Pathophysiology of Cancer

Pathophysiology of Cancer Gauri Sabnis, PhD Reading: Katzung et al, Basic and Clinical Pharmacology, Chapter 54 Goodman and Gilman’s Pharmacological Basis of Therapeutics, Chapter 37 Golan D. et al. Principals of Pharmacology, Chapter 39, 40 1 Cancer Epidemiology ✥Second leading cause of death in the US ✵Leading cause of death in Hispanic Americans ✥Risk of developing cancer increases with age ✵Except childhood cancers ✥People in the lower socioeconomic strata, racial minorities, and people without adequate health-care resources are at a higher risk of dying from cancer ✵Preventive care and regular screenings can help improve cancer mortality by Ensuring preventable cancers can be prevented Cancer can be diagnosed in its early stages when its curable • Pharmacists in the community can contribute 3 ✥ Ager Risk Factors es ✵ The most significant risk factor for most cancers z Lifestyle ✥ ✵ Obesity (Diet and lack of physical activity) ✵ Alcohol consumption ✵ Tobacco (smoking yourself) ✥ Environmental factors ✵ Tobacco (being exposed to smoking) ✵ Ionizing radiation (including sunlight) ✵ Infections (HPV, HIV, H. Pylori) ✥ Hormone related factors Risk Factors (NCI) 4 Obesity and Cancer ✥Certain types of cancers’ risk is increased in overweight/obese patients ✵Hormonal influence ✵Chronic low-grade inflammation leading to immune system exhaustion 5 Smoking and Cancer ✥Smoking increases risk of cancers - not just lung cancer ✥Risk goes higher with continued smoking, but stays the same if you quit ✵Does not go back down to non-smoker level ✥Smoking cessation and relapse 6 What is cancer? ✥Uncontrolled growth of cells ✥Normal (non-cancerous) cells have ✵Contact inhibition ✵Cellular senescence Hayflick limit: Number of time a normal cell can divide 7 What is cancer? ✥Uncontrolled growth of cells ✥Normal (non cancerous) cells have ✵Contact inhibition ✵Cellular senescence Hayflick limit: Number of time a normal cell can divide Cancer: A wound that never heals 8 set Senescence ✥Senescence is irreversible End proliferation of cell proliferation ✥Normal cells divide for a finite number of times ✵Hayflick limit ✵Cells cease to divide and are arrested Due to telomere shortening 9 Telomeres and Cellular Senescence ✥Telomeres: Short repeat sequence (TTAGGG) at the end of chromosomes 3’ ssDNA overhangs and invades dsDNA Shaped, folded to form a tloop Provides a “cap” over the DNA ends that protects chromosome ends ✵DNA polymerase unable to replicate this part of the DNA End replication problem ✵Progressive shortening of telomeres with each cell division 10 shortening fix the to Telomeres ✥Telomere shortening leads to GE of telomeric caps ✵ Disruption ✵ Activation of DNA checkpoint ✵ State of cell cycle arrest – senescence ☀ p53 (tumor suppressor gene) ✵ Responsible for aging and genomic instabilityt ☀ Oncogenesis ✥Telomerase, a reverse transcriptase, ✵ Uses RNA template to synthesize the TTAGGG repeats ✵ Allows cells to restore telomere length and divide indefinitely ✵ Maintains the presence of 3’ overhang ✵ Telomerase activation seem in stem cells, germ-line cells ✵ Also essential for tumor formation and maintenance 11 Chromosome maintenance and immortalization as BEEstewor Mytiple Apoptosis: Programmed cell death 12 Apoptosis shivinkingofchromatin Programmed Cell Death sky ✥Apoptosis f aside ✵Cell shrinkage, pyknosis, blebbing and budding OKIE✵Fragmentation: Formation of apoptotic bodies followed by phagocytosis by macrophages and/or surrounding cells 13 external not Apoptosis vs. necrosis no t justhappenswhensomething'susers easycleanupprocess compeartoneysis tragmalptgaigon to Intwiscaseis sayingoftle call get's biggerinsize andburstopen releaseacythy insidetrecell Thentimmore responseinflord Informationin tantara 14 Apoptosis vs. necrosis Normal Apoptosis Necrosis 15 Cellular Transformation ✥Benign vs. malignant ✵Cellular or oncogenic transformation Accumulated mutations Ability to divide unlimited number of times Visible only under the microscope ✵Pathologist is the only one who can diagnose cancer so 16 Carcinogenesis/oncogenesis ✥Development of cancer is a multi-step process ✵Infections and malignant transformation ✵Viruses, bacteria and parasites Disrupt normal signaling controlling cell growth, DNA repair Weakened immune system Cause chronic inflammation 18 Viral oncogenesis awl 20 Cellular Transformation 21 andDIRIA theyaremostlysinglestand Oncogenic infectious agents j use mornadeosis canleadtocancer Viruses Infectious Agent Parasites Bacteria (1) Cancers caused Spread through Prevention Certain types of lymphoma, Epstein-Barr Virus (EBV) cancers of nose and throat Saliva (kissing, sharing toothbrushes, drinking glasses); sexual contact, blood transfusions, organ transplant Hepatitis B and C Virus (HBV & HCV) Blood (transfusion, sharing needles, mother to baby); sexual contact Liver cancer Human Kaposi sarcoma, lymphoma (nonImmunodeficiency Virus HL and HL), cancers of cervix, (HIV) anus, lung, liver and throat keep possible Cervical cancer; most anal cancers Human Papillomaviruses and many oropharyngeal, vaginal, (HPVs) vulvar, and penile cancers Human T-Cell Leukemia/ Aggressive type of non-Hodgkin Lymphoma Virus Type 1 lymphoma called adult T-cell (HTLV-1) leukemia/lymphoma (ATLL) giggling Sexual transmission; Blood (transfusion, sharing needles) High-risk HPVs spread easily through direct sexual contact, including vaginal, oral, and anal sex Blood (sharing needles, transfusions), sexual contact, mother to child in the womb or via breastfeeding Helicobacter pylori (H. pylori) Noncardia gastric cancer and gastric MALT lymphoma Opisthorchis viverrini cholangiocarcinoma (cancer of the Eating raw or undercooked freshwater fish that bile ducts in the liver) contain the larvae Schistosoma hematobium Bladder cancer Vaccine (elderly, healthcare workers) Viral load Vaccine (starting at 9) All blood screened in US Consumption of contaminated food or water and direct mouth-to-mouth contact I Antiparasitic infectious free-swimming flatworm larvae burrow drugs into skin when comes in contact with contaminated fresh water (1) NCI 19 Oncogenes ✥Genes with known properties that can result in transformation of a normal cell to a cancer cell. no ourbody✵Proto-oncogene is a gene that can become oncogene hafts i get Nomatgenehave 3copiescmonDad multiple Concergenchore 22 ow JI Tumor Suppressor Genes Epiest ✥Loss of function of genes that regulate transformed cells or cellular transformation ✵Pro-apototic genes ✵DNA repair genes ✥Example - TP53 gene that encodes p53 protein mutated/deactivated in ~70% human cancers ✵Activates DNA repair ✵Cell cycle regulation ✵Senescence response to short telomeres ✵Activates pro-apoptotic genes 23 Oncogenesis ✥Cancer initiators: Transform normal cells into cancer cells ✵Oncogenes ✵Deactivating mutations in tumor suppressor genes Normal Cell ✥Cancer promoters: No cancer promoter Immune activity Apoptosis Transformed Cell DIII Cancer promoter Cell proliferation Further stimulate the growth of malignant cells ✵Hormones such as estrogens, testosterone, growth hormone, IGF etc. Cancer 24 Cell Cycle synthesis OfDIA terete Seal spread 25 Phases of Cell Cycle ✥G1 - synthesis of components needed for DNA synthesis ✵ Gap 1 ✵ 40% of the time spent in cell cycle is spent in G1 phase ✥S - DNA synthesis (DNA replication) ✵ 39% of the time spent in cell cycle is spent in S phase 2 ✥G2 - synthesis of components needed for mitosis ✵ Gap 2 ✵ 19% of the time spent in cell cycle is spent in G2 phase 3 ✥M - includes mitosis ✵ Only 2% of the time spent in cell cycle is spent in M phase In ✥After mitosis, cells enter quiescent phase (G0) or undergo differentiation 26 DNA replication in S phase ✥DNA is replicated (new DNA