Lecture 6: Genetics of Cancer (PDF)

For classroom use only. THE GENETICS OF CANCER An Overview Christian Joseph N. Ong, M.Sc., LPT Instructor/Professor Department of Biology College of Science Bulacan State University 1 Cancer S...

For classroom use only. THE GENETICS OF CANCER An Overview Christian Joseph N. Ong, M.Sc., LPT Instructor/Professor Department of Biology College of Science Bulacan State University 1 Cancer Statistics 2 For the 1st time, female breast cancer has become the most commonly diagnosed cancer, surpassing lung cancer, in particular due to high prevalence in low- and middle-income countries (LMICs). Lung cancer remains the leading cause of cancer deaths in the world. 3 4 Percentage (%) of the population in which the frequency of cancer is measured on each continent in children (age 0-14 years) and adolescents (age 15-19 years) The true burden of cancer in children is unknown in most low-income countries. Childhood cancer varies by region, with the highest incidence in more developed regions. 5  Cancer Statistics in the Philippines Number of New cases in 2020 (both sexes) Cancer (both sexes) Total # of cases/Percentage (%) Breast 27,163 (17.7) Lung 19,180 (12.5) Colon/Rectum 17,364 (11.3%) Liver 10,594 (6.9%) Prostate 8,242 (5.4%) Other Cancers 71,208 (46.3%) 6  Breast Cancer “In 2016, the Philippines topped 197 countries with the most number of cases of breast cancer” Data released by Philippine Obstetrical and Gynecological Society 7  Highlights from Reports Cervical cancer is the 2nd leading cancer site among women. 99.7% of women with cervical cancer are positive for Human papiloma virus (HPV) An average of 12 Filipino women die of cervical cancer daily. Eight Filipinos die each hour from smoking- related diseases Obesity exposes a person to several cancers 8  Highlights on cancers The following cancers can be detected early and when treated properly can be cured - BREAST, CERVIX, COLON, RECTUM, ORAL, THYROID, PROSTATE. These comprise 42% of all cancers, 27% of cancers in males, and 58% of cancers among females. 9  Cancer Treatment “The right drug in the right dose and route given at the right time will relieve cancer pain”. 10 “Cancer is genetic, but usually not inherited”. 11  Cancer A Genetic (DNA) disease Disease of genes A multi-step process Involves changes/mutations in genes/ chromosomes Changes in chromatin structure that alters gene expression (Epigenetics) 12 Differences between normal and cancer cells 13 http://archive.cnx.org/resources 14  Normal vs Cancer cells Normal Cancer Mortal (Cells die after 50 cell Immortal (Divide divisions indefinitely) Contact inhibition Loss of contact inhibition Stay bound together Can easily detach from each other Controlled cell death Unresponsive to Apoptosis (Apoptosis) Controlled Cell Division Increase rate of cell division Control of angiogenesis Unregulated angiogenesis Mature to functional cells Do not mature to functional cells 15  Types of Cancer Type Incidence Sporadic 70-90% Familial 10-20% Hereditary 5-10% 16  Sporadic Cancer Accounts for most cancers -75-80% Gene mutations that cause cancer are acquired Gene mutations occur only in tumor cells 17  Familial type of Cancer Cancers that affect more one member of the family “clustering of cancer “ in a family generally, late onset of CA 18  Mechanisms of Cancer 1. Gene Mutation 2. Chromosome alteration: –Change in chromosome number –Change in chromosome structure 3. Epigenetics 19  Genes Commonly Associated with Cancer 1. Oncogenes (onc) 2. Tumor Suppressor Genes (TSG) 3. Mutator Genes 20 Mutation Overexpression Under expression Loss of Function New function Changes in gene expression Gene Protein Normal gene expression Normal function 21  Proteins in Cancer I. Growth Factors II. Growth Factors Receptors III. Signal Transduction Proteins IV. Transcription factors V. Pro or anti-apoptotic proteins VI. Cell Cycle control proteins VII. DNA Repair proteins Note: refer to this numbering in next slide. 22 The seven types of proteins that participate in controlling cell growth 23  Proteins and Cancer genes Mutations changing the structure or expression of proteins in classes I – IV generally give rise to dominantly active oncogenes. The class VI proteins mainly act as tumor suppressors; mutations in the genes encoding these proteins act recessively to release cells from control and surveillance, greatly increasing the probability that the mutant cells will become tumor cells. 24  Proteins and cancer genes Class VII mutations greatly increase the probability of mutations in the other classes. These are usually the mutator genes (e.g. mismatch repair genes) Virus-encoded proteins that activate growth- factor receptors (Ia) also can induce cancer. 