Fall 2022 Cancer Biology Exam 2 Study Guide PDF

Exam 2 Study Guide Fall 2022 Cancer Biology (BIOL4472/5472) The exam will cover chapters 5-9 of the MBC textbook. It will consist of multiple choice, short, drawn, matching and essay style questions. There will be at least one question for each of the items on this list (notice...

Exam 2 Study Guide Fall 2022 Cancer Biology (BIOL4472/5472) The exam will cover chapters 5-9 of the MBC textbook. It will consist of multiple choice, short, drawn, matching and essay style questions. There will be at least one question for each of the items on this list (notice that some things overlap). Some questions will assess multiple items on the list. Suggestions of what to focus on for each topic will be provided in the study/review session. Be prepared to ask questions about what you need clarification on during the review session. 1. The cell cycle a. Phases of the cell cycle i. G0 phase: most cells exist in this phase. Terminally differentiated, do not usually re-enter the cycle. (ex: neurons, rbcs, muscle cells). Stem and quiescent cells can be reactivated by mitogens or growth factors. Cell will not fully re-enter cell cycle until it passes G1. ii. G1 phase: Cell growth checkpoint-checks whether the cell is big enough and has made the proper proteins for synthesis phase. If not, it goes back to G0. Monitors internal and external cell environments. Cell growth, protein synthesis, and preparation for DNA replication iii. S phase: DNA synthesis checkpoint-checks whether DNA has been replicated correctly, if so it goes to M phase. iv. G2 phase: blocks entry to M phase, detection of DNA damage halts the cell cycle and activates DNA repair mechanisms. Environmental ans cellular stress can also trigger the checkpoint. Synthesizes proteins necessary for mitosis, including spindle fibers, and to check for any DNA damage before cell division. v. M phase: mitosis checkpoint-checks whether mitosis is complete. If so, the cell divides. 1. Mitosis (What are the stages? What happens during each stage?) Prophase: chromosome condensation, nuclear membrane breakdown, separation of duplicated centrosomes, and assembly of mitotic checkpoint proteins at the centromeres. Metaphase: aligning of the chromosomes on the metaphase plate, assembly of microtubules to form the mitotic spindle. Microtubule capture of both centromere regions of a chromatid pair silences the checkpoint. When the last pair attaches to the spindle, metaphase is complete. Anaphase: spindle pulling apart and separating chromatid pairs Telophase: accumulation of chromosomes at their respective poles, re-forming of the nuclear membrane, chromosome decondensation, and cytokinesis.  2. Cytokinesis: separation into two daughter cells b. Cycle checkpoints (What are they? What does each checkpoint do?) Made of biochemical signaling, trigger cell cycle arrest in response to problems with the cell. Maintain the integrity of the genome. Have 3 parts: sensors, signal transducers, and effectors. Sensors are proteins that detect a problem and activate the signal transducer. Signal transducers amplify and relay the signal, pushing the cell to make a decision about its fate. Effector proteins carry out the decision made by the cell. The decision usually serve to protect the cell. If damage is beyond repair, a different set of effectors can be activated which induce different response like cell death Cell growth checkpoint- Occurs near the end of G1. Checks whether the cell is big enough and has made the proper proteins for synthesis phase. If not, it goes back to G0. DNA synthesis checkpoint- Occurs in S phase. checks whether DNA has been replicated correctly, if so it goes to M phase. Mitosis (spindle assembly) checkpoint- Occurs in M phase. checks whether mitosis is complete. Determibes whether all sister chromatids have successfully attached to the mitotic spindle and that the spindle is intact/functional. If so, the cell divides. c. Bistability The transition from one phase to another occurs as a bistable switch. Regulated by feedback loops. Positive amplifies the system, and negative inhibits the system. The resting state switch is off, stimulus breaks the status quo and turns the switch on. These switches are tightly controlled by cyclin-CDK complexes. d. CDKs & cyclins i. Mechanism of regulation Phosphorylation and dephosphorylation by kinases and phosphatases. Specific proteolytic degradation is mediated by protein ubiquitination and subsequent targeting of the proteasome for degradation. 