synthesized) in the S- phase of the cell cycle TCG AG and pyrimidines ✥Purines are bases that form the code of the DNA ✵Purines: adenine, guanine ✵Pyrimidines: cytosine, thymine, uracil Nucleosides are derivatives of purines and pyrimidines that are conjugated to ribose or deoxyribose Nucleotides are mono- di- or tri-phosphate esters of corresponding nucleosides DID A II 27 PHAR-602 28 G 1 Phase is ✥Nucleotide synthesis involves 3 sets of sequential reactions Is ✵Synthesis of ribonucleotides 2 ✵Reduction of ribonucleotides to deoxyribonucleotides ☀ All ribonucleotides are reduced to deoxyribonucleotides by enzyme ribonucleotide reductase 3 ✵Conversion of deoxyribouridylate to deoxyribothymidylate 29 Synthesis of Nucleotides and DNA ✥Basic pyrimidine ring, orotateis assembled from carbomoyl phosphate and aspartate Moneta ✵ Pyrimidine ring is made before adding on the sugar Pyrimidine synthesis ✥Orotate reacts with Aspartate phophoribosyl pyrophosphate (PRPP) to form uridylate Carbamoyl phosphate Orotate uridine MIGHTUMP phosphoribosylpyrophosphate RNA j PRPP UTP Ribonucleotide reductase MTHF dUMP Thymidylate synthase THF DHFR DHF Ribonucleotide reductase RNA dTMP dingdff'd Lhydroflatarth dTTP CTP dCTP dCTP and dUTP by the enzyme ribonucleotide reductase ✥Methylation of dUMP to dTMP is catalyzed by thymidylate synthase ✥Methyltetrahydrofolate (MTHF) DNA W'deoxy thymidine triphosphate DNA PHAR-602 ✥CTP and UTP are reduced to provides the methyl group ✥MTHF synthesized from tetrahydrofolate (THF) by the enzyme DHFR 30 Synthesis of nucleotides and DNA RNA Purine synthesis GTP GMP Ribonucleotide reductase IMP dehydrogenase (IMPDH) XMP Inosine Monophosphate (IMP) ATP Adenyol succinate RNA AMP Ribonucleotide reductase dGMP dAMP dGTP dATP DNA DNA ✥ Inosine monophosphate (IMP) central to purine synthesis ✥ IMP oxidized to xanthylate (XMP), which is converted to guanosine monophosphate ✥ IMP can be aminated to AMP (then to ATP) ✥ GMP and AMP are reduced to dGMP and dAMP, resp. by ribonucleotide reductase 31 DNA Synthesis (S phase) Replication Proteins stabilize the unwound parental DNA. (ssDNA binding proteins) Enzymes unwind the parental double helix. (Topoisomerase I DNA helicase) The leading strand is synthesized continuously by DNA polymerase. 3' DNA polymerase 5' and Replication fork creatnice RNA primer smooth Primase 5' DNA polymerase 3' Parental strand Okazaki fragment DNA polymerase DNA ligase Bottone 3' 5' The lagging strand is synthesized discontinuously. Primase, an RNA polymerase, synthesizes a short RNA primer, which is then extended by DNA polymerase. © 2016 Pearson Education, Inc. DNA polymerase digests RNA primer and replaces it with DNA. DNA ligase joins the discontinuous fragments of the lagging strand. 32 A C A G C T A T C T G C A T A A T G T C G A T A G A C G T A T 5’ T A T G C A A C G A T G A T A C G T A G C T A T T A T G A G T T C C T T C A A A T T G C C A G G T A A A C C G A A C T T T 3’ 5’ DNA synthesis (S phase) ✥Enzymes of the S-phase ✵Topoisomerase I - creates single strand breaks to help unwind the DNA ✵DNA helicase - unwinds the DNA ✵DNA polymerase catalyzes DNA synthesis Template ✵DNA ligase - to seal adjacent bases 3’ 5’ 5’ 5’ G Daughter T Daughter A Template 3’ 3’ 3’ T 3’ 5’ Parent DNA double helix 33 Mitosis ✥M-phase of cell cycle ✵Mitosis - Nuclear division ✵Cytokinesis – Cellular separation 34 mi separatingz copiesofchromatin Microtubules and mitosis ✥Cylindrical hollow fibers composed of polymers of tubulin I d ✵Heterodimeric protein of α and β subunits ✵β - tubulin also has GTPase activity ✥Originate from a central microtubule organizing centrosome 35 Microtubule polymerization owto 36 Microtubules ✥Central event of mitosis - chromosome segregation depends on mitotic spindle ✥Spindle is a bipolar array of microtubules - pull sister chromatids apart in anaphase ✵ Minus end focused at the poles ✵ Plus end radiates outwards ✥Plus ends of microtubules interact with plus ends of microtubule from the other pole arguidetosee ✵ Interpolar microtubules wherethey ✥Plus ends of microtubules segue interact with sister chromatid pogrowin 2 Kinetochore microtubules ✵ ✥Plus ends radiate outwardsvergthingen and connect to the cell cortex to position the spindle arattitte ✵sAstral microtubules YEH 37 ✥ Microtubules are dynamic structures that nucleate from Dynamic Instability S happensin mostly Aphase centrosomes ✥ In mitosis, they align at the opposite ends of the cell ✥ They are extremely dynamic during M phase of the cell cycle ✵ Grow and shrink at a much greater during this phase ✵ Allows them to locate and attach to the chromosomes ✵ Upon attaching to the chromosomes, they act as motors and align with the equators ✵ Once they align with the equators, they shorten ✵ This separates the diploid pair of chromosomes into each half of the cell ✵ Finally cytokinesis occurs ✥ Disruption of microtubules freezes the cells in M phase, leading to activation of apoptosis 38 Cell Cycle Checkpoints ✥To monitor progress of cell cycle ✵G1-S checkpoint Start/restriction point ✵G2-M checkpoint as Beforecall goesto ✵Metaphase to anaphase mitosis checkpoint Spindle checkpoint ✥Control system blocks progression if a problem is detected outside or inside the cell ✥Error is fixed  cell cycle progression ✥Error is not fixed/fixable, cell undergoes apoptosis ✵G0 for G1-S checkpoint 39 Cell cycle control we of DNA integrity ✥To monitor integrity ✵ Checkpoints ✥At each transition point in cell cycle, specific proteins such as p53, chk-1 monitor integrity of DNA ✵ Upon detection of DNA damage, may initiate DNA repair or send the cells towards apoptosis (if damage is too severe) 40 Cyclins/Cdks ✥Two key types of regulatory proteins determine the fate of the cell cycle ✵Cyclins and Cdks (cyclin-dependent kinase) ✥Activities of Cdks rises and falls as a cell progresses through cell cycle ✥Main regulator of Cdks are cyclins ✵Unless tightly bound to a cyclin, Cdk has no activity ✵Cyclins undergo a cycle of synthesis and degradation in each cell cycle (Cdk levels are relatively constant ) ✵This results in cyclical assembly and disassembly of cyclin-Cdk complex; which in turn triggers cell cycle ✥Each cyclin-Cdk complex phosphorylates substrate proteins 41 Cell cycle Cyclin-Cdk Cyclin complex Cdk Function Cyclin D1, D2, D3 Cdk 4, Cdk 6 Detect outside signals such as growth factors and other mitogens G1-S Cyclin Cyclin E Cdk 2 Cyclin E activates Cdk 2 in late G1-phase to help trigger progression through start – commitment to cell cycle entry S – Cyclin Cyclin A Cdk 2, Cdk 1 Elevated until mitosis – activate early mitotic events – targets helicases and polymerases M – Cyclin Cyclin B Cdk 1 Stimulates entry into mitosis at G2-M checkpoint G1-Cyclin 42 ✥Activity of cyclins is regulated G 1 -S transition by other cell cycle regulatory proteins ✥Cell cycle control also depends Hormones activate of transcriptional control cyclinI ✵ Changes in cyclin gene transcription ✥Transcription of S-phase genes activate this such as cyclin E, DNA polymerase and enzymes required for nucleotide synthesis 7 ✥CKIs- Cdk inhibitory proteins she ✵ p16 – suppress G1 Cdk activity didn't (frequently inactivated in cancer) tank✵ p21 – suppress G1-S and S Cdk abat DIApolymerase activities following DNA damage then✵ p27 - suppress G1-S and S Cdk activities when they terminally differentiate 43 G 2 -M Checkpoint ✥M-cyclin activates Cdk that stimulates entry into mitosis at G2-M checkpoint (Cdk1) a jaw ✵Induce assembly of mitotic spindle 2 ✵Ensure that each chromatid is attached to opposite pole of the spindle 3✵Triggers Arty chromosome condensation 9✵Promotes breakdown of nuclear envelop, rearrangement of actin cytoskeleton and Golgi ✥DNA damage and chromosomal integrity 44 DNA Damage