25  Proteins and Cancer Class of Proteins Remarks Type of Gene Classes I-IV Mutation in these classes of Oncogenes genes generally result to dominantly active oncogenes Class V Pro or anti apoptotic Tumor suppressor proteins (e.g Bcl2 proteins) genes/oncogenes Class VI These proteins mainly act to Tumor suppressor genes stop or prevent growth of tumors. Class VII Mutation in these genes Mutator genes greatly increase the probably of mutation in other genes 26 27 7 major Description Examples classes of proteins Structural Spider silk, human hair, bone, tendon, Actin ligament Contractile Provide muscle movement Myosin Storage Ovalbumin (egg white) Defensive Antibodies; help protect the body Immunoglobulin against pathogens, antigens Transport Hemoglobin in blood Ferritin Signal Transmit signals and coordinate Growth hormones, biological processes between different glucagon, melatonin cells, tissues and organs. Enzymes Carry out numerous chemical Phenylalanine reactions in the cell; some serve as hydroxylase chemical catalysts 28  PROTO-ONCOGENES A normal gene that could become an oncogene due to mutation or change in gene expression. Genes that encode proteins are normally involve in cell proliferation Active where and when high rates of cell division are necessary such as in wound healing and in the developing embryo. 29  ONCOGENES Genes whose proteins hike/increase active production of growth factors and promote mitosis activation other than wound sites and in the developing embryo Genes that predispose cells to become cancerous and form TUMOR. Gene whose products are transcription factors that bind to some proto-oncogenes and activate transcription. Products (proteins) of these activated genes contribute to the characteristics of the tumor. Dominant mode of Inheritance 30  How proto-oncogenes becomes oncogenes PROBLEM arises when the turning ON (activation) occurs at the wrong time and or place. Genetic mechanisms of protooncogene activation 1. Chromosome rearrangements – 1.1 Inversion – 1.2 Translocation: involvement of an antibody – 1.2 Translocation: formation of gene fusion and chimeric (fusion)protein 2. Gene Amplification 3. Point mutation 4. Insertional mutagenesis 31  1. Chromosomal Rearrangements 1. Chromosome Inversion Inversion paved the was for an increased expression of a gene at a new location. Example: Parathyroid cancer 2. Translocation Gene fusion results to the formation of fusion proteins with increased expression. Example: CML 32  1.1 Increased expression at a new location 1. Cancer of the parathyroid gland Inversion of chromosome 11 Proto-oncogene (Cyclin D1 or CCDN1) sits besides a gene controlling transcription of a parathyroid hormone (PTH) gene. As the gland synthesizes the hormone, the oncogene is expressed. (See next slide) Arnold A et al., 1989. Molecular Cloning and chromosomal mapping of DNA rearranged with parathyroid hormone gene in a parathyroid adenoma.J. Clin. Investig 33  PTH PTH coding gene region 11p15.3 11p15-q13 PTH 5’ regulatory Pericentric region inversion CCDN 0 11q13 Cyclin D 1 Chromosome 11 The paricentric inversion of chromosome 11p15q13 resulted to the translocation of the PTH 5’ coding region directly upstream of CCDN1 proto-oncogene, inducing over 34 expression of cyclin D1 protein.  1.2 Translocation: Role of the Immune system 1.2 Translocation of a protooncogene next to an antibody (Ig) gene Burkitt lymphoma. The basis for the malignant transformation of lymphocytes. C-myc gene is normally located on chromosome 8. When it translocates to chromosome 14, it sits right next to the Immunoglobulin heavy (IgH chain gene. The immunoglobulin gene is activated in B lymphocytes and acts as a promoter for the c- myc gene. 35  Burkitt lymphoma cells are found to carry a reciprocal translocation between chromosomes 8 and 14. C-myc can also translocated in chromosome 2 and 22. Dalla-Favera et al. 1982. Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. PNAS. 36 1.3 Fusion Proteins with New Functions Protooncogene moves next to another gene site. – Fusion transcript – Transcription and translation of gene pair – FUSION protein – Activates or Lifts control of cell division Example: BCR-ABL gene fusion in CML 37 t(9;22)(q34;q11.2) 38  BCR ABL e1 b2 a2 BCR BCR-ABL RNA (q11) fusion Derivative 22 Normal 22 PROTEIN ABL ABL-BCR (q34) fusion (p210) Normal 9 Derivative 9 A B Fig. 2. A. Reciprocal translocation in chromosomes 9 and 22; t(9;22)(q34;q11) producing the Philadelphia (Ph) chromosome. The fused gene in the Ph chromosome produces the BCR-ABL protein which cause white blood cells to actively divide causing the malignancy. 39 ABL1 Protooncogene Encodes for a nonreceptor tyrosine kinase, a homolog of the oncogenic v-abl gene carried by the Abelson murine leukemia virus Consists of 11 exons, with 2 first alternative exons i.e. exons la and 1b Expressed either as a 6 or 7 kb mRNA transcript with alternatively spliced first exons 40  ABL protein A tyrosine kinase ABL Can be found both in the cytoplasm and nucleus; but primarily in the cytoplasm Cytoplasmic ABL has been implicated in G1/S checkpoint regulation; cell adhesion, stress response Nuclear ABL inhibits binding of the DNA repair protein RAD51 to sites of DNA damage. The activity of ABL1 protein is negatively regulated by its SH3 domain and deletion of the SH3 domain turns ABL1 into an oncogene. 