1. Association with cyclins 2. Addition of phosphate groups that activate CDKs 3. Association with CKIs (p16, p21) 4. Addition of phosphate groups that inhibit activity ii. Commonly mutated CDKs and cyclins in cancer P53, p21, CDKs, RB. Missense mutation in CDK4 blocks its inhibition by INK4 CKIs (melanomas). Chromosomal translocations causing over expression of CDK6 (leukemias). Amplification of cyclins D & E (breast cancers, skin cancers). CKI p16 INK4A deletion (lung, pancreatic, and colon cancers)) iii. CDK inhibitors  First gen CDK inhibitors are non-selective. Ex: Flavopiridol inhibits CDKs 1,2,4,6,7, & 9. Induces cell cycle arrest at the G1S ad G2/M phases. Was found in a plant from India. Next gen CKIs are more selective. Seliciclib inhibits CDKs 2, 7, 9. Palbociclib inhibits CDKs 4, 6. e. Basic principle (of the cell cycle): transfer a complete and precise copy of the genome from one generation to the next. Cancer cells violate this! f. Cell cycle cancer drugs: G0= cell cycle independent drug. Platinum/alkylating agents: Busulfan, Cyclophosphamide, Ifosfamide, Nitrosoureas. G1= hormonal agents: tamoxifen, megestrol acetate. Antitumor antibiotics: dactinomycin, doxorubicin, doxorubicin liposome. G1 restriction point= RB, p53 modulate S= Antimetabolites: Azathiioprine, Cladribine, Cytarabine. 5-fluorouracil, hydroxyurea. Topoisomerase inhibitors: Etoposide, teniposide, Irinotecan, topotecan. G2= Topoisomerase inhibitors: Etoposide, teniposide, Irinotecan, topotecan, bleomycin. M= Microtubule inhibitors: Paclitaxel, vinblastine, vincristine, eribulin g. Mitotic spindle inhibitors: Paclitaxel (stabilizes microtubules). Vinblastine/Vincristine (inhibit microtubule assembly). Ispinesib: targets kinesin spindle protein (KSP) and prevents mitotic spindle pole separation. 2. Tumor suppressor genes (Know the ones that were discussed in class.) a. RB protein i. Role in cell cycle regulation Regulates activity of E2F transcription factor family, critical for cyclin expression and genes needed in S phase. Cyclin-CDK complexes regulate RB activity via phosphorylation. Product of RB1 tumor suppressor gene. Chromatin associated protein. Regulator of TFA. Inhibits G1 to S phase transition. Indirectly regulates transcription of genes affecting cell proliferation and differentiation (E2F, chromatin modeling enzymes). ii. Protein structure & binding partners Nuclear RB protein, p107, and p130 are “pocket proteins”. Binding of HDAC and E2F to the pocket is important for its function. LXCXE motif of HDAC binds domain B. E2F and its associated subunit DP, recognize a conserved sequence at the interface of the domains. iii. Types of mutations in cancer Cancer requires loss of BOTH Rb1 alleles. Most mutations are deletions, frameshift, or nonsense mutations that result in defective RB function. Mutations of Ser567 are found in cancer and may disrupt the normal regulation E2F activity. RB1 is in all tissue but only some retinoblastomas, small-cell lung carcinomas, osteosarcomas and a few other types of cancer have RB1 mutations. Inactivation promotes chromosome instability, angiogenesis, errors in chromosome segregation, aneuploidy. b. p53 protein i. Role in the cell cycle Guardian of the genome. Main part of a cell’s tumor suppressive mechanism. Inhibits the cell cycle. Triggered by induction of CDKN1A gene (p21). Decision- repair or senescence. Two homologs: TP73 and TP63. It is one of the most highly connected proteins to other signaling pathways and can recurve numerous stress signals and respond by diverse actions. In the absence of cell stress, low p53 induces antioxidant activity with combats ROS. ii. Role in apoptosis Can be induced intrinsically or extriniscally. Pro-apoptoptic genes are induced, anti-apoptotic genes are repressed. iii. Upstream signals & molecular pathways that activate p53 DNA damage (ATM/CHK2), aberrant growth signals , oncogene activation (p14ARF/MDM2), cell stress (hypoxia, nucleotide depletion, ATR/Casein kinase II). Disruption of the p52-MDM2 interaction is vital for p53 activation. iv. Downstream molecular mechanisms, targets and effects of p53 activation Cell cycle arrest or senescence, DNA repair, apoptosis, inhibition of angiogenesis. 