and Chromosome Maintenance ✥DNA damage happens all the time - spontaneously a Gop✵Endogenous DNA damage ☀Reactive oxygen free radicals 2 ✵Exogenous DNA damage ☀Radiation including UV, x-rays, γ-rays ☀Environmental toxins like smoke, soot op ✥DNA damage can be repaired - various pathways ✵Fewer than 1 in 1000 accidental changes result in mutation ✵If not repaired can give rise to mutations that will then multiply ☀Dysfunctional DNA damage repair can give rise to cancer me ✥dsDNA makes it easy to repair damage - a complimentary copy of the DNA is available to fix any mistakes im to rapid rate of ✵ssDNA contained in very few organisms - susceptible F mutations vyse✥Un-repaired damage can take the cell towards apoptosis or senescence ✥DNA damage delays progression of cell cycle ✥Inherited defects in the DNA repair pathways is generally associated with inherited cancer risk BII III 45 DNA Repair and Chromosome Maintenance ✥DNA damage and repair ✵aMismatch repair ✵2Base excision repair 3 Nucleotide excision repair ✵ a Double strand break repair ✵ 46 Replication Error and Mismatch Repair j ✥Replication errors happen when DNA is being replicated but usually get fixed immediately following DNA replication (S phase) ✵ DNA proofreading A-G and C-T mismatches Insertion/deletion loops ✥ Cells with abnormal MMR accumulate errors and cause formation of microsatellite fragments ✵ Repeated of DNA sequences ✵ Repeating units of one to six base pairs in length. ✵ Most common GT/CA repeats ✵ Identifiable by PCR ✥ Microsatellite instability (MSI) results in frame-shift mutations ✵ Presence of MSI in several cancers including colorectal, ovarian, endometrial, gastric and brain (glioblastoma) – susceptibility to immunotherapy ✥ MSI-H status - presence of Lynch syndrome (hereditary nonpolyposis colon cancer) ✵ Autosomal dominant with CA repeats ✵ 80% life-time risk of developing colon cancer 47 mo regnal hismysmalch a p (1) Mismatch detected in newly replicated DNA MSH2-MSH6 Heterodimer Mis-match Daughter 5’ 3’ T A C A G C T A C C T G C A T A A T G T C G A T A G A C G T A T Template 3’ 5’ Excision Nuclease (2) Incorrectly replicated DNA strand is cut and part of the strand with incorrect base and its neighbors are removed 5’ 3’ Daughter T A C A G C A T G T C G A T A G T G C A T A A C G T A T Template 3’ 5’ ✥ Steps in MMR: (3) Missing bases are replaced with correct bases by DNA polymerase 5’ 3’ Daughter T A C A G C T A T C T G C A T A A T G T C G A T A G A C G T A T Template 3’ 5’ DNA ligase (4) DNA ligase seals the gap in the DNA backbone 5’ 3’ Seal the Gaps Daughter T A C A G C T A T C T G C A T A A T G T C G A T A G A C G T A T Template ✥ MMR happens immediately after replication ✥ Specific repeats of dinucleotides is unstable DNA polymerase Replacement DNA Mismatch Repair I (MMR) Pathway 3’ 5’ ✵ Mismatch recognition ๏ MSH2 and MSH6 heterodimer - single base mismatch ๏ MSH2 and MSH3 - insertion-deletion to loops ✵ Recruitment of additional MMR factors and excinucleases ✵ Degradation of newly synthesized strand by excision exonuclease ✵ Re-synthesis and then ligation ✥ Germline mutations in MLH1 and MSH2 gene account for ~½ of HNPCC cases 48 Base Excision Repair ✥Base excision repair (BER) pathway primarily responsible for small, non-distorting chemical alterations to the DNA ✵ Cellular respiration, superoxides, peroxides ✵ Methylation, hydroxylation, deamination, alkylation O- and N-linked alkylation Abnormal bases AP sites (abasic) Interstrand cross-linking Eb transcription and replication but frequently ✥May or may not impede miscode ✵ Important pathway to prevent mutagenesis ✵ Fixed before DNA replicates (G1 phase) ✥Highly important pathway - inactivation of BER core proteins leads to embryonic lethality ✵ Specific polymorphism of XRCC1 (complexes with DNA ligase) associated with certain lung cancers ✵ Overlap with TCR pathway and SSB