41  Tyrosine kinase A subclass of kinases An enzyme that can transfer a phosphate group from ATP to a protein in a cell. Acts as an "on" or "off" switch in many cellular functions The phosphate group is attached to the amino acid tyrosine on the protein. 42  Breakpoint Cluster Region (BCR) gene Protein coding gene; Has 23 exons Occupies a region of about 135 kb on chromosome 22 Protein’s function is not completely understood May act as GTPase activating protein (GAP) GAPS turn off (inactivate) proteins called Rho GTPases (proteins which play important role in chemical signaling within cells Rho GTPase is said to play important roles in neurite growth and axonal guidance. 43  ABL-BCR Gene Fusion The t(9;22) translocation results in the head- to-tail fusion of the BCR and ABL1 genes; BCR- ABL gene fusion The fusion occurs between the 5′ part of BCR gene, normally located on chromosome 22, and the 3′ part of the ABL gene This fusion gene is transcribed and then translated into a hybrid protein. 44 Diagram shows breakpoints in ABL gene as well as on BCR gene. BCR-ABL gene fusion results to 3 Major protein variants because breakpoints in the BCR gene may vary. 45  BCR-ABL Gene Fusion/Protein Hall mark of 90-95% of CML Various breakpoints at BCR gene Constant breakpoint at ABL gene Various protein products (size) based on the breakpoints: p230, p210, p190, p185, 130 P210 is generally found in CML P130 in chronic neutrophilic Lk P185 is more common in ALL (children) P190 (isoform) splice variant of p210 46  BCR-ABL Protein Tyrosine kinase translated from BCR-ABL gene fusion can speed up cell division through inhibiting DNA repair, causing genomic instability and consequently blast crisis in CML. 47  Classification of breakpoints in the BCR gene Type of breakpoint Breakpoint in the Protein produced /fusion Gene BCR gene from the BCR-ABL Fusion Major breakpoint Either exon 13 or 14 P210 b2a2 or e13a2 B3a2 or e14a2 Minor breakpoint Exon 1 185 e1a2 Micro breakpoint Exon 19 P230 e19a2 Note: In the ABL gene it is always exon 2 that is involved in the gene fusion. 48  ABL gene/Protein “Overall, it appears that the Abl protein serves a complex role as a cellular module that integrates signals from various extracellular and intracellular sources and influences decisions in regard to cell cycle and apoptosis.” Ren R. 2005. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer. Mar;5(3):172-83. 49 Routine Sample needed for BCR-ABL test is bone marrow aspirate (3-5 ml) 50 2. Gene Amplification: Receiving a too long Division Signal 25% of women breast cancer is caused by overexpression of Her2/neu Mutation in the HER 2 gene causes an overexpression of its protein. Affected Cells have millions of copies of a cell surface protein called Her2/neu (a receptor for epidermal growth factor (EGF) Epidermal growth factor receptors (EGFR) are transmembrane molecules 51  Her2/neu One end of the transmembrane receptors dips into the cytoplasm Functions as a tyrosine kinase Growth factor (EGF) binds to its receptor (Her2/neu); it picks up a phosphate group, sends signal into the cell that activates transcription of genes that stimulates cell division. 52  Her2/neu or ERBB2 gene Other names of the gene: HER2 or HER2/neu (a proto-oncogene) Protein: Human receptor growth factor 2, tyrosine- protein kinase erb2; also called CD340 (cluster of differentiation 340) A member of the human epidermal growth factor receptor family (transcription factors) Other members include: EGF2, 3 and 4 53  Her2/neu Clear causes of (Her2/neu) breast cancer: (1) Too many tyrosine kinase receptors (2) Too many signals for cell division This type of breast cancer usually strikes early and spreads quickly. Treatment of breast cancer with Her2/neu motation: Monoclonal antibody based drug: Herceptin The drug binds to the receptors to block the signal 54  Testing for HER2 Status ▪ An accurate HER-2 assessment can enable the most appropriate therapy decision. ▪ Test should measure either gene copy number or presence of protein receptors. ▪ Use of formalin-fixed, paraffin-embedded breast cancer tissue. ▪ Trastuzumab by Genentech is a targeted monoclonal antibody treatment for women with HER-2 positive metastatic breast cancer. 55 Assessment of Her2/neu either by immunohistochemistry or Fluorescence in situ hybridization A probe can label various region(s) of the DNA 56 57  Count HER-2 and Chromosome 17 signals in 20 nuclei (2 alleles; 2 centromeres of chromosome 17) The ratio of HER-2 to Chromosome 17 is calculated. If value is 2 or greater, the patient is considered to have an amplified HER-2 or HER2 positive. Normal gene count = HER2 negative FISH allows the viewer to literally count the copy of genes/alleles per cell. DNA is a very stable material. Tissue preparation has very little effect Sauter G, et al. Guidelines for Human Epidermal Growth Factor Receptor 2 on test outcome. Testing: Biologic and Methodologic Considerations. J Clin Oncol 27:1323- 1333, 2009. 