1. 1 Genetics of Cancer Ömer Faruk Bayrak

2. “Cancer is, in essence, a genetic disease. Although cancer is complex, and environmental and other nongenetic factors clearly play a role in many stages of the neoplastic process, the tremendous progress made in understanding tumorigenesis in large part is owing to the discovery of the genes, that when mutated, lead to cancer.” Bert Vogelstein (1988) NEJM 1988; 319:525-532.

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4. Cancer: review of molecular genetics Cancer cells Genetic basis for cancers Types of cancer Causes of cancer Cancer warning signs Prevention, detection, treatment

5. Definition of Cancer Cancer is a disease characterized by the uncontrolled proliferation of cells. The normal mechanisms that regulate cellular growth and division break down. This breakdown results from mutations that overcome the normal limits to the number of cell divisions that can take place before a cell dies.

6. Cancer 1.Oncogenesis may be due to: a.Spontaneous genetic changes, such as spontaneous gene or chromosome mutations. b.Exposure to mutagens or radiation. c.The action of genes introduced by tumor viruses.

9. The 3 phases in the development of cancer cells Initiation – a single cell undergoes a mutation that causes it to divide repeatedly Promotion – a tumor develops and cells within the tumor mutate Progression – a cell mutates in such a way that allows it to invade surrounding tissue

10. Stages of Cancer Progression Primary cells Immortalization (Benign) Transformation Metastasis

11. Relationship of the Cell Cycle to Cancer Regulation of Cell Division in Normal Cells 1.Cell differentiation occurs as cells proliferate to form tissues. a.Cell differentiation correlates with loss of ability to proliferate, with the most highly specialized cells terminally differentiated. b.Terminally differentiated cells have a finite life span, and are replaced with new cells produced from stem cells. c.Stem cells are capable of self-renewal. d.Proliferation of eukaryotic cells is described by the cell cycle: i. M is mitotic phase. The rest of the cell cycle is interphase. ii. During G1 the cell monitors its size and environment. (1) If conditions are appropriate, it moves into S phase (DNA synthesis), and completes the cycle with G 2 and M. (2) A cell that does not commit to DNA replication may enter G 0 for a long period, then reenter the cell cycle and proliferate.

12. 2.Normal cell cycle is controlled in several ways. Most important are signal transduction pathways. a.Extracellular factors bind to surface receptors, transmembrane proteins that relay signals into the cell. b.Factors include (Figure 18.2): i. Growth factors that stimulate cell division. ii. Growth-inhibiting factors that inhibit cell division. c.Healthy cells produce progeny only when the balance of stimulatory and inhibitory signals favors cell division. d.Neoplastic cells reproduce without constraint, sometimes because of mutations in inhibitory or stimulatory factor genes.

13. Chapter 18 slide 13 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 18.2a General events for regulation of cell division in normal cells

14. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 18.2b General events for regulation of cell division in normal cells

15. Characteristics of cancer cells  Lack differentiation and do not contribute to body functioning  Have abnormal nuclei that are enlarged and may have an abnormal number of chromosomes  Unlimited ability to divide one way is through turning on the telomerase gene that allows telomeres on chromosomes to continually be built thus allowing a cell to divide over and over again  Form tumors Benign tumors are usually encapsulated and do not invade adjacent tissue while a cancerous tumor usually is not encapsulated and eventually invades surrounding tissue  Can divide without growth factors  Become abnormal gradually through a multistage process  Undergo angiogenesis and metastasis

16. Cancer Spreads Step-by-Step

17. Cancer is a Genetic Disease Cancer is a genetic disease that develops in a predictable sequence of steps  Carcinogenesis Transformation of a normal cell into a cancerous cell Step-by-step transformation

18. A Common Type of Colorectal Cancer May Develop by These Steps

19. Colon cancer results from genetic alterations in multiple genes Inherited mutations in the APC gene dramatically increase risk of colon cancer

20. Chapter 18 slide 20 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 18.15 A multistep molecular event model for the development of hereditary adenomatous polyposis (FAP), a colorectal cancer