1. Apoptotic gene targets (Are they intrinsic or extrinsic apoptosis?) Intrinsic pathway: BAX, NOXA, PUMA, p53AIP1 Extrinsic pathway: FAS, IGFBP3, DR5, PIDD, PERP 2. Cell cycle targets Repression of cyclin A, cyclin B1, cyclin B2, cdc2/cdk1, Cdc25A, Cdc25C, Rad51, PLK1/2 and BRCA1. P21 too. 3. Other targets (DNA repair, angiogenesis, others) XPC gene (NER), Thrombospondin (angiogenesis inhibitor), GPX1 & SESN genes (antioxidants), TIGAR, (glycolysis inhibition) Inhibition of SNAIL by miR-3A (prevents cancer cell migration) v. Protein structure (domains and mutation hotspots) Transactivation domain/MDM2 binding site, DNA binding domain, Tetramerization domain, Regulatory domain. Mutational hotspots in DNA binding domain: 245, 249, 282  vi. Regulation by MDM2, MDMX, and HAUSP MDM2: ubiquitin ligase, main regulator of p53, modifies the carboxy-terminal domain of p53 by ubiquinating it. (proteolysis, occurs in proteosomes) Binds to and inhibits the p53 transactivating domain at the amino terminus. MDMX: can also inhibit p53 HAUSP: REMOVES ubiquitin from p53 vii. Regulation of apoptosis and arrest (How does it decide?) Promotor selectivity and p53 binding affinity influence cell fate. Response element sequence, position of response element in the promoter, post translational modifications of p53, absolute levels of p53, p53 activation kinetics, subcellular p53 location (TF independent roles), cell type, epigenetic state, and binding partner presence. Threshold needs to be reached to induce apoptosis. viii. Mutations in cancer P53 is the most frequently mutated gene in cancer. Mutations cause genomic instability. High frequency of mutations is due to selective pressure favoring mutant cells that escape tumor suppression. MORE than 75% of mutations are missense (90% are in DNA binding domain, 30% of those affect 6 codons aka hotspots). Many mutants are more stable than wild type p53, allowing for build up of mutated protein that may show a gain of function (Ex: histone modification to allow increased cell growth) ix. Li-Fraumeni syndrome Germline deletion of TP53, young age of patients, frequent occurrence of multiple primary tumors. Autosomal dominant disease (50% chance of passing it to kids). 25-fold increased risk of cancer before 50 yrs. Tumor types: sarcomas, breast, leukemia, and brain. Not always a complete loss of WT TP53 allele (haplosufficiency is enough for tumor formation). DOES NOT fit Knudson’s two-hit hypothesis (tumor suppressor “dose” may play a role in cancer development aka dominant negative scenario. This leads to abnormal oncogenic activities) x. Mechanisms and drugs correcting p53 VIRAL- Advexin: gene therapy -> p53 restored. Onyx 015 virus: Viral replication/ Cell lysis. CHEMICAL- APR-246/PRIMA-1: Restores p53 conformation/ Aberrant folding. Ataluren: Read through. Nutlins/MDM2: target p53 inhibitor MDM2/activates p53. Pifithrin-a: Inhibits p53 transcription/may be used to limit side effects of chemotherapy in normal cells. c. PTEN Phosphatase and tensin homolog on chromosome 10. Tumor suppressor & phosphatase frequently mutated in cancer. Dual specificity (acts like a protein and lipid phosphatase). Dephosphorylates PIP3 to form PIP2 (blocks activation of the PI3K pathway which helps cells avoid apoptosis and promotes cell growth) Cowden syndrome- germline deletion of one PTEN allele.  