58  HERCEPTIN Acts only when there are extra receptors rather than from extra transcription from of a single her2/neu gene 59 60 Growth factor Inhibitors 61 How Herceptin interacts with cancer cells 62 Trastuzumab (Herceptin) mechanism of action. Trastuzumab is a molecularly targeted drug for breast cancer. Herceptin binds to the extra cellular region of HER@ and is able to prevent downstream activation of cell signals that induce cell proliferation and angiogenesis The action of the drug is specific only for the overexpression of HER2/neu gene. 63 3. Cancer caused by Infectious agents (bacteria and viruses) 1. Cancer associated with viral infection Viruses can lead to cancer by associating with their host proteins, they proliferate when the human genome is weakened and then hijack proliferating cells. Tumor viruses are unusual because the generally infect not kill their host cells 64  HPV and Cervical Cancer Cancer registries do not collect data on the presence or absence of HPV in cancer tissue at the time of diagnosis. Global studies : about 90-99% of cervical and anal cancers, about 70% of vulvar and more than 60% of penile cancers are caused by HPV infection. About 85% of the global burden are found in the less developed regions, where it accounts for almost 12% of all female malignancies. It is second to breast cancer as the most common cause of cancer death among women worldwide 65  HPV and Cancer More than 200 HPV types recognized based on DNA sequence data showing genomic differences; many of these are harmless Classification : low-risk HPV types and high risk HPV types HPV genome: Single circular, double stranded DNA with the ORF protein coding sequences confined on one strand Its genome has 3 regions: 1. non-coding upstream regulatory region/long control region (LCR) 2. early region (E) 3. late region (L) 66 The genome structure of HPV showing the 3 major regions: LCR, E and L. LCR region is the most variable region of the genome and contains the p97 core promoter, enhancer and silencer sequences that control ORFs transcription in the regulation of DNA replication. Note the function of genes found in E and L regions. C.M. D’Abramo and J. Archambault, 201167  HPV and Cervical cancer HPV-associated cancers can contain either (1) integrated HPV DNA, (2) extrachromosomal viral DNA, or (3) a mix of both Integrated HPV pathway is reported in more than 80% of HPV positive cervical cancer; predominantly in HPV16 and HPV18 types. This integration event is thought to drive oncogenesis by dysregulating expression of the E6 and E7 viral oncogenes, leading to inactivation of critical cell cycle checkpoints and increased genetic instability in the host. 68 Where do HPV integrate in the human genome? In so-called genomic “hotspots” are highly correlated with common fragile sites Transcriptionally active regions of the genome Regions of microhomology (1–10 bp) between viral and human genomic sequences as these may contain AT-rich regions of the genome that have the potential to form stem—loop structures that promote the formation of stalled replication forks during replication Within cancer-associated genes or pathways (ex. C- myc), McBride and Warburton, 2017 69  Infectious agents that can cause cancer Infectious Agent Cancer Human Papilloma Virus Cervical Cancer, Anal Carcinoma, Oropharyngeal Carcinoma, Penile Carcinoma Epstein Barr Virus Burkitt’s Lymphoma, Hodgkin’s Disease, and Nasopharyngeal Carcinoma H pylori Gastric adenocarcinoma Hepatitis B virus (DNA) Hepatocellular carcinoma Hepatitis C virus (RNA) Hepatocellular carcinoma Human Adult T-cell Leukemia Virus Type 1 T-cell leukemia 70 4. Point Mutation A single base substitution in the DNA chain results in a miscoded protein. For example, Point mutation of the ras oncogene is found in approximately 30% of common human cancers, such as carcinoma of the lung, large intestine, and pancreas. 