21. The Multistep Nature of Cancer 1.Cancer induction may require accumulation of 6–7 independent mutations over several decades, typically involving: a.Conversion of proto-oncogenes to oncogenes. b.Inactivation of tumor suppressor genes. 2.An example is Vogelstein’s model for a form of colorectal cancer, hereditary FAP (Figure 18.15). a.Mutation of both alleles of a tumor suppressor gene on chromosome 5, APC (adenomatous polyposis coli), causes increased cell growth. b.Hypomethylation of the DNA leads to a benign tumor (adenoma class I). c.Mutation of the chromosome 12 ras proto-oncogene allows cells to form a larger benign tumor (adenoma class II). d.If both copies of DCC, a tumor suppressor gene on chromosome 18, are lost, an even larger adenoma class III results. e.Mutation of both p53 alleles on chromosome 17 results in conversion to a carcinoma. f.Other gene losses result in the cancer metastasizing. g.Other paths are possible, but in all cases deletions of APC and mutations of ras occur before deletions of DCC and p53.

22. The Two-Hit Mutation Model for Cancer 1.Cancers can be caused by viruses, but most result from mutations in cellular genes. Usually these mutations have accumulated over time, and research has identified the genes involved. 2.The incidence of cancer falls into two categories: a.Sporadic cancers, the more frequent type, do not appear to have an hereditary cause. b.Familial (hereditary) cancers run in families. Retinoblastoma provides an example (Figure 18.3). i. Retinoblastoma is the most common eye tumor in children birth to 4 years. Early treatment (usually gamma radiation) is over 90% effective. ii. Retinoblastoma has two forms: (1) Sporadic retinoblastoma (60%) develops in children with no family history of retinoblastoma, and occurs in one eye (unilateral tumor). (2) Hereditary retinoblastoma (40%) patients typically develop multiple tumors involving both eyes (bilateral tumors). (a)Onset is usually earlier in the hereditary form. (b)Siblings and offspring often develop the same type of tumor. (c)Pedigrees of affected families are consistent with a single gene responsible for retinoblastoma.

23. 3.Knudson (1971) proposed the 2-hit mutational model, that two mutations were required for development of retinoblastoma (Figure 18.4). a.In sporadic retinoblastoma, the child starts with two wild-type alleles (RB + /RB + ). i. Both alleles must mutate to produce the disease genotype (RB/RB). ii. The probability of both mutations occurring in the same cell is low, so only one tumor forms. b.In hereditary retinoblastoma, the child starts out heterozygous (RB/RB + ). i. Only one mutation is needed for tumor formation (RB/RB). ii. Mutations resulting in loss of heterozygosity (LOH) are likely in rapidly dividing cells, and multiple tumors occur. 4.In Knudson’s model: a.Retinoblastoma alleles are recessive, because only homozygotes (RB/RB) develop tumors. b.However, in pedigree analysis, the disease appears to be dominant. This is because: i. Heterozygous individuals (RB/RB + ) are predisposed to the cancer, since only one mutation is required for the neoplasm. Families with one allele already mutated will have a significant incidence of the disease. ii. Homozygous dominant individuals (RB + /RB + ) develop the cancer only when both alleles in the same cell are mutated. Therefore, most children in the general population do not develop the disease. 5.This hypothesis is supported by later studies of the chromosomes of retinoblastoma patients, which: a.Mapped the gene to 13q14.1-q14.2 (long arm of chromosome 13). b.Showed that the gene encodes a growth inhibitory factor (tumor suppressor). 6.Retinoblastoma is rare among cancers because a single gene is critical for its development. In most cases, cancers result from a series of mutations in different genes for growth and division.