d. P21 A tumor suppressor gene that regulates the cell cycle and prevents DNA damage from turning cancerous. Inhibits several cyclin-CDK complexes leading to G1-S and G2-M cell cycle pauses. Promotes PCNA which is involved in DNA synthesis and repair. miR-34a (p53 target) has similar cell cycle arrest and senescence inducing properties 3. Aneuploidy The occurrence of one or more extra or missing chromosomes in a cell or organism. Aneuploidy refers to any chromosome number that is not an exact multiple of the haploid number of chromosomes (which is 23 in humans). Ex: Down Syndrome 4. Knudson’s two-hit hypothesis Two separate mutations are needed to inactivate the two allelic copies of a tumor suppressor gene to induce cancer. a. Retinoblastoma – how/why does it fit the hypothesis? Retinoblastoma is caused by a mutation/loss of RB1. 1 germline (from parents) and 1 somatic mutation (mitotic recombination). Fits hypothesis since two mutations are required for cancer. 5. Continuum model of tumor suppression Knudson’s two-hit hypothesis +exceptions. Subtle dosage effects of tumor suppressors as a result of changes in either levels of expression or protein activity, can play an important role in carcinogenesis. (PTEN, PAX5, & TP53 are affected by dosage). In the continuum model, both tumor suppressors and oncogenes are considered. For tumor suppressor genes (TSGs), their expression is correlated with reduced malignancy or tumor formation (this is a negative correlation, light green line). When a complete loss of the TSG occurs, a fail safe can be triggered that affect tumor development to some extent (dark green line). With oncogenes, their expression is positively correlated with tumor development (more oncogene expression = more tumors or higher chance of tumor formation. Red line). If too much or over expression of oncogenes occurs, this can also trigger fail safe systems to reduce the impact of the overexpression leading to a negative correlation with tumor development (orange line). 6. Viruses that affect cancer a. Viruses that promote cancer development (oncogenic viruses) Papovaviruses, Adenoviruses, herpesviruses, Hepatitis B, HPV  b. Viral protein effects on p53 protein Inactivators: Adenovirus E1B, Papillomavirus E6, SV40 large T antigen c. Viral protein effects on RB protein Inactivators: Adenovirus E1A, Papillomavirus E7, SV40 large T antigen E1B, E6, and E7 can trigger degradation of p53 or RB by attaching ubiquitin to the tumor suppressor proteins. 7. Apoptosis a. Process/steps Controls cell numbers and eliminates damaged cells. Important mediator of tumor suppression. Ensures cells with excessive damage can’t proliferate. Mechanisms to evade cell death is a cancer hallmark, so inhibition of apoptosis is a high priority for tumor cells. Intrinsic, active, organized, ends in phagocytosis by macrophages and neighbor cells. 1. Cell shrinks/chromatin condenses 2. Membrane blebbing/organelles disintegrate 3. Nucleus and organelles collapse/more membrane blebbing 4. Apoptotic bodies form 5. Macrophages phagocytose apoptotic bodies b. Key features (SUMO differences to necrosis) c. Intrinsic and extrinsic pathways (know how the pathways converge too) Intrinsic: does not depend on external stimuli (death factors). Activated by DNA damage and cell stress via BCL2 family on the mitochondrial membrane (can be pro or anti apoptotic). Permeabilization of the mitochondrial membrane and release of pro-apoptosis factors is required. Extrinsic: triggered by death factor ligands. Adaptor proteins (FADD/TRADD): bind receptor death domains, transfer death signal from receptors to caspases, recruit procaspase-8 via DEDs. Death inducing signaling complex (DISC). cFLIP can inactivate the pathway by binding DEDs or FADD (catalytically inactive caspase-8 homolog) Convergence of pathways: Converge at the activation of the downstream caspases. Both pathways converge when caspase 3 (executioner caspase) is activated, resulting in cell death. Example: Caspase-8 cleaves BID (a pro-apoptotic BCL2 family member). BID directly activates Bax and Bak which allows cytochrome C to be released from the mitochondria. Downstream caspases are activated. d. Caspases Cysteine-rich aspartate proteases. Molecular scissors that cut intracellular proteins at aspartate residues. Help break down cellular components