71  Point Mutation: Ras Gene The RAS gene family consists of three small G proteins (isoforms):Ha, N and Ki-ras RAS proteins are anchored on the cytoplasmic side of the cell membrane and play a central role in cell signaling They mediate signal transduction downstream from tyrosine kinase membrane receptors to a variety of effector molecules, stimulating a cascade of parallel phosphorylation reaction pathways that ultimately culminate with the activation of nuclear transcription factors 72  Ras genes and cancer The three main effector pathways that are activated downstream of RAS – – RAF/MEK – MAPK, PI3K/AKT, and – RAL–GDS – When RAS genes are mutated, cells grow uncontrollably and evade death signals RAS mutations also make cells resistant to some available cancer therapies 73 When stimulated by upstream signaling molecules, wild type RAS proteins interact with guanine nucleotide exchange factors to replace GDP with GTP, resulting in an activated protein conformation. RAS activity is terminated by interaction with GTPase activating protein, which stimulates the GTPase activity of the protein and converts GTP back to GDP, thereby restoring the inactive form of RAS 74 Tumor Suppressor Genes 75  TUMOR Suppressor Genes (TSGs) Genes that encode proteins that normally function to regulate cell division keeping it in check Suppress tumor growth-inhibiting signals When a TSG is mutated (e.g. deletion) it does not produce the protein or the protein produced does not function properly and this results in uncontrolled cell division. Such mutations may contribute to the development of cancer. To remove the function of TSG both alleles/copies of the gene must be mutated 76  Well Studied TSGs 1. Wilms tumor gene (WT1) 2. Retinoblastoma gene (Rb) 3. P53 gene 4.BRCA1 77  Wilms Tumor (Nephroblastoma) An embryonal malignancy of the kidney, is the most common renal tumor of childhood Characterized by an abdominal mass in an otherwise apparently healthy child. 25-30% of children suffer from abdominal pain, fever, anemia, hematuria, and hypertension Definitive diagnosis is based on histology 10-15% of cases have genetic predisposition or an epigenetic alteration 78  WT gene / WT protein WT gene encodes for WT protein In children with Wilms tumor children, the WT gene that normally functions to stop mitosis is absent. The WT protein has four C-terminal Zn-fingers. Protein functions as a transcriptional regulator and a tumor suppressor WT1 Gene is mapped on chromosome 11p13 About 90% of Wilms tumor cases are somatic mutations 79  Wilms tumor A Child kidneys still develops retaining many pockets of cells (as in the fetus) eventually forming a tumor out 5%-10% of individuals with Wilms tumor have bilateral or multicentric tumors The prevalence of both kidneys affected by the tumor, is high among individuals with genetic predisposition or an epigenetic alteration occurring early during embryogenesis Turner et al., 2022 80  WILMS TUMOR www.physio.pedia.com/koutiala kids.blogspot.com 81  Wilms tumor Recent studies support the claim that the tumor suppressor function of AT1 is correlated with transcriptional activation It appears likely that WT1 promotes growth suppression by activating the transcription of genes encoding proteins that inhibit cell proliferation. WT1 can also induce apoptosis or inhibit P53 mediated apoptosis 82  Retinoblastoma A cancer that starts in the retina, the very back part of the eye. It is the most common type of eye cancer in children. Caused by mutation in Rb gene Chromosome 13q14.1-q14.2 83 Retinoblastoma https://ghr.nlm.nih.gov/art/large/retinoblastoma-eye.jpeg 84  Knudson’s Hypothesis/Two-hit hypothesis A “driver” in the development of CANCER GENETICS Postulates the recessive nature of tumor‐initiating gene If one is born with a mutated copy (first genetic hit) of a TSG (in this case RB gene), only one more hit is needed for tumorigenesis to occur. Even with one copy the TSG gene is still functioning. The second genetic hit alters the Rb gene and makes it nonfunctional. This 2nd hit are commonly large deletions. Cells that carry these 2 copies of mutated Rb gene would have a loss of function.. 85  Knudson’s Two Hit Hypothesis On the other hand, if one is born with a normal pair of TSG, two acquired mutations are needed for tumorigenesis to occur. With both copies of the Rb gene functionally lost, Retinoblastoma (tumor) was initiated. 86 RETINOBLASTOMA: The Two Hit Hypothesis 87  P53 Gene P53 gene is a TSG; Encodes p53 protein If a cell losses p53 or if the gene mutates or malfunctions, a cell with damaged DNA is PERMITTED to divide and CANCER may be the result. Involve in Half or > of human cancers Location: Chromosome 17 Types of mutation in P53 gene can be different in different cancers: TRANSITION or TRANSVERSION Usually mutated in the late stage of cancer and often indicates cancer metastasis 88  P53 gene (‘guardian of the genome’) A tumor suppressor gene: – Growth arrest – DNA repair – Apoptosis 1 2 3 4 5 6 7 8 9 10 11 Transactivation Proline-rich DNA binding Oligomerisation Regulation (I 1-42; 43-62) II (63-97) (102-292) (323-356) (363-393) N- P P P P P P P Ac P P APc -C P Phosphorylation site Acetylation site 89 Ac  P 53 protein The p53 protein is located in the nucleus of cells throughout the body, where it attaches (binds) directly to DNA. Arrests the cell cycle when there is DNA damage Stimulates apoptosis in the presence of DNA damage. 