24. Chapter 18 slide 24 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 18.4 Knudson’s two-hit mutation model

25. Some Tumors Are Cancer, Others Are Not Hyperplasmia Cells in a tissue overgrow Resulting defined mass: tumor (neoplasm) – Benign, e.g., moles Slow growth Expands in the same tissue; does not spread Cells look nearly normal – Malignant Rapid growth Invades surrounding tissue and metastasizes Cell differentiation usually poor

26. Dysplasia Abnormal change in the size, shape, and organization of cells in a tissue Often an early step toward cancer – Microscopic characteristics of cancer cells – Behave differently from normal cells Some Tumors Are Cancer, Others Are Not

27. Cancer Cells Are Abnormal in Their Growth and Appearance

28. 28 Telomeres Chromosome tips TTAGGG repeats Shorten with each cell division Nerve cells have short telomeres – Do not divide very often Gametes have long telomeres – Must divide many, many times – Telomerase adds TTAGGG repeats

29. 29 Cancer Cells Produce telomerase Immortal

30. 30 Henrietta Lacks Died of cervical cancer in 1951 Biopsy of her cancer is still alive! Cultures of her cancer cells in labs world wide Called HeLa cells

31. 31 HeLa Cells Used to develop vaccine for polio Divide every 24 hours Often contaminate research labs New species evolved from humans – One celled microorganism Reproduces on its own Has all the characteristics of every other living species

32. 32 Cancer Cells Mutated cells Do not respond to cell cycle control signals – Do not repair DNA damage in interphase Grow continuously Transplantable – Can inject into an animal and it will continue to grow

33. 33 Cancer Cells (cont.) Different appearance – Some are more round Heritable – Offspring of CA cells are also cancerous Dedifferentiated – Less specialized than the cells they arose from. Loss of contact inhibition – Do not stop dividing when they crowd other cells

34. 34 Cancer Cells (cont.) Invasive – Secrete chemical to cut paths through healthy tissue Angiogenesis – Stimulate blood vessels to grow and feed CA Metastasize – Travel by bloodstream or lymphatic system to start new tumors

35. Normal Moles Are Common Examples of Benign Growths

36. Main Features of Benign and Malignant Tumors

37. A Cancer Cell’s Structure Is Abnormal Cancer is a result of a series of mutations in the cell’s genes – Larger cell nucleus and less cytoplasm – Loss of structural specialization – Cytoskeleton shrinks – Plasma membrane proteins could be lost or altered – New plasma membrane proteins may appear – Changes passed on to cell’s descendants

38. Genes and Cancer 1.Three classes of genes are mutated frequently in cancer: a.Proto-oncogenes, whose products normally stimulate cell proliferation. b.Tumor suppressor genes, whose products normally inhibit proliferation. c.Mutator genes, whose products ensure accurate replication and maintenance of the genome.

39. Proto-oncogenes Cellular homologues of viral oncogenes (a.k.a. normal cellular oncogenes, c-onc) e.g, v-src and c-src; very similar genes (few a.a. different) c-onc genes a lot of conservation in structure among species c-onc’s have introns; v-onc’s do not

40. Oncogenes 1.Tumor viruses induce infected cells to proliferate and produce a tumor. There are two types, based on the viral genome: a.RNA tumor viruses transform cells by introducing viral oncogenes. (An oncogene is any gene that stimulates unregulated proliferation.) b.DNA tumor viruses do not carry oncogenes, and use other mechanisms to transform the cell.

41. 41 Oncogenes are usually dominant (gain of function) cellular proto-oncogenes that have been mutated (and “activated”) cellular proto-oncogenes that have been captured by retroviruses and have been mutated in the process (and “activated”) virus-specific genes that behave like cellular proto- oncogenes that have been mutated to oncogenes (i.e., “activated”)

42. 42 Class I: Growth Factors Class II: Receptors for Growth Factors and Hormones Class III: Intracellular Signal Transducers Class IV: Nuclear Transcription Factors Class V: Cell-Cycle Control Proteins Five types of proteins encoded by proto-oncogenes participate in control of cell growth:

43. 43 amino acid position Ras gene 12 59 61 Tumor c-ras (H, K, N)GlyAlaGlnnormal cells H-rasGlyAlaLeulung carcinoma ValAlaGlnbladder carcinoma K-rasCysAlaGlnlung carcinoma ArgAlaGlnlung carcinoma ValAlaGlncolon carcinoma N-rasGlyAlaLysneuroblastoma GlyAlaArglung carcinoma Murine sarcoma virus H-rasArgThrGlnHarvey strain K-rasSerThrGlnKirsten strain Amino acid substitutions in Ras family proteins (inactivates GTPase)