for disposal. Synthesized in their inactive form-procaspases (2% the proteolytic activity of active caspases/ activated by cleavage at aspartate residues). Domino effect: only a few caspases are needed to rapidly convert all procaspases to their active forms (amplification of apoptotic signal) e. BCL2 proteins 25 members that contain at least 1 BCL homology (BH) domain. The BH3-only pro-apoptotic proteins (i.e. BIM), (Induce activity of pro-apoptotic molecules) (BH3-only activators) (Inhibit anti-apoptotic BCL2 proteins) (BH3-only sensitizers). Pro-apoptotic members are tumor suppressors. Anti-apoptotic genes are oncogenes f. MOMP Mitochondrial outer membrane permeabilization. Process of pro-apoptotic BCL2 family member regulated release of apoptotic mediators from the mitochondrial compartment. Initiates by BID and DIM (BH3 only proteins) activating BAX. BAX changes conformation which increases permeability of the outer mitochondrial membrane. Pores formed due to permeability release apoptotic mediators (i.e. cytochrome C and caspase-9) g. IAPs & SMAC/DIABLO IAPs: XIAP inhibits caspases 3 & 7 once they have been processed by binding to their active sites. XIAP can also inhibit caspase 9 by changing the conformation so the active site is unavailable NF-κB (a transcription factor) is a potent inhibitor of apoptosis that induces transcription of IAPs SMAC/DIABLO: Eliminates inhibition by IAPs. Competes with activated caspase 9 to bind XIAP h. Defects that affect tumor development Extrinsic pathway defects FAS pathway mutations, Caspase-8 suppression (deletions, missense mutations, hypermethylation) Intrinsic pathway defects  p53 mutations, ATM, CHK2, MDM2 mutations, Oncogenic activation of BCL2 due to chromosomal translocation i. Drugs that activate apoptosis SAHA Most chemotherapeutics induce DNA damage (Intrinsic pathway stimuli), Some chemotherapeutics induce TNF (Extrinsic pathway stimuli), Loss of p53 makes cells resistant to apoptosis, Increased expression of anti-apoptotic proteins (BAX) increases drug resistance. 8. Necrosis a. Process/steps Cells can sometimes recover before fully committing, Passive process, “Untidy” or “Messy”, Ends with the death of many cells – like a bomb going off b. Key features (SUMO differences to apoptosis) 9. Pyroptosis a. Activation Dependent on caspase or granzyme activation of GSDM (forms pores in cell membrane). Can be induced by chemotherapeutic drugs, activated immune cells, and inflammatory signals (cytokines like IL-1β and TNF-α) b. Key mediator proteins Pore-forming Gasdermin (GSDM) proteins. GSDMB, GSDMC, GSDME. 10. Alternative death pathways Necroptosis Similar to necrosis, Active, regulated from of cell death, Requires receptor-interactingserine/threonine-protein kinase 3 (RIPK3) Mitotic catastrophe Caused by aberrant mitosis, Defects in mitosis genes (i.e. BECN1) can cause tumorigenesis Autophagy (means “eating oneself”) Recycling system for the cell, Excessive autophagy triggers non-apoptotic cell death 11. Autophagy a. Steps (initiation, nucleation-elongation-maturation, fusion, degradation) Initiation: Pre-autophagosomal structure (PAS) and ULK1 compex Nucleation-elongation-maturation: Phagophore to Autophagosome membrane isolation, Nucleation: PI3K compex, ATG9A system, Elongation: ATG12-conj. System, LC3-conj. system Fusion: Autophagolysosome Degradation: Autolysosome b. Benefits to cancer Cancer cells upregulate autophagy and use the breakdown products to sustain nucleotide pools and energy homeostasis and, thus, their own growth and survival. In early cancer stages, efficient Autophagy causes tumor survival. Inhibited autophagy: apoptosis/ tumor regression & necrosis/inflammation. Enforced autophagy: PCDII and tumor regression. 