90 The p53 also upregulates the p21 protein, which blocks the formation of the cyclin CD/Cdk4 complex, thereby preventing the phosphorylation of RB and, in turn, halting cell cycle progression by preventing the activation of E2F. 91  P53 Reflects environmental insults: ✓ Viruses ✓ Toxins (cigarettes, alcohol etc..) ✓ Radiation In addition, other factors such as abnormal metabolic activity, genetic damage and advanced ageing can also trigger the protein’s activation, enabling p53 to directly regulate the expression of hundreds of genes to initiate an appropriate response. 92  Metastasis: Epithelial to Mesenchymal Transition Epithelial-Mesenchymal transition (EMT) is one of the critical event for metastasis in carcinoma. During EMT, epithelial cells lose their plasma membrane polarities, break their intercellular tight junctions, and degrade basement membrane extracellular matrix (EMC)components to become migratory mesenchymal cells (Qiagen website). 93  Mesenchymal cells Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differentiate into a variety of cell types, including: – osteoblasts (bone cells), (cartilage cells), – myocytes (muscle cells) and – adipocytes (fat cells which give rise to marrow adipose tissue). 94 BREAST Cancer 95  Breast Cancer The Philippines has the highest prevalence of breast cancer in Asia, and the 9th highest in the world today. According to the Philippine Statistics of Authority and the Department of Health, it shows that 3 in every 100 Filipina women will be diagnosed with breast cancer in their lifetime. 96  BRCA1 and 2 Mutations in these genes are rare in the general mutations In USA about 1 in 400 people have a BRCA1/2 mutation However, prevalence varies by ethnic group. Among Ashkenazi Jewish women and men, about 1 in 40 carry a BRCA1/2 mutation. 97  Hereditary types of Breast Cancer Genes most commonly mutated in hereditary breast and ovarian cancer: – BRCA 1 gene (Chromosome 17 – BRCA 2 gene (Chromosome 13 Increases the risk of breast cancer Both of these genes function as TSG 98  Genes in Breast Cancer ❑ Listed in the pie chart are known predisposition genes which when mutated predisposes a person to breast cancer. These genes account for 45 percent of all familial breast cancer. ❑ The other 55 percent are unexplained. https://discoverysedge.mayo.edu/2015/10/07/breast-cancer-predicting-individual-risk/ 99 100 Gene Mutations increase susceptibility to Breast Cancer Chromosome 13 Chromosome 17 101  Angelina Jolie: ‘Why I’m getting my ovaries and fallopian tubes removed’ March 25, 2015 | New York Times A simple blood test had revealed that I carried a mutation in the BRCA1 gene. It gave me an estimated 87 percent risk of breast cancer and a 50 percent risk of ovarian cancer. In my case, the Eastern and Western doctors I met agreed that surgery to remove my tubes and ovaries was the best option, because on top of the BRCA gene, three women in my family have died from cancer. My doctors indicated I should have preventive surgery about a decade before the earliest onset of cancer in my female relatives. My mother’s ovarian cancer was diagnosed when she was 49. I’m 39. Last week, I had the procedure: a laparoscopic bilateral salpingo-oophorectomy. There was a small benign tumor on one ovary, but no signs of cancer in any of the tissues. It is not easy to make these decisions. But it is possible to take control and tackle head-on any health issue. You can seek advice, learn about the options and make choices that are right for you. Knowledge is power. 102 Mutator Genes 103  Mutator Genes/Mismatch Repair Genes Genes that are involved in repairing the errors made during DNA reliectioon Mutation in these genes causes the increase of mutation rate in one or more genes. Genes that elevate the genomic mutation rate are likely to induce deleterious mutations and thus suffer an indirect selective disadvantage; First studied in Bacteria (those carrying them can increase in frequency only by generating beneficial mutations at other loci). Tanaka and Levine, Genetics 2003. 104  Mismatch Repair Genes Phenotype is caused by at least 5 different genes – MSH2 (16 exons) – MLH1 (19 exons) – PMS 2 (and ?1) – MSH6 – MSH3 SCOBEC and Birmingham Cancer Study Day 6th June 2007 105  DNA mismatch Repair (MMR) Genes Important genome caretaker system Ensure genomic stability by correcting mismatches that occur during DNA replication and recombination Suppress homologous recombination Act as a damage sensor that signals apoptosis in cells with severe DNA damage 106  Mismatch Repair Pathway This highly conserved pathway occurs in 4 (1) Recognition of a mismatch by the MSHs, (2) recruitment of the MLHs by ATP-bound MSHs that then connect the mismatch recognition signal to the distant DNA strand scission where excision begins, (3) excision of the DNA strand containing the wrong nucleotide and (4) resynthesis of the excision gap by the replicative DNA polymerase using the remaining DNA strand as a template. 107 The figure summarizes the 4 steps by which MMR genes perform their mismatch repair function. 