44. 44 Activation mechanisms of proto-oncogenes proto-oncogene --> oncogene

45. 45 CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS NeoplasmTranslocationProto-oncogene Burkitt lymphoma t(8;14)80% of casesc-myc 1 t(8;22) 15% of cases t(2;8) 5% of cases Chronic myelogenoust(9;22)90-95% of cases bcr-abl 2 leukemia Acute lymphocytict(9;22)10-15% of cases bcr-abl 2 Leukemia 1 c-myc is translocated to the IgG locus, which results in its activated expression 2 bcr-abl fusion protein is produced, which results in a constitutively active abl kinase

46. 46 GENE AMPLIFICATION OncogeneAmplification Source of tumor c-myc ~20-foldleukemia and lung carcinoma N-myc 5-1,000-foldneuroblastoma retinoblastoma L-myc 10-20-foldsmall-cell lung cancer c-abl ~5-foldchronic myoloid leukemia c-myb 5-10-foldacute myeloid leukemia colon carcinoma c-erbB ~30-foldepidermoid carcinoma K-ras 4-20-foldcolon carcinoma 30-60-foldadrenocortical carcinoma

47. 47 The result: Overproduction of growth factors Flooding of the cell with replication signals Uncontrolled stimulation in the intermediary pathways Cell growth by elevated levels of transcription factors

48. 48 Tumor suppressor genes Normal function - inhibit cell proliferation Absence/inactivation of inhibitor --> cancer Both gene copies must be defective

49. 49 KNUDSON TWO HIT HYPOTHESIS IN FAMILIAL CASES RB rb RB Familial RB (%30) Tumor cellsNormal cells Inactivation of a tumor suppressor gene requires two mutations, inherited mutation and somatic mutation. RB LOH

50. 50 RB LOH RB Mutation Normal Cells Tumor cells KNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASES RB Inactivation of a tumor suppressor gene requires two somatic mutations.

51. 51 TUMOR SUPPRESSOR GENES Disorders in which gene is affected Gene (locus) Function Familial Sporadic DCC (18q) cell surface unknowncolorectal interactions cancer WT1 (11p) transcription Wilm’s tumorlung cancer Rb1 (13q) transcription retinoblastomasmall-cell lung carcinoma p53 (17p) transcription Li-Fraumeni breast, colon, syndrome & lung cancer BRCA1(17q) transcriptionalbreast cancerbreast/ovarian tumors BRCA2 (13q) regulator/DNA repair

52. 52 CELL CYCLE Daugther cell Mitosis DNA replication Control Point Gateway Growth Factors Cell cycle inhibitors CELL CYCLE S

53. 53 Rb gene Rb protein controls cell cycle moving past G1 checkpoint Rb protein binds regulatory transcription factor E2F E2F required for synthesis of replication enzymes E2F - Rb bound = no transcription/replication Growth factor --> Ras pathway --> G1Cdk-cyclin synthesized Active G1 Cdk-cyclin kinase phosphorylates Rb Phosphorylated Rb cannot bind E2F --> S phase – Disruption/deletion of Rb gene – Inactivation of Rb protein --> uncontrolled cell proliferation --> cancer

54. 54 p53 Phosphyorylated p53 activates transcription of p21 gene p21 Cdk inhibitor (binds Cdk-cyclin complex --> inhibits kinase activity) Cell cycle arrested to allow DNA to be repaired If damage cannot be repaired --> cell death (apoptosis) Disruption/deletion of p53 gene Inactivation of p53 protein --> uncorrected DNA damage --> uncontrolled cell proliferation --> cancer

55. p53 Most mutations in DBD cannot bind to target genes, so targets not transcribed recessive loss-of-function mutations also important in cellular stress response normal p53 important in DNA damage repair