repair pathway 49 (1) Incorrect base in a newly replicated DNA 5’ 3’ Deaminated C Daughter T A C A G C T A U C T G C A T A A T G T C G A T G G A C G T A T 3’ 2 5’ Template Uracil DNA Glycosylase (2) Incorrect base is removed (excised) leaving a baseless nucleotide "missing tooth” 5’ 3’ Uracil removed and baseless nucleotide left behind Daughter T A C A G C T A A T G T C G A T Template G C T G C A T A G A C G T A T 3’ 5’ AP Endonuclease Phosphodiesterase (3) The nucleotide is removed leaving a small hole 5’ 3’ Nucleotide removed Daughter T A C A G C T A A T G T C G A T Template G C T G C A T A G A C G T A T 3’ 5’ PARP DNA polymerase 5’ 3’ Replacement Nucleotide Daughter T A C A G C T A C C T G C A T A A T G T C G A T G G A C G T A T Template 3’ 5’ (5) DNA ligase seals the gap in the DNA backbone 5’ 3’ Seal the Gaps T A C A G C T A C C T G C A T A A T G T C G A T G G A C G T A T Template ✵ Recognize specific altered bases ✵ “Base Flipping” ✵ Catalyze hydrolytic removal of the base from sugar ✥ AP endonuclease ✵ Recognize and remove “Missing tooth” ๏ AP - apurinic/apyrimidinic Endo - nuclease that cleaves inside the polynucleotide chain ✥ Phosphodiesterase to remove sugar phosphate ✥ ss breaks are recognized by PARP enzyme DNA ligase Daughter ✥ DNA glycosylases ๏ (4) Missing nucleotide is replaced by DNA polymerase Base excision repair 3’ 5’ ✵ PARP1 polyADP ribosylates variety of targets ✵ Recruits additional proteins (XRCC1), which recruits DNA polymerase 50 Nucleotide Excision Repair ✥Nucleotide excision repair (NER) pathways deals with helix distorting lesions that ✵Interfere with normal base pairing ✵Obstruct transcription and replication ✥2 types - global genome NER and transcription coupled (TCR) ✥Implicated in xeroderma pigmentosa, Cockayne syndrome and trichothiodystrophy ✵Rare photosensitivity disorders - increases risk of skin cancers ✥Example: pyrimidine dimers (Bulky adducts) ✵Caused by UV light, polycyclic aromatic hydrocarbons such as Benzo[a]pyreneThe son causeDNAdomage ✵Other bulky adducts: DNA intercalates 51 (1) Thymine dimer formed due to UV rays, smoking T A C A G C A A T G T C G T T A T 3’ Thymine Dimer Daughter 5’ C T G C A T A A G A C G T A T Template 3’ 5’ Nucleotide Excision Repair 3 Excinuclease (2) DNA surrounding the dimer is opened to form a bubble. Enzymes cut out the bubble. based 29mer A G C 5’ 3’ T T G C DNA helices C Daughter T A C A G A T G T C G T A A G A C G A T A T A T Template 3’ 5’ DNA polymerase (3) Missing bases are replaced with correct bases by DNA polymerase 5’ 3’ Replacement DNA Daughter T A C A G C T A A C T G C A T A A T G T C G A T A G A C G T A T Template 3’ 5’ (5) DNA ligase seals the gap in the DNA backbone 5’ 3’ Seal the Gaps T A C A G C T A A C T G C A T A A T G T C G A T A G A C G T A T Template ✵ Chemical reaction due to UV rays, smoking - thymine dimer ✥Excinuclease - UV specific excision nuclease cleaves DNA in 2 places ✵ Remove 27-29 nucleotides (6th from 3’ and 22nd on 5’ side) ✥Gap filled by DNA DNA ligase Daughter ✥Used to repair damaged 3’ 5’ polymerase and sealed by DNA ligase 52 Theyarethemos typo damaging Double Strand Break Repairs DNAforge ✥DNA ds breaks (DSBs) induced by X-rays and other ionizing radiation as well as, free radicals, and certain anti-cancer drugs ✵ If a DNA with SSB undergoes replication ✥DSBs pose problems during mitosis ✵ Intact chromosome a prerequisite for proper chromosomal segregation before cell division ✵ Once detected, cell-cycle is halted, breaks repaired; otherwise cell death ✵ Arrest in G1 phase is mediated by p53 ✥The most dangerous type of DNA damage is ds breaks ✵ If left un-repaired, will lead to breakdown of chromosomes ✥Frequently induce various chromosomal aberrations such as Aneuploidy Deletion (loss of heterozygosity) Chromosomal translocation Associated with Carcinogenesis ✥Cells with specialized DNA recombination