12. Stem cells a. Types of division Classical model: stem cell division is intrinsic and asymmetric (one daughter cell maintains stem cell characteristics, and the other shows characteristics of differentiation. Neutral competition model: stem cell division is dependent on the niche and the niche size. A parental stem cell can produce 2 or 2 new stem cells depending on the competition of the daughter cells to occupy the stem cell niche. b. Difference between regular stem cells and CSCs BOTH CAN: Self-renewal & ability to phenotypically diverse cancer cells with limited proliferation potential (aka regular cancer cells) Use both the classical and the neutral competition model of stem cell division KEY DIFF: CSCs can produce new tumors if transplanted into host animals like mice c. Cellular plasticity (broad concept, not specific details or examples) Homeostasis and Regeneration. Hierarchic stem cell model/ Dynamic stem cell model. Migration, de-differentiation. After damage, cells can de-differentiate back into their stem cell forms. d. Self-renewal Is the ability to regenerate or be re-born. Stem cells self-renew by producing daughter cells which maintain the stem cell characteristics. Relevance to cancer: Self-renewal provides increased opportunities for mutations to occur. Altered regulation of self-renewal directly contributes to carcinogenesis. i. WNT signaling pathway 1. Inactive and active Inactive: kept inactive by the degradation complex (AXIN, APC, GSK3B, & CKI). AXIN & APC for the scaffolding for GSK3B & CKI which act as serine/threonine kinases to inactivate β-catenin and flag it for degradation. Inability of β-catenin to bind the TCF/LEF transcription factors allows Groucho to bind and inhibit them. Active: Porcupine (PORCN) attaches palmitoleic acid to WNT, modifying it so that it can bind Frizzled and its LRP co-receptor. Phosphorylation of LRP by CKI and GSK3B recruits AXIN away from the degradation complex. β-catenin can then go to the nucleus and bind the TCF/LEF transcription factors to induce target genes. β-catenin sometimes needs co-activators (i.e. BCL9 and Pygopus) to activate the transcription factors. Some target genes provide feedback to the WNT pathway Negative: RNF43 & ZNF3 Positive: R-spondin 2. Inhibitors Best inhibitors block interaction of β-catenin with TCF transcription factors. Drugs at this stage counter the inactivating mutations of APC, GSK3B, & Axin as well as activating mutations of β-catenin. PRI-724 prevents β-catenin binding with its co-activator, CBF. For tumors with normal APC Inhibition of WNT secretion would inhibit CSC self-renewal LGK974 – PORCN inhibitor Ipafricet – WNT decoy protein Vantictumab – Frizzled antibody Rosamantuzumab – R-spondin antibody 3. Defects in cancer Mutations that activate WNT signaling have been identified in many cancers. Most inactivate APC or activate β-catenin ii. HH signaling pathway 1. Inactive and active Generally inactive in adults except during tissue repair and maintenance. 3 primary proteins (Sonic, Desert, and Indian). 2 main receptors (Patched & Smoothened). Patched can inhibit Smoothened when HH proteins are absent.GLI proteins are the transcription factors of the HH pathway. When smoothened is inactive, SUFU CK1, and GSK3B form a complex that degrades GLI When smoothened is active, it teams up with EVC and EVC2 to dissociate the complex that degrades GLI. This allows GLI to be transported to the nucleus and get activated. 2. Benefits to cancer Many tumors have increased HH signaling. Activating mutations in Smoothened. Increased expression of GLI and other HH family  members. Patched is a tumor suppressor gene. Inhibiting mutations are associated with increased cancer risk in many tissues. 3. Inhibitors Vismodegib & Sonidegib for BCC. Glasdegib for AML (these drugs inhibit Smoothened). Prevention of Sonic HH from binding Patched. Robotnikinin blocks interaction of Sonic HH and Patched. 