Biology 318 Fall 2024 Midterm Exam Study Guide PDF

Biology 318 Fall 2024 Midterm Exam Study Guide Introduction to Cancer (lecture #1): What is cancer & how it can kill. ○ Unregulated growth of tissue, tumors arise from tissue ○ Cause of death: destruc...

Biology 318 Fall 2024 Midterm Exam Study Guide Introduction to Cancer (lecture #1): What is cancer & how it can kill. ○ Unregulated growth of tissue, tumors arise from tissue ○ Cause of death: destruction of critical organs, infection due to impaired immune responses easting (cachexia), hemorrhage Understand the concept that cancer is a group of diseases that affect multicellular species. Differences b/w invasiveness & metastasis. ○ Invasiveness: Cancer cells growing past normal stromal boundaries; enclosed in connective tissue capsules ○ Metastasis: Spread to & growth in foreign sites; central feature in malignancies ut a few cancers can develop the ability to metastasize Exceptions to metastasis: gliomas and basal cell carcinomas of skill, both of which are highly invasive Know the terms presented on slides 7 & 8. ○ Hyperplasia: growth of excessive number of cells; cells look normal and are able to form normal looking tissue despite deregulated proliferation ○ Metaplasia: one type of cell layer is replaced by another cell type not normally found on that particular site of the body; ex: barret’s esophagus ○ Dysplasia: cytologically abnormal; changes in size/cell shape of the nucleus, increased mitotic activity, lack of normal cytoplasmic features present in differentiated cells; result in major changes in tissue architecture; transitional state between benign and premalignant growth ○ Neoplasia: abnormal, disorganized growth that leads to formation of a distinct mass; refers to both malignant and nonmalignant growth ○ Anaplasia: tumor cells that have “dedifferentiated”. No longer possible to use histopathological criteria to identify tissue of origin. ○ -oma: usually benign Staging of cancers and its relationship to prognosis. ○ Stage 1 (Survival: 75-90%) ○ Stage 2 (Survival 45-55%) ○ Stage 3 (Survival 15-25%)  ○ Stage 4 (Survival under 5%) For major types of cancers and examples of those that don’t fall into any of these four categories. (1) Carcinoma: Most common type; cancer of epithelial origin (2) Sarcoma: cancer of mesenchymal/connective tissue origin (3) Leukemias and lymphomas: cancer of blood (hematopoietic) cells (4) Neuroectodermal: glioblastomas, neuroblastomas, Schwannomas Cancer as a genetic disease and the relationship b/w cancer & aging. ○ As a genetic disease: inherited mutations may play a role but development of cancer require additional hits ○ Relationship between cancer & aging: strong age association increase (ex: colon cancer 100x higher for 8- year old man than 30 year old); cancer usually takes time to develop (ex: smoking leads to lung cancer 20-30 years later) Cancer as a monoclonal disease and the examples given. ○ Monoclonal: deriving from one cell Two Examples: Leiomyomas: benign tumors of the uterine wall; observe pattern of X-chromosome inactivation ○ Expresses only one of two variant form of the enzyme, glucose-6-phosphate dehydrogenase (G6PD) but not both Myelomas: cancers involving B cell precursors Understand cancers as they affect human populations: causes, variations in frequencies of specific cancers in different parts of the world, etc. ○ Factors: Viral: tumor viruses Carcinogens: physical; chemical Lifestyle Ames Test: what it is, strengths & limitations. ○ What it is: measures the frequency of “back mutations” in strains of salmonella unable to grow without histidine ○ Strengths: simplicity & efficiency, sensitivity to mutagens, use of metabolic activation systems ○ Weaknesses: limited to prokaryotic cells, false positives and negatives, limited scope of mutations, does not address non-mutagenic carcinogens  Hallmarks of cancer, reductionist vs. heterotypic view of tumor biology (importance of stromal environment). ○ Hallmarks of cancer (1) Loss of growth factor dependence (2) Loss of responses to anti-growth signals and differentiated state (3) Resistance to apoptosis (4) Limitless replicative potential (5) Recruitment of body/lymph (6) Invasion of metastasis (7) Genetic instability Oncogenes (lecture #2): Definitions, general features of oncogenes and tumor suppressors. ○ Oncogenes: gain of function mutations, “always on” hence tumorigenesis ○ Tumor suppressors: loss of function mutation, inactive tumor suppressor hence tumorigenesis Mechanisms of protooncogene oncogene transformation and examples of