56. 56 These are genes that ensure each strand of genetic information is accurately copied during cell division of the cell cycle. Mutations in DNA repair genes lead to an increase in the frequency of mutations in other genes, such as proto-oncogenes and tumor suppressor genes. i.e. Breast cancer susceptibility genes (BRCA1 and BRCA2) Hereditary non-polyposis colon cancer susceptibility genes (MSH2, MLH1, PMS1, PMS2) have DNA repair functions. Their mutation will cause tumorigenesis. DNA REPAIR GENES

57. 57 Van Gent et al, 2001 Molecular mechanisms of DNA double strand break repair BRCA1/2

58. Chromosomal rearrangements and cancer CML- chronic myelogenous leukemia Philadelphia chromosome - reciprocal translocation between chromosomes 9 and 22 c-alb oncogene involved (on chromosome 9); ber gene on chromosome 22

59. pBRCA1 and pBRCA2 Mutant forms of these TS genes implicated in breast and ovarian cancer brca1- map to ch 17; brca 2 - map to ch 13 220-350 kd proteins in nucleus - putative transcription factors mutations in these about 7% of all breast cancers and 10% of ovarian cancers carriers high probability of disease

60. Comparing these genes in normal and cancer cells

61. Types of cancer Oncology – study of cancer Carcinomas: cancers of the epithelial tissue Adenocarcinomas: cancers of glandular epithelial cells Sarcomas: cancers of muscle and connective tissues Leukemias: cancers of the blood Lymphoma: cancers of lymphatic tissues

62. Genetic causes of cancer Examples of genes associated with cancer: – BRCA1 and BRCA2 – tumor-suppressor genes that are associated with breast cancer – RB – a tumor-suppressor gene that is associated with an eye tumor – RET – proto-oncogene that is associated with thyroid cancer Mutations of these genes predispose individuals to certain cancers but it takes at least one more acquired mutation during their lifetime to develop cancer

63. Environmental causes of cancer Radiation: – Environmental factors such as UV light (in sunlight or tanning lights) and x-rays can cause mutation in DNA Organic chemicals: – Tobacco smoke: increases cancer of lungs, mouth, larynx and others – Pollutants: substances such as metals, dust, chemicals and pesticides increase the risk of cancer Viruses: – Hepatitis B & C: virus that can cause liver cancer – Epstein-Barr virus: can cause Burkitt’s lymphoma – Human papillomavirus: can cause cervical cancer CANCER QUICK COURSE—WHAT CAN YOU DO TO PREVENT/TREAT?

64. Seven warning signs of cancer Change in bowel or bladder habits A sore that does not heal Unusual bleeding or discharge Thickening or lump in breast or elsewhere Indigestion or difficulty in swallowing Obvious change in wart or mole Nagging cough or hoarseness

65. Other ways to detect cancer Tumor marker tests – blood tests for tumor antigens/antibodies – CEA (carcinoembryonic antigen) antigen can be detected in someone with colon cancer – PSA (prostate-specific antigen) test for prostate cancer Genetic tests – tests for mutations in proto-oncogenes and tumor-suppressor genes – RET gene (thyroid cancer) – P16 gene (associated with melanoma) – BRCA1 (breast cancer) A diagnosis of cancer can be confirmed by performing a biopsy

66. Standard cancer treatments Surgery – removal of small cancers Radiation therapy – localized therapy that causes chromosomal breakage and disrupts the cell cycle Chemotherapy – drugs that treat the whole body that kills cells by damaging their DNA or interfering with DNA synthesis Bone marrow transplants – transplant bone marrow from one individual to another

67. Newer cancer therapies Immunotherapy – inject immune cells that are genetically engineered to bear the tumor’s antigens Passive immunotherapy – antibodies that are linked to radioactive isotopes or chemotherapeutic drugs are injected into the body p53 gene therapy – a retrovirus in clinical trial that is injected into the body where it will infect and kill only tumor cells (cells that lack p53 = tumor cells) Angiogenesis inhibition - Angiostatin and endostatin are drugs in clinical trials that appear to inhibit angiogenesis CANCER QUICK COURSE—WHAT CAN YOU DO TO PREVENT/TREAT?