5E1 antibody inhibits Sonic HH. Inhbitiing HH transcription. GNAT61 prevents GLI from binding DNA. iii. PcG proteins Polycomb group proteins. Repress transcription of specific genes by epigenetic mechanisms (i.e. histone modifications, gene methylation). Repress developmental proteins and the p53 pathway. Cannot bind specific DNA motifs. They are recruited by transcription factors and/or lncRNA (i.e. HOTAIR). Nicknamed the “guardians of stemness” 1. Role in cancer and stem cells Suppress differentiation. Promote self-renewal.Over expression of PcG proteins or their partners are known to occur in cancer, (BM1 (PcG repressor), EZH2, SUZ12.) Behave as oncogenes 2. Inhibitors Inhibitors of PcG proteins affect the self-renewal potential of CSCs. Inhibition BMI1 using PTC-209 (Shown to inhibit colorectal cancer CSC growth in vitro. Inhibited tumor growth in mice) 13. Lineage specific transcription factors in leukemia Lineage-specific transcription factors (Activate lineage-specific genes, Inhibit the cell cycle in terminally differentiated cells). Important for cellular differentiation. Disruption of transcription factor function can lead to cancer development. Examples: AML & CLL a. In tumor development b. RUNX1 RUNX1 (aka AML1) is a TF important for almost all HSC lineages c. PML-RAR APL Generally caused by a chromosome translocation (15;17), PML-RAR hybrid protein d. Leukemia differentiation therapies Aim to change malignant cells into benign, normal cells. ATRA (all trans retinoic acid) treatment yields complete remission of APL. Converts PML-RARA from repressor to activator to induce proteolysis. Relapse occurs quickly due to resistant CSCs. Combination with ATO (arsenic trioxide) cured patients. ATO induces degradation of PML and PML-RARA by flagging PML for degradation 14. EMT a. Role in self-renewal The epithelial–mesenchymal transition (EMT) process can give cells the ability to self-renew b. Role in metastasis  EMT is a developmental program that causes cells to lose their epithelial characteristics and gain mesenchymal features, which makes them more invasive and migratory c. Benefits to cancer (i.e. drug resistance, metastasis, etc.) Tumor immune-escape, stemness, resistance to therapy. d. Induction of EMT Epithelial–mesenchymal transition (EMT) is a process that occurs when cancer cells lose their epithelial properties and develop mesenchymal properties. Instigated by signals from the tumor stroma Ligands: HGF, EGF, PDGF TGF-β Kinase receptors: MET receptor, EGFR, PDGFR, and TGFR Signal transduction pathways: MAPK and PI3K Transcription factors: SNAIL, SLUG, ZEB1, TWIST, and FOXC2 Epithelial genes are turned off. E-cadherin, β-catenin, claudin-1, occuldin, type IV collagen Mesenchymal genes are turned on. N-cadherin, SLUG, TWIST, ZEB1, fibronectin, vimentin, S100A4, MMPs e. Epithelial cell characteristics Inflammatory microenviornment, soluble factors, CAFs, ECM, hypoxia and low ph, immunoediting, antitumor drugs, alternative splicing. f. Mesenchymal cell characteristics Poor prognosis, metastasis, invasion, tumor progression, Tumor immune-escape, stemness, resistance to therapy. 15. Metastasis Process by which tumor cells from a primary site invade and migrate to other parts of the body It is impossible to remove all metastases from patients by surgery a. Types of metastasis i. Spreading – Monoclonal: seeded by one cell or subclone Polyclonal: seeded by 2 or more subclones ii. Pattern – Linear: from primary tumor to metastasis Branched: one primary tumor seeds 2 or more metastases iii. Combinations of spreading and patterns Monoclonal linear Monoclonal branched Polyclonal linear Polyclonal branched iv. Cross-seeding one metastasis seeding 1 or more other metastases alone or with cells from the primary tumor. b. Influencers tumor microenvironment, CSC subpopulation, Interaction of the tumor with distant locations via signaling molecules. c. Metastatic cascade/steps Invasion, Intravasation, Transport, Extravasation, Metastatic colonization 1. Cell types required and their roles/functions/contributions Entry of the tumor cell into a blood or lymphatic vessel Requires the meeting of 3 cell types: Perivascular macrophage (TIE2high/VEGFhigh) Tumor cell over expressing MENA Endothelial cell Combined, the cells make the TMEM doorway 2. 