each. ○ Quantitative: gene amplification - Myc, HER2 ○ Qualitative: point mutation - Ras Know all details concerning Ras and Myc: what these proteins are, how they work, signaling pathways these proteins are involved in, how they’re involved in cancer. DON’T need to know all pathways outlined in slide 17, however, you DO need to know the MAP kinase pathway. ○ Ras What is it: Small (21kD) protein that binds guanine and nucleotides (GDP, GTP) hence “G protein” that resides in plasma membrane (farnesyl palmitoyl anchors - potential targets for therapy) ○ 3 Forms K-Ras: associated with pancreatic, lung, and colon cancer H-Ras: associated with bladder, urinary tract cancer N-Ras: associated with leukemia How do they work: ○ Ras-GDP is inactive whereas Ras-GTP is active ○ Ras can hydrolyze GTP to GDP thereby inactivating itself ○ Ras acts as a molecular switch, alternation between GDP and GTP bound forms when signaling is received  ○ Initiated through GF binding to RTKs Signaling pathways involved in: ○ Raf-MAPK ○ RI3K-Rac ○ RalGDS ○ PLCE How it’s involved in cancer / Ras pathways components in cancer: ○ Ras: mutations that abolish GTPase activity and lock Ras in an active state ○ RTKs: overexpression of Her2/neu subunit leads to increased signaling ○ Raf: overexpression associates with lung and liver tumors ○ GAPs: aka neurofibromin is deficient making Ras bound to GTP, hence active ○ RalGDS, PI3K, PLCE: role downstream of Ras in cancers most likely aka other effectors ○ Myc What is it: bHLH-Zip DNA binding protein; a transcription factors that turns on other genes and regulates many genes including those linked to cell growth and proliferation How do they work: Arises due to ○ Amplification ○ Translocation of Myc gene resulting in deregulation of gene activity Binds DNA only as heterodimer with Max (highly related protein) ○ Max: a general factor that interacts with Mad which repressed many genes that are activated by Myx::Max; also interacts with Mga, Mnt How it’s involved in cancer: When it’s over expressed in signaling cells… ○ Reduces dependence on exogenous growth factor: blocks exit from the cell cycle, increases cell size, accelerates progression through the cell cycle ○ When Myc induction is blocked in cells stimulated by serum, cells do not enter the cell cycle ○ Blocks terminal differentiation  MAP kinase pathway Mutations that affect other components in MAP kinase pathway (Erbb2, Her2, Raf, NF-1, etc.). ○ Erbb2 aka Her2: breast cancers ○ Raf: thyroid, colorectal, lung cancers ○ NF-1: neurofibromatosis type 1 (mutation leads to persistent Ras activation) Targeted therapies: FTIs and Ras. ○ FTIs: inhibits ras activity in cancers, keeps Ras away from membrane and inactive Same deal for Myc: know it! Genes regulated by Myc. ○ Highly controversial Activity on reporter genes in transient transfection week (2-5X) Effects on real target genes also small, not always same as transients Activation of some targets cell type-specific Some genes repressed by Myc Recent studies show that Myc DNA-binding activity does not correlate directly with target gene expression - large # of gene promoters bind Myc, only a fraction are known to be regulated by Myc Know, especially, what cancers are linked to Myc and how this occurs (i.e. chromosomal translocation, amplification).  ○ C-myc: Burkitt’s lymphoma (B cell tumor) by reciprocal translocation between c-Myc and IgG locus ○ N-myc: neuroblastomas ○ Multiple Myeloma ○ Small Lung Cancer ○ Breast Cancer ○ Colorectal Cancer ○ Ovarian Cancer ○ Acute Myeloid Leukemia (AML) ○ Medulloblastoma What does Myc activate and repress? ○ Activates: expression of cyclinD, cdk4; “pushes” cell cycle forward ○ Represses: p21, a CDK inhibitor Tumor Suppressors (lecture #3): Know what are LOH & the Two-Hit Hypothesis. ○ LOH: aka loss of heterozygosity; ○ Two-Hit-Hypothesis: Suggested by Alfred Knudson What it is: mechanism that explains inherited retinoblastoma. It correctly predicted a general role for LOH in tumor suppressor genes and explained basis of much more rate non-hereditary retinoblastoma; two hits are needed in both Rb First hit occurs (early in development or later) can determine the # of cells involved and the severity of Rb Why the inherited and spontaneous forms of retinoblastoma are different in terms of incidence and severity. ○ Rb loss-of-function mutations are seen in a large number of cancers acting in conjunction with other genetic lesions ○ Two types of retinoblastomas: Hereditary retinoblastoma: when Rb heterozygotes loss function of remaining WT allele in retina, tumors arise. Somatic loss of Wt allele occurs with sufficient frequency that approximately 90% if Rb heterozygotes develop tumors. Mice homozygous for loss of Rb die during embryogenesis. Sporadic retinoblastoma: not hereditary; caused by spontaneous loss of both Rb allele in one sell consequently much less likely to occur than hereditary form of Rb How Tumor suppressors are inactivated (recombination, missegregation, etc.).  