activities (B and T cells) more sensitive to DSBs ✵ Why lymphomas and leukemias are the most common secondary malignancies 53 ✥Non-homologous end joining u (NHEJ) ✵ No intact template to aid in repair ✵ G1 phase (1) Non-homologous end joining High energy radiation ✵ Loss (sometimes addition) of 1 or 2 nucleotides at the site of joining ๏ Quick and dirty ✵ Can result in mutation ✵ Mostly in the non-coding region of the DNA ✥By age 70, human DNA may show ~200 such “scars” Chromosome is “glued” back together with a small mutation at the sticky end ✥Broken ends are recognized by Ku heterodimer proteins, brought together and joined by DNA ligase Double Strand Break Repair 54 Double strand break Sister Chromatids Exonuclease degrades 5’ ends Strand invasion Branch point DNA synthesis and migration of branch point Homologous Recombination ✥More accurate than NHEJ ✥Uses sister chromatid to copy and fix the damaged DNA ✥Happens in newly replicated DNA ✵ S and G2 phase ✥Takes place between DNA Continued branch migration, DNA synthesis and pairing of newly synthesized DNA duplexes that have extensive regions of sequence similarity ✵ Homology DNA ligase ✥Strand invasion and homology recognition requires ATM kinase and other key proteins 55 High energy radiation Double Strand Breaks DSB Repair Using Homologous 3’ Recombination (1) Histone Modification P ATM H2AX ✥Ataxia telangiectasia (ATM) kinase 3’ generates γH2AX by phosphorylation (2) MRN recruitment H2AX P MRE11 NBS1 RAD50 ATM ✵ Presence of γH2AX on the DNA is indicative of DNA DSB ✵ Helps open up the chromatin to allow additional proteins access to the DNA 3’ (3) Nuclease mediated resection RAD52 ✥γH2AX with MDC1 recruit MRN 3’ P (4) Strand invasion BRCA1 BRCA2 ATM, CHK2 BRCA1 RAD51 (5) DNA Synthesis – branch migration DNA polymerase DNA ligase complex to the site fwmI ✥RAd52 is recruited - nuclease mediated DNA resection ✥BRCA1 is recruited to the site and phosphorylated by ATM and CHK2 kinase ✥Together with Rad51, & BRCA2, phosphorylated BRCA1 facilitates strand invasion 56 BRCA1 and BRCA2 ✥Breast and ovarian cancer susceptibility gene product (BRCA1 and BRCA2) ✵ 2 proteins are not related; not analogs or paralogs ✥Involved in the process of DSB repair using homologous recombination ✥Mutations in these genes linked to hereditary breast and ovarian cancer ✵ Altered BRCA1 (women) - 50-85% risk of breast cancer by age 70 and 40-60% risk of developing ovarian cancer by age 85 ~30% women will get a second breast or ovarian cancer within 10 years Men have a slightly higher risk of prostate cancer ✵ Altered BRCA2 (men and women) - same risk as BRCA1 for women 15-25% risk of prostate cancer for men (7X normal) ✵ Mutations in these genes have also been linked to melanoma and pancreatic cancer (men and women) ✥Genetic testing for BRCA mutations (Association for Molecular Pathology v. Myriad Genetics; US Supreme Court 2013) ✥Low BRCA functionality and chemotherapy ✥Synthetic lethality and BRCA function 57 DNA damage versus mutation DNA Damage ✥ Damage physically changes the DNA ✵ Single or double strand breaks ✵ DNA adducts ✵ DNA intercalation ✥ Not inherited itself but can lead to mutations, which can be inherited ✥ Incorrect base incorporated in the DNA ✵ Mismatch between the two strands ✵ DNA unable to transcribe ✵ Translation prevented ✵ DNA unable to replicate ๏ Cell may die Mutation ✥ Changes in the genetic code that can be inherited ✵ Incorrectly transcribed mRNA ✵ Incorrectly translated protein ๏ Non-functional protein ๏ Mis-functional protein • Reduced affinity for the ligand • Reduced binding to substrates ๏ Constitutively active protein • Does not require ligand to activate 58 Mutations 59 60 DNA Damage and Diseases ✥Defects in DNA repair has been associated with increase in cancer risk as well as inherited cancer risk 61 Sia ok Synthetic Lethality ✥Type of genetic interaction