3 steps of intravasation Streaming migration chemotaxis mediated co-migration Requires macrophage CSF1R and tumor cell EGFR expression Can be blocked by using CSF1R inhibitors Vascular permeability Requires macrophage VEGF, tumor cell MENA, and MMPs (from other cells) Inhibiting MENA reduces the number of circulating tumor cells Transendothelial migration Passing of tumor cell between endothelial cells and into the bloodstream iii. Transport Is one-way (in the direction of blood flow) Is harsh on migrating tumor cells Cells need to resist immune cell attack and anoikis as well as survive without ECM Expression of oncogenes like EGFR helps tumor cells survive transport Tumor cells may form clumps with platelets that secrete TGF-β that inhibits immune cells and enforces EMT Only ~0.01% of circulating tumor cells are successful at metastasizing iv. Extravasation 1. 3 mechanisms of extravasation Initial attachment and rolling, arrest and ahesion, diapedesis/transmigration. Stepwise process, Rupture of the vessel due to cancer cell proliferation, Induction of necroptosis 2. Steps of non-traumatic extravasation tethering/rolling, integrin activation, firm adhesion, transendothelial migration. v. Metastatic colonization Depends on the location of extravasation Ability of the tumor cell to adapt to the new microenvironment Is the establishment of a progressively growing tumor at a distant site, involving the formation of new blood vessels (to provide nutrients)  Disseminated tumor cells (DTCs) can spread to other tissues early during carcinogenesis, but do not initiate growth of a new tumor CSCs or CSC-like cells are required to initiate a new tumor Can remain dormant for years as a micrometastasis in quiescence (non-proliferating state that is reversible) 1. DTCs Can spread to other tissues early during carcinogenesis, but do not initiate growth of a new tumor CSCs or CSC-like cells are required to initiate a new tumor Can remain dormant for years as a micrometastasis in quiescence (non-proliferating state that is reversible) 2. Dependencies d. Pre-metastatic niche i. “Seed and soil” theory Metastases selectively colonize specific organs because of a “match” between the migrating tumor cell and a “suitable” environment BM cells respond to systemic factors (released by tumor cells) by migrating to the pre-metastatic niche and re involved in preparing a favorable environment for the cancer cells to colonize ii. How is it formed Bone marrow (BM) cells express VEGFR1 and VLA-4 to help them bind to fibronectin rich environments Tumor type-specific factors from the primary tumor change the environment before the tumor cells arrive The factors help create the niche in selective locations iii. Characteristics iv. Key proteins/requirements e. Exosomes Small vesicles (30-100 nm diameter) that carry nucleotides and proteins Formed by invagination of endosomal membranes and cytosol samples creating multivesicular bodies Multivesicular bodies fuse with the cell membrane to release exosomes from the cell Exosomes contain DNA, RNA, lipids, and proteins f. Metastasis suppressor genes Known as metastasis suppressor genes because they have low expression in tumor metastases. Inhibit overt metastasis without affecting primary tumor growth Can maintain/keep DTCs in their dormant state ~23 known genes/miRNAs NM23 (inhibits MAPK signaling) MKK4 (inhibits metastatic colonization through apoptosis induction) miRNA-335 & miR-126 Other genes also regulate MAPK signaling as well as gap junction communication g. Metastasis susceptibility genes Can be cancer type specific  Are influenced by pre-existing genetic makeup. For example, gene polymorphisms Genetic makeup can influence cancer progression, its metastatic potential, and where the tumor might metastasize to Germline variants of APOE APOE4 – induces a favorable outcome by inducing anti-tumor immunity APOE2 – induces a poor outcome by facilitating tumor invasion h. MPI and EMT blockers MPIs (no current drugs with FDA approval). Marimastat & Neovastat (MMP inhibitors) failed clinical trials. Poor trial design (given in late-stage cancer instead of early). Unexpected side effects (pain) MET inhibitors: In many cases are similar to EGFR inhibitors. Cabozantinib – small molecule that prevents receptor activation. Inhibits MET receptor and VEGFR What does RIPK3 induce? Necrosis Cancer vs normal antibiotics Anti-cancer antibiotics CANNOT inhibit bacterial cells Focus on Chapters 5 and 6