Know how Rb & p53 work, what components of the cell cycle machinery are affected by these tumor suppressors. ○ Rb: Mutations in Rb cause inherited childhood retinoblastomas Mutation in Rb lead to loss of function, not gain of function Loss of function accelerates passage through cell cycle by increasing free E2F levels, confers selective advantage to transformed cells Key regulator of cell cycle progression (controls restriction point), important target for cyclin-CDK complexes What makes Rb a tumor suppressor, how it works: ○ In G0, Rb binds tightly to a series of DNA-binding transcription factors called E2FS E2Fs: activate genes necessary for cell progression but are not functional when bound to Rb; can be bound to DNA with or without Rb E2F 1-3: activate gene transcription E2F 4,5: repress gene transcription ○ When Rb is bound to E2F it recruits HDACs, which can keep histones de-acetylated and chromatin in a closed state, characterized by tightly packed nucleosomes. ○ When P-Rb dissociates then E2F can interact with HATS, which acelates histones disrupting nucleosomes and “opening” chromatin structure to allow access to other transcription factors ○ Cyclin/Cdk complexes phosphorylate Rb which leads to a conformational change that releases E2Fs, which can activate the expression of gene targets E2F Targets: DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, cdk1, cyclin E, cyclin A, ARF, securim, E2F, p73 - promote either DNA synthesis or cell cycle progression or differentiation What happens when Rb is inactivated: ○ Immortalizes cells Vital oncoproteins can immortalize cells by binding and sequestering Rb ○ P53: Famous tumor suppressor that was mistaken for an oncogene Is a DNA-binding transcription factor Acts as a “watchdog”; can activate cell cycle arrest or apoptosis in cells exposed to many different stresses (such as DNA damage or oncogene aviation >50% of all human tumors have inactivating mutations in p53 - probably others have mutations elsewhere in p53 pathway; dysregulation of p53 is an advantage to tumor cells Not required for growth and development Problem: Has the characteristics of both oncogene and tumor suppressor High p53 protein levels are seen in tumors caused by viruses when wild type p53 was transfected into cells it did not immortalize, it had the opposite effect; did not allow oncogene formation How does it work: Works as a tetramer; inactivation of one allele through missense mutation can inactivate almost all p53 complexes in cell If one gene is a mutant and disrupts function then only one complex is a wildtype If one gene is lost through mutation, silencing, etc. then only one complex is formed Different affects of missense vs. null mutations involving p53. Missense mutations: mutant proteins can still assembly buy p53 function is effectively ablated - these mutations are dominant negatives (this explains why these mutations make p53 look like activated proto-oncogenes) Null mutations: also loss of function but these mutations do not produce any proteins What genes does p53 regulate and what effects do they have? P21 cyclin-cdk inhibitor: activation by p53 can lead to cell cycle arrest at G1/S and G2/M checkpoints GADD45: induced by DNA damage, binds to nuclear proteins involved in DNA nucleotide excision repair and can cause cell cycle arrest IGF-BP3: insulin-like growth factor binding protein -3; blocks signaling of a mitogenic growth factor Bax: pro-apoptotic protein MDM2: oncoprotein involved in controlling p53’s own stability  What mechanisms regulate p53 activity? **unregulated p53 activity can be lethal; regulating p53 is complex** Protein stability: much of p53’s activity is controlled by regulating amount of total p53 in cell Nuclear localization: shuttling in and out of nucleus regulates p53’s access to target genes p53 activation - post-translational modifications that modulate p53’s transcriptional activity Are p53 levels in normal cells high or low? Why? Low to very rapid degradation of constitutively expressed protein. Under condition of stress, degradation is inhibited and p53 protein accumulates - process regulated