where the co-occurrence of 2 genetic events results in cell death ✥Synthetic lethal interactions may be dependent on ✵Genetic background ✵Hypoxia or metabolic changes ✵Extrinsic conditions such as treatment with DNA-damaging agents ✥Examples of specific drugs used in specific sub-types of cancers: ✵PARP inhibitor for BRCA mutant breast and ovarian cancer ✵Immune checkpoint inhibitors for MSI-H colorectal cancer ✥Identifying the adaptation that the cancer cell has made and attacking that pathway 62 Different ioncallsDONI ✥Organized formcancer arrangement of cells within a tissue Cellular Differentiation ✥Specific function to cells ✥Generation of an organism from a zygote ✥Most differentiated cells do not divide ✥Differentiated cells will not form cancer 63 Differentiation of cells 64 Ii Cancer Stem Cell ✥ Tissues maintain a store of pluripotent stem cells ✥ Cancer cells acquire a “stem-like” phenotype ✵ Ability to multiply unlimited number of times ✵ Asymmetrical cell division ✵ Self-renewal ✵ Epithelial to mesenchymal transition (EMT) ✥ Cadherin switch e ✵ Epithelial cells express tight junctions, and these are characterized by expression of E-cadherins ✵ Mesenchymal cells are less differentiated (multipotent stem-like) and express N-cadherin ✥ Embryogenic process – neural tube formation ✥ Cancer cells have shown the ability to undergo cadherin switch 65 Relapse and progression ✥A single cancer cell left behind after therapy (or surgery) is capable of recapitulating the tumor W E of signs and ✵Remission – decrease or disappearance symptoms of cancer (cancer may still be present in the body)* ✵Relapse – return of a disease or the signs and symptoms of It a disease after a period of improvement ✵Recurrence – return of cancer either recur at the original site, nearby in the same tissue, adjacent tissue or at a distant site ✥Angiogenesis ✥Metastasis *NCI dictionary We spreadofcancercallfrom theiroriginal 66 Angiogenesis ✥Development of new blood vessels ✵Migration, growth and differentiation of endothelial cells ✥Tumors can not grow beyond a certain size without proper blood supply ✵Tumors (interior) tend to be hypoxic ✥Angiogenesis helps tumors receive nutrients as well as for metastatic spread 67 Regulators of angiogenesis ✥Complex process involving multiple regulators ✥VEGF (vascular endothelial growth factor) and VEGF receptors ✵VEGFR1 and VEGFR2 are expressed on endothelial cells ✵VEGF3 key for lymphangiogenesis ✥Activation of VEGFR is mediated by stimuli such as hypoxia, cytokines, growth factors, oncogenes and hormones ✥Cytokines such as IL-1, IL-6 as well as COX-2 activation stimulates VEGF production. ✵IGF-1R, BCR-Abl and inactivation of p53, PTEN can result in VEGF production 68 02 SI Hypoxia and angiogenesis ✥Low oxygen conditions allow (hypoxia inducible factor) HIF1α to accumulate ✵In O2 rich environment, HIF-1α is hydroxylated and degraded ✥HIF-1α is a transcription factor that translocates to the nucleus and activates transcription of hypoxia induced genes such as VEGF, EPO 69 Metastasis ✥Spread of tumor cells from the primary site to distant sites ✵Hematogenous ☀ Use the circulatory system to spread through the body ✵Lymphatic ☀ Use the lymphatic fluids as carrier ✥Clinically visible distant metastasis are seldom curable ❊Metastasis is the reason for cancer-related mortality 70 Metastasis smh ✥Up to 60% of patients with invasive cancers have overt or A occult metastasis at diagnosis ✥Acquisition of invasive phenotype is an early event in cancer progression ✥Millions of tumor cells are shed daily into the circulation ✥Less than 0.01% of circulating tumor cells initiate a metastasis focus ✥Circulating tumors cells can be detected in patients who do not develop overt metastasis ✥Angiogenesis is ubiquitous and early event that is Is paramount and responsible for metastatic dissemination 71 Metastasis cascade 72