC involved in p27 degradation. MYC transcriptionally regulates CUL1 CAK then phosphorylates CDK2 leading to active CyclinE/CDK2- reauired

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for Rb hyperphosphorylation and E2F release. MYC is able to induce expression of E2F1, E2F2, E2F3, trascription factors. MYC binds to MIZ1 and leads to reduced p15 and p21 CDKIs expressions

p53 Lecture: Safety break of the cell cycle. Li-Fraumeni syndrome: Rare inheirited disorder, one normal and one abnormal allele of p53 Overview: Highly conserved tumor suppressor gene DNA binding protein, regulates expression of genes involved in DNA repair, cell cycle arrest and apoptosis. -maintains genome stability -gets activated by various stresses and damages -continuously removes genetically damaged cells (reduced incidence of cancer) -acts as transcription factor -inhibts cell proliferation and transformation Mutant p53 is responsible for its immortalising and transforming effects i.e. oncogene Wild type is a tumor suppressor Sequence specific transcription factor. Exists as a homotetramer Direct DNA bidnding. P53 inactive in normal cells and is located in the nucleus, 20 min half life, MDM2 main regulator of p53: inhibits transactivation activity, binds to molecule and initiates degradation. Autoregulatory feedback loop involving MDM2 Transcription factor: main function of p53 – activates some genes (central domain of protein) - Represses other genes ( C-terminal domain of protein) P53 is a transcriptional activator - Regulates transcription positively - functions as a transcription facotr that halts cell cycle response to DNA damage and attempts to aid in repair process. - Transcriptional activation of = p21- target of p53 GADD45 (role in repair DNA damage) BAX,Bid, APAF1 (role = apoptosis) Thrombospondin-1 (anti-angiogenisis) MDM2 (role =p53’s negative regulator p53 activation: In response to DNA damage Accumulates in nucleus Decrease in degradation rate Change of protein conformation leads to increased functional activity

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Forms tetramers Main activity cell cycle arrest of apoptosis. p53 can induce cell cycle arrest at G1 or G2 (Delay progression through the cell cycle. Function: Allows cellular damage to be repaired and prevents transmission of damaged genetic information from one cell generation to another If damage is detected in G1 then p53 will halt cell cycle progression through transcriptional ipregulation of CDKI p21 (increase in p21). DNA machinery then activates p53 targets CyclinB-CDK1: It targets 14-3-3 protein sequesters cuclinBCDK1 complex in the cytoplasm, prevents it from moving to the nucleus – prevents mitosis until successful DNA repair. GADD45 (p53 target) binds to and dissociates CDK1-cyclinB kinase. p21CDKI p53 and apoptosis: Activates death receptors (Fas). Inhibits transcription of anti apoptotic Bcl-2 and upregulates the pro apoptotic BAX Extrinsin vs Intrinsic pathways Clinical classifications of Breast Cancer: ER positive: ER/PR positive - LuminalA/B HER2 positive: HER2 overexpressed/amplified – HER2/Luminal B Triple negative: ER/PR negative – HER2 not overexpressed/amplified – Basal-like Receptor Tyrosine Kinase: 3 domains: Extracellular : binds ligand Transmembrane – anchors the recpetor to the cell surface Intracellular – transmits a signal to the cell, contains the kinase domain Normal vs cancer cells : signal transduction is normally tightly regulated In cancer cells constitutive activation of signalling pathways leads to dysregulation of cell division, growth and cell death Constitutive activation is caused by mutation and overexpression HER2 positive breast cancer: HER2 is over expressed in 20-25% of breast cancers Amplification of HER2 gene on chromosome 17q -increased receptor expression on cell surface - Increased phosphorylation of HER2 - Increased activation of Akt and MAPK pathways - Increased proliferation and decreased sensitivity to treatment Breast cancer patients with HER2 over-expression have a poorer prognosis compared to HER2 negative patients. Trastuzumab – Mechanism: Humanised monoclonal antibody with high specificity and strong affinity for HER2. - Down regulation of HER2 - Inhibits Dimerisation - Inhibits proliferation - Antibody dependant cellular cytotoxicity - Promotes apoptosis. Trastuzumab has two mechanisms for action 1) Inihibits HER2 signalling and 2) engages the immune system through antibody dependent cell-mediated cytotoxicity Pertuzumab – IgG1 mAb. HER2 dimerisation inhibitor Pretuzumab and Trastuzumab together overcame trastuzumab resistance in phase 2 trial. Lapatinib – small molecule tyrosine kinase inhibitor. Reversible, ATPcompetitive inhibitor of EGFR and HER2