by MDM2, which binds directly to p53. ○ What is the process of ubiquitination of regulatory proteins? E1 - ub activating enzyme binds to Ub and transfers it to: E2 - Ub conjugation enzyme - can pass Ub to E3 ligase or directly to target in presence of E3 ligase E3 ligase - binds to target protein and (in conjunction with E2) transfers Ub to lysine residue. Formation of poly-Ub chain targets protein for degradation. Specificity of reaction lies with E3 ligases. ○ p53 accumulates when interaction with MDM2 is destabilized - how is that event regulated? Phosphorylation of p53 on N-terminal residues (S15, S20) by number of different kinases impairs ability to bind MDM2. Kinases include ATM, ATR, Chk1, Chk2 - all of these are activated by genotoxic stress (DNA damage) What are other oncogenes that mediate the interaction of p53 and MDM2? MDM2 - (oncoprotein when overexpressed) - decreases p53 levels p19ARF - tumor suppressor - increased o53 levels by inhibiting MDM2 Myc, Ras - proliferative signals checked by upregulating ARF (and p53) - oncogenic forms confer strong selective pressure for loss of p53 function Cancer Stem Cells (lecture #4): Stem cells ○ What makes a stem cell a stem cell? Can divide without limit (or life of organism)  Capable of self-renewal - when a stem cell divides, each daughter cell can either retain its stem cell identity, or can become restricted with regard to proliferation and possible differentiated outcomes (commitment) Non-stem progeny cells are committed progenitors that can terminally differentiate into multiple lineages (pluripotent) Adult stem cells thought to arise from embryonic stem cells, but have more limited potential Transient amplifying cell, embryonic vs adult stem cells (features of each esp. in terms of replicative potential). ○ Embryonic stem cells: derived from blastocysts, totipotent (can make tissues in vivo, can be primed to differentiate into an increasing number of cells types in vitro) ○ Adult stem cells: multipotent Specific signals that maintain “stemness” (or not). ○ Mutations in c-kit receptor tyrosine kinase or steel (stem cell factor - SCF) ligand lead to multiple phenotypes - defects of melanocytes hematopoietic lineages neural structures - reflecting defects in stem cells. How do stem cells divide? ○ Environmental asymmetry: loss of molecular signals from critical stromal cells could disrupt “stem identity” - consistent with piebald mutations ○ Divisional asymmetry: genetic evidence of Numb in Drosophila nerve differentiation: Numb blocks Notch signaling; cell with Numb because neuronal. Hematopoietic stem cells as a model system to understand stem cells: ○ Found in bone marrow in adults From where would cancer stem cells originate? ○ Alteration of normal stem cells through mutation, conferring partial “transformed” character ○ Alternative - “dedifferentiation” of a committed progenitor in which it gains self-renewal capacity Fluorescence-activated cell sorter (FACS) analysis, adoptive transfer as experimental approaches to isolate and study stem cells. ○ Adoptive transfer: can separate marrow cells by virtue of the cell-surface proteins they express (FACS sorting). By testing these separate cells they can identify those that produce long-term reconstitution (CD34); very few of these cells in marrow How cancer stem cells are isolated and studied (example covered in class: slides 13 & 14). ○ Non-hematopoietic cancer stem cells: cells from 9 breast cancer patients either purified directly or passaged once in mice  Authors purified a subpopulation of tumor cells that have high levels of CD44 expression, low levels of CD24 Only these cells (~15% of total) reproducibly formed tumors when injected into the food pads of mice ~50 fold enrichment in tumorigenic activity What is different about cancer stem cells and normal stem cells? ○ Traditional therapies may be best at “debulking” majority of cancer cells in tumors, but may be less effective at eradicating cancer stem cells Apoptosis & Cancer (lecture #5): Features of apoptosis & cell necrosis, similarities & differences of each. ○ Apoptosis: highly ordered, ATP-dependent process ○ Necrosis: cell lysist triggered by damage, ischemia, release of cellular components Features associated with the intrinsic & extrinsic pathways. Know proteins of each pathway (i.e., Fas, BcL-2, Bax, Bak, BH3-only proteins, etc.), how they influence apoptotic signals, how they work. ○ Extrinsic (Death Receptor) Pathway: receptor mediated Death Receptors: Fas, TNF, R3M TRAIL-R1 & R2 Fas - when bound to ligand (FasL) on