108  Microsatellites Stretches of DNA consisting of repeated units of two, three or four nucleotides: TGTG…TG; CAACAA..CAA; AAT Because of their repetitive nature, these are regions in the DNA strand are prone to replication errors. Errors that are not repaired can cause genomic instability 109  Microsatellite Instability(MSI) A change that occurs in certain cells (such as cancer cells) in which the number of repeated DNA bases in a microsatellite (a short, repeated sequence of DNA) is different from what it was when the microsatellite was inherited. Hallmark of underlying mutation in one or more MMR genes. 110 The presence of high level of MSI (MSI-H) is normally associated within a mutation of the hMLH1 and hMSH2 genes Low level of MSI appears largely due to mutations in the hMSH6 gene (10 %), and the hPMS2 gene (5 %). Epigenetic silencing (methylation) of MMR gene Expression leads to Sporadic Cancers. 111  MSI MSI in these genes results in altered signaling transduction, apoptosis, DNA repair, transcriptional regulation, protein translocation and modifications, and immune monitoring 112  Genes involved in COLORECTAL CANCER 113  Gene Mutation: Colorectal Cancer (CRC) Types of CRC 1. Sporadic 2. Familial 3. Hereditary 114  Genes Mutated in CRC Oncogenes (Onc) e.g. KRAS, HRAS, NRAS Tumor Suppressor Genes (TSG) e.g. p53, DCC, APC Mismatch Repair Genes (MMR) e.g. MLH1, hMSH2 115  Gene Mutation When genes are mutated they produce altered proteins, with the following changes: Gain of function Loss of function Novel function 116  Bert Vogelstein (1949- ) is Clayton Professor of Oncology and Pathology A Howard Hughes Medical Institute Investigator at the Johns Hopkins University Genes involved in Cancer: Mountains Hills (low frequency) 117  Fearon and Vogelstein Model CRC is the result of changes (mutations) of genes with important functions in regulating cell proliferation or repair of DNA damages, mutations in more than one gene are required, the sequence of mutations is important in determining the eventual formation of CRC 118  Colorectal Cancer (Fearon and Vogelstein Model , 1990) Bert Volestein 119 120  Molecular genetic paradigm of Colorectal Cancer The genetic paradigm of colorectal cancer. The genetic changes that accompany the stepwise transformation of normal colonic mucosal tissues to carcinoma are depicted in the model. Both mutational inactivation of tumor suppressor genes 121  Tumor Staging TNM Staging System Classify solid tumors based on extent of: ✓ bowel wall penetration or size of primary lesion (T = primary tumor) ✓lymph node involvement (N = regional lymph nodes) ✓presence of distant metastases (M = metastases) Important predictor of patients’ prognosis patient selection for adjuvant therapy 122 123  Risks Factors Associated w/ CRC Diet (fats, red meat) Age over 50 yrs Smoking & heavy alcohol consumption Exposure to carcinogenic agents Heredity Inflammatory bowel disease & presence of colorectal polyps 124  Role of KRAS in Signal Transduction acts as a “switch” between an inactive GDP- bound & active GTP- bound relays message from cell membrane to the nucleus Point mutation necessary to activate the gene 125  KRAS Gene Structure G domain G-1 (residue 5-20) Effector binding Switch I 2 (residue 32-40) GTP binding Switch II 3 (residue 53-62) 4 (residue 112-119) Hypervariable domain 5 (residue 144-146) (Friday and Adjei, 2005) 126 Contribution of Gene Mutations SCOBEC and Birmingham Cancer Study Day 6th June 2007 127  Mutator Genes: Mismatch repair (MMR) Genes Encode for proteins that ensure fidelity of DNA replication Genes: hMLH1, hMSH2, hMSH6, hPMS2, hPMSI, hMLH3 Mutation in one or more of these genes result to genetic instability 128  Hereditary types CRC Genes associated with CRC) (1)Familial adenomatous polyposis (FAP) Gene: Adenomatous polyposis coli (APC) (2) Hereditary nonpolyposis Colorectal Cancer (HNPCC) Genes: MMR genes ---- MSI 129 FAP vs HNPCC SCOBEC and Birmingham Cancer Study Day 6th June 2007 130 131  HNPCC: Amsterdam Criteria at least 3 relatives must have colon cancer, with at least one of them being the first degree relative of the other two, at least two successive generations must have affected individuals, at least one family member had colorectal cancer before the age of 50, FAP should be excluded as the basis of the disorder 132  Molecular Genetics of HNPCC 6 MMR genes: hMSH2, hMLH1, hMSH6, hPMS1, hPMS2, hMLH3 Germline Mutation: Deficient DNA mismatch repair (MMR) system Microsatellite instability (MSI) pathway 133  HNPCC: MMR Genes MSH2 MSH6 MSH1 PMSL2 PMSL1 Chromosome 2 Chromosome 3 Chromosome 7 134  MISMATCH REPAIR (MMR) GENES hMSH2 hMLH1 Chrom. 