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Mechanisms of resistance: Trastuzumab: -Alternative HER signalling -PI3K/Akt/mTOR signalling -ER signalling Trastuzumab: - IGF-IR -p-95 -MUC1 Laptinib: -AXL overexpression -MCL-1 overexpression -SRC upregulation -Overexpression of XIAP

Apoptosis Lecture:   

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Normal function: Tissue homeostasis – balance between cell death and proliferation. Apoptosis – Disease: Cell Death > proliferation: excessive apoptosis leads to degenerative diseases such as alzheimers and Parkinson disease. Suppression of apoptosis leads to cancer. Hallmarks of Apoptosis: - Cell Shrinkage - Chromatin condensation - Nuclear and Cell fragmentation - Membrane blebbing - Formation of Apoptotic bodies - Movement of membrane lipid phosphatidylserine from inner to outer side of plasma membrane- acts as recognition signal for phagocytic cells to engulf apoptotic cells. Triggers apoptosis: Radiation,cytotoxic durgs, poisons/toixns, nutrient deprivation, cell displacement Intrinsic pathway: Mitochonrial pathway, involves release of cytochrome C from mitochondria Extrinsic pathway: ‘Death receptor pathway’ Triggered by engagement of death receptors on cell surface. Both pathways result in activation of enzymes – Caspases, which lead to cell death. Caspases: Expressed as larger inactive pro-enzymes, amino terminal domain. Caspases have a large and small subunit Cleaved after aspartic acid Small and large subunited form a heterodimer or tetramer Act in an enzymatic cascade. Inhibitor procaspases: Caspases 8, 9 and 10 are initiator caspases, integrating different apoptotic signals. Prodomain has DED (death effector domain) found in procaspase 8 or 10 or CARD domain (caspase recruitment domain) in procaspase 9. DED or CARD allow for homophilic interactions with their adaptor proteins.

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Executioner procaspases : Executioner procaspases (3 and 7), activation requires proteolytic cleavage, mediated by initiator caspases. Executioner procaspase is cleaved at aspartate residues to give a short domain, a large and small subunit-form heterodimer-2. Heterodimers form the proteolytic tetramer. Effect/executioner pathway common to both intrinsic and extrinsic. Executioner Caspases kill via direct disassembly of cell structures. Caspases kill via inactivation of apoptotic inhibitors e.g. i-CAD Deregulation of other protein activity kills the cell-caspase3 Effector caspases: a) Cleave structural components – actin, lamins, cytokeratins b) Phospohotidylserine exposed on outside of cellular membrane c) Counteract BCL-Xl and BCL-2 d) Inhibit genes that regulate repair of DNA lesions during cell cycle (MDM2/Rb) e) Inactivate enzymes responsible for stability, integrity and repair of DNA (PARP/DNA-PK) f) Cleave iCAD – DNA degradation Intrinsic pathway of apoptosis: Activated by a variety of cellular stresses eg DNA damage caused by chemo and radiotherapies, hypoxia, depleted gf’s or deregulated oncogenes Cellular stresse signal is relayed to mt, leading to MOMP (mt outer membrane permeabilizaiton) channels open in the outer mt membrane – release of apoptotic proteins including cytochromeC and SMAC (Diablo) Cytochrome C release and role of anti-apoptotic proteins of BCL2 family: CytochromeC in the cytosol associates with other proteins (Apaf-1) triggers cascade leading to apoptosis. Smac (Diablo) also relased from mt, inactivates group of anti-apoptotic proteins called IAP’s (inhibitors of apoptosis). BCL-2 family members are key players – BCL2 and BCL-XL(anti apoptotic) work to keep the channels closed keeping cytochrome c in the mt. BAX and BAK are pro apoptotic. CytochromeC activates the apoptosome BCL-2 Family Members: 3 groups Prosurvival members; BCL-2, BCL-XL, MCL1, BCL-w and A1 (essential for cell survival Proapoptotic (BAX family): BAX, BAK essential for MOMP Proapoptotic; BH3 only family: BIM, BAD, NOXA, PUMA, BID – Required for apoptosis initiation. BCL-2 Family Members – Regulators of Apoptosis: BH3- only proteins thought to activate BAX/BAK either through direct binding [ tBID,BIM, PUMA] and or indirectly by binding to their repressors. BCL-2 family of proteins in Cancer: Upregulation of anti-apoptotic proteins and inactivation of pro-apoptotic proteins e.g. BAX is inactivated by mutation in over 50% of colon cancers.