other cells, trimeric receptor recruits FADD protein (Fas-associated-death-domain) through homotypic interactions between death Domains in both receptor and FADD DEDs on FADD then recruit procaspase 8, which auto activates and triggers effector caspases downstream Autoactivation may be simply a result of bringing two molecules of pro-caspase 8 into proximity What are the advantages of death receptors? Fas expressed on may cells FasL expression tightly regulated To clear unneeded lymphocytes post-infection, lymphocytes upregulated both Fas and FasL to stimulate each other’s demise Cytotoxic T cells, natural killer cells express FasL and can induce apoptosis in specific targets What are the negative death receptor signaling mechanisms? FLIP - protein similar structure to pro-caspase 8, but lacks proteolytic cleavage site, Docks with FADD but can't be cleaved blocks access of bona fide pro-caspase 8. Induce expression of NF-kB, a transcription factor associated with differentiation and protection of apoptosis. Seems  counterproductive - may be a mechanism whereby memory immune cells are retained after infection ○ Intrinsic (mitochondrial) Pathway: mitochondrial homeostasis; release of cytochrome c Prosurvival Bcl proteins maintain outer mitochondrial membrane (OMM) integrity (preventing Bak oligomers from forming) In response to apoptotic stimuli, BH3-only proteins bind to Bcl-2 and Bax is recruited to the OMM Bax/Bak oligomers can then form , producing pores in the OMM and releasing cytochrome c, Smac/Diablo - ultimately resulting in caspase activation Overexpression of pro-survival Bcl family members can block apoptosis, whereas overexpression of BH3 (or Bax/Bak proteins) can promote apoptosis ○ Growth factors provide permission to take up glucose and metabolize ○ Loss of GF signaling reduces glucose transport and glycolysis, despite lots of exogenous glucose - ultimately leads to apoptosis ○ Warburg effect Tumor cells have higher rates of metabolic activity (vs non-transformed cells) Cancer cells are highly glycolytic, producing lots of pyruvate (lactate). Now realize these as cells have artificially activated GF signaling pathways (Activated proto-oncogenes) Advantages - (1) need lots of pyruvate as molecular building block (2) lots of ATP produced (3) high glycolytic activity protects cell from apoptosis ○ Cellular energy production linked to apoptosis through Cytochrome c, part of the ETC, and located in mitochondrial intermembrane space. Maintenance of inner mitochondrial membrane potential critical for ATP production and organelle integrity. ○ Akt Akt acts downstream of GF receptors to stimulate glucose metabolism, cell growth and can inhibit apoptosis. Constitutively active Akt acts as an oncogene. ○ What are some new opportunities for treatments? Inhibiting pro-survival Bcl proteins (small molecule inhibitors, antisense or RNAi) BH3-only mimetics - treatments that activate/release BH3-only proteins Engaging death receptors on tumor cells (normal cells quite refractory) ○ How does p53 differentially activate arrest instead od apoptosis?  One model: p53 may bind regulatory sites in “arrest” genes with hgiher affinity than sites in “apoptotic” genes. Would predict arrest first, then apoptosis with high enough p53 levels (greater stress) Second model: p53 interacts with other proteins that distinguish arrest targets from apoptotic targets JMY interacts with p300 (HAT) to increase binding of p53 to Bax promotes, but not pp21 promoter P53 mediated apoptosis requires presence of highly related p53 family members p63 or p73 ○ Caspase substrates ○ Endogenous regulators of caspases - IAPs (inhibitors of apoptotic proteins) Cytochrome c release very protein inducer of apoptosis binds to Apaf-1 and leads to caspase 9 and caspase 3 activation in “apoptosome” ○ Apaf-1, pro-caspase 0,3, - present in cytosol, but inactivate, without signal  Cytochrome c signal augmented by Smac/Diablo which inhibits IAP function Key steps in apoptosis regulated by Bcl proteins in mitochondrial outer membrane ○ Apoptotic signals result in Bax relocation from cytosol to mitochondrial outer membrane. ○ BH3-only proteins bind to and inhibit pro-survival Bcl-2 proteins ○ Bax/Bak homo-oligomers create pores that release cytochrome c Effects of mutations in Bcl-2 proteins ○ Bcl-2: mice are viable, but with renal defects, premature loss of lymphocytes and melanocytes ○ Bcl-XL: - die in embryogenesis, massive apoptosis seen in CNS, liver, and hematopoietic lineages ○ Bax or Bak single KO - not much phenotype, but the double KO…. ○