2p16 Chrom 3p23 16 exons 19 exons Protein (MutS) protein (MutL) recognizes base catalyzes the mismatch (exon 12-14) downstream reaction of MMR Promotes binding of MutH to MutS for repair to proceed 135  Inactivation of MMR genes encoding these activities leads to genome-wide instability, particularly in simple repetitive sequences. Predisposition to certain types of cancer like HNPCC 136 Positions of mutations identified 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Deletions (MLPA) hMSH2 Duplications (MLPA) Splice site Frameshift/nonsense Missense SCOBEC and Birmingham Cancer Study Day 6th June 2007 137  hMLH1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Deletions (MLPA) Missense Splice site SCOBEC and Birmingham Cancer Study Day Frameshift/nonsense 138 6th June 2007 The Bethesda Panel of Markers: BAT26 D2S123 BAT25 D17S250 D5S346 4 2 5 17 2 The Bethesda Panel of Markers used and their location in the chromosomes. 139  MSI: 5 Bethesda Markers Validated and recommended as reference panel for research: BAT 25 BAT 26 D2S123 D5S346 D17S250 Classification: MSI-H, MSH-L 140  A Filipino HNPCC Family 4 generations; 115 individuals Proband (III-9); 41 yrs. Old Moderately differentiated adenocarcinoma 26 family members included Excluded < 18 years old members Screening for 2 MMR genes only 141  Mutation detection Two genes analysed – hMLH1 – 19 exons – hMSH2 – 16 exons hMSH6 in development Point mutations (missense, nonsense, splice site) Small insertions and deletions Large deletions and duplications SCOBEC and Birmingham Cancer Study Day 6th June 2007 142 143  Chromosomal Instability and Aneuploidy in Cancer 144  Chromosomal instability (CIN) A form of genomic instability (GIN) that involves frequent cytogenetic changes leading to changes in chromosome copy number (aneuploidy). While both CIN and aneuploidy are common characteristics of cancer cells, their roles in tumor initiation and progression are unclear. 145  CIN chromosomes are unstable, such that either whole chromosomes or parts of chromosomes are duplicated or deleted.. Daughter cells do not have the same number of chromosomes as the cell they originated from. 146  Aneuploidy Theory in Cancer David Rasnick and Peter Duesberg 35,000- 40,000 genes in the human genome Linear gene arrangement along the 46 chromosomes Aneuploidy – abnormal chromosome number: monosomy or trisomy Ex. Down syndrome (trisomy 21) 147  Aneuploidy theory of Cancer Aneuploidy Monosomy Trisomy Which is the first event? gene mutation? or chromosomal abnormality? 148  Aneuploidy -- Cancer When a nucleus has one or more extra or missing chromosomes—the cell almost always fails to develop or function properly. Cancer cells, which are overwhelmingly aneuploid, are an exception. They thrive and divide with fury. Why? Contradiction? Megan Scudellari, 2014 149  Aneuploidy and Cancer Aneuploidy (no matter which chromosome is affected) causes genomic instability Instability predisposes the cell to mutations that cause cancer. Aneuploidy may help cancer cells adapt quickly to stress in the body and nudge cells toward mutations that directly promote tumorigenesis. 150  Aneuploidy and Cancer Aneuploidy promotes structural abnormalities known to cause cancer. “That cancer genomes select for extra chromosomes that contain potent oncogenes, and select against extra chromosomes rich in tumor suppressors” Elledge and colleagues at Harvard University The Method in Cancer’s Madness,” Spring 2014. HHMI Bulletin). 151  Aneuploidy Destabilizes a cell in much the same way that a dent disrupts the symmetry of a wheel. With each revolution the wheel becomes more distorted. As aneuploid cells divide, their genomes become increasingly disorganized to the point where most of these cells stop dividing and die. 152 153  Aneuploidy But rarely, and disastrously, an aneuploid cell with the right number and combination of extra chromosomes wins the genetic lottery and keeps right on going. Then it has become a cancer cell. 154  Aneuploidy Aneuploid cells contain an imbalance in the complement of genes and chromosomes compared to the normal or "diploid" cell. This imbalance in the chromosomes leads to a wide variety of problems, one of which is cancer 155  Aneuploidy chromosomal imbalance disrupts the normal balance and interactions of many thousands of genes, because just one chromosome may contain several thousand genes. A cancer cell may have several copies of a given chromosome. Aneuploidy is likely to be far more devastating to the life of a cell than a small handful of gene mutations 156  Aneuplopidy Bert Vogelstein of Johns Hopkins University has said that "at least 90 percent of human cancers are aneuploid." Argument: “Gene mutations must initiate the aneuploidy or that aneuploidy must be a consequence of gene mutations” 157 “Many cancers carryabnormal chromosome number as well as structural anomalies.” 158 Karyotype: Composite: 46-52, XY, del(3) (q27or 28 🠆ter), t(6;13) (q25;q14), t(9;22) (q34;q11), t(12;14) (p13;q24), +15,+18,+19,+20,+21, del(22)(q11 🠆ter) 159 Numerous structural and numerical abnormalities in a tumor cell. 160 An accurate gene test can tell if mutation is present; but the finding does not guarantee that the disease will develop. Not all mutations are pathogenic. 161 THANK YOU 162