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Inhibitors of apoptosis: 8 proteins include c-IAP1, c-IAP2, XIAP and survivin X-IAP inhibits caspase 3 and 9 c-IAP1 ubiquitinates caspase 3 and 7 – proteosomal IAPs inhibited by SMAC (diablo) Increased Akt activity (kinase) found in many cancers, Inhibits apoptosis – several targets : Akt targets BAD (inactivates) – BAD connected to intrinsic Suppression of pro apoptotic BH3-proteins BIM and PUMA through phosphorylation of FOXO3a Bid-Akt has been found to prevent cleavage of Bid (tBid) from extrinsic pahtway which connects to the mitochondrial pathway Promotes p53 degradation by stabilising MDM2 p53 and Apoptosis: p53 activated by broad range of cytotoxic stress singals. In unstressed cells p53 protein levels are LOW – Increase in response to stress signals p53 and BCL-2 Family: To induce apoptosis, p53 directly transcriptionally up-regulates expression of BCL-2 family members, the proapoptotic BH3-only proteins, PUMA, NOXA, and multi-BH domain protein BAX. PUMA most important for p53-induced apoptosis. p53 activates diverse pathways, not clear which are criticla for tumor supression. p53 and Apoptosis Extrinsic: p53 increases transcription of Fas p53 enhances Fas transport to cell surface Intrinsic: p53 induces BAX, PUMA and NOXA (pro-apoptotic proteins_ p53 induces APAF-1 Inhibits BCL2 amd BCL2-XL (direct binding) Extrinsic Pathway of Apoptosis. Induction brought about by signaling such as ligand binding to a specific plasma-membrane receptor Death receptors-ligands are members of TNF family of proteins (TNF-a, TRAIL,FasL) Role in normal mass tissue homeostasis unclear. Primarily involved in immune cell killing of viral infected cells and cells in other pathological states. Ligands Death Receptors TRAIL DR4 TNF related apoptosis inducing ligand DR5 TNF TNF RI Fas Ligand (CD95L) Fas (APO-1/CD95

Receptors TNF-R1, DR4, DR5 and Fas contain intracellular domain called the Death Domain (DD) Receptor Trimerization brings 3 death domains into close proximity Aggregation of the DD orients the DD in a conformation that recruits the adaptor protein FADD (Fas associated death domain) to form a complex called the Death Inducing Signalling Complex (DISC)

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DISC then recruits and activates the initiator caspase, procaspase 8. The DISC triggers the self-cleavage of the caspases – now active protease CD8+ T cell killing of Tumor cell through Death Receptor and Perforin/Granzyme mediated Pathways Some Tumor cells evade this host response by 1. Down-regulation of Fas receptor on tumor cells 2. Expression of non-functioning Fas receptor 3. Secretion of soluble for Fas receptor Killing (Fas mediated) 1. Extrinsic Pathway TCR engagement activates FAS ligand Killing via the extrinsic pathway Apoptosis triggered 2. Perforin/Granzyme Pathway TCR engagement activates the T-cell killing apparatus Lytic granules are delivered to Target CD8+ cells kill antigen-bearing cells

Immunosurveillance 

Tumor Immune Profile: Bad Vs Good M2 Macrophages Cross-presenting APCs Myeloid Derived Suppressor Cells ...