Student Learning Outcomes: 18 Cancer Chapter 18 Cancer: Student Learning Outcomes: Explain causes, development of cancer (clonal): Chemicals, radiation, virus, spontaneous Decribe diversity of tumor viruses (model systems) Explain essential features of Oncogenes: Derived from proto-oncogenes, examples Explain essential features of tumor suppressors: Normal cell functions, examples Describe molecular approaches to cancer treatment

Introduction 18.1 Cancer cells have abnormalities in multiple cell regulatory systems. Breakdown of regulatory mechanisms that govern normal cell behavior: Grow, divide in uncontrolled manner, Spread throughout body Interfere with function of normal tissues, organs Understand cancer cells at molecular, cellular levels. Studies of cancer cells illuminate mechanisms of normal cell behavior

The Development and Causes of Cancer Tumor: abnormal proliferation of cells: Benign tumor confined to original location, not invade normal tissue, not spread to distant sites Malignant tumor invades surrounding normal tissue, spreads through body via circulation or lymphatics (metastasis) termed cancers. Fig. 18.1Pancreatic cancer (purple stained nuclei) in normal

Development and Causes of Cancer Most cancers three main groups: Carcinomas: malignancies of epithelial cells (about 90% of human cancers). Sarcomas (rare in humans): solid tumors of connective tissue, (muscle, bone, cartilage, fibrous tissue) Leukemias and lymphomas: blood-forming cells and cells of immune system, respectively. Tumors are further classified according to tissue of origin and type of cell involved.

Note most common cancers; most lethal Cell5e-Table-18-01-0.jpg

Most cancers develop late in life Fig 18.2 Tumor clonality Tumor clonality Fundamental feature of cancer Tumors develop from single cell that proliferates abnormally (evidence from X-inactivation pattern) Most cancers develop late in life Cancer is multistep process: Cells gradually become malignant through progressive alterations Multiple abnormalities accumulate Selection for growth advantage Ex: colon cancer increases with age. Cell5e-Fig-18-02-0.jpg Fig. 18.2 clonality Fig. 18.3 age and colon cancer

Fig 18.4 Stages of tumor development Tumor initiation: genetic alteration → abnormal proliferation of single cell, → population of clonal tumor cells. Tumor progression: additional mutations occur within cells of population Growth advantage, Survival Invasion Metastasis Single-cell origin from analysis of X chromosome inactivation (female cancers). Fig. 18.4

Fig 18.5 Development of colon carcinomas EX. Colon cancer Proliferation of epithelial cells → small benign neoplasm (adenoma or polyp). Clonal selection → Growth of adenomas of increasing size, proliferative potential Becomes carcinoma metastasis Cell5e-Fig-18-05-0.jpg Fig. 18.5

The Development and Causes of Cancer Carcinogens - substances that cause cancer. Radiation, chemicals, viruses can damage DNA, induce mutations. Solar UV radiation is major cause of skin cancer. Aflatoxin produced by some molds that contaminate peanuts and other grains Viruses can cause cancer People can inherit cancer-susceptibility genes (oncogenes, damaged tumor suppressor genes)] Tobacco smoke carcinogenic chemicals: benzo(a)pyrene, dimethylnitrosamine, nickel compounds (nearly 1/3 cancer deaths). Fig. 18.6

The Development and Causes of Cancer Tumor promoters stimulate cell proliferation:. Hormones, particularly estrogens, are tumor promoters in some human cancers. exposure to excess estrogen increases likelihood woman will develop uterine cancer. Some viruses cause cancer: liver (HBV), cervical carcinoma (HPV) Bacterium Heliobacter pylori causes stomach cancer. * Studies of tumor viruses identify molecular events in development of cancers.

Properties of cancer cells Characteristic properties of Cancer cells: 1. lack density-dependent inhibition of proliferation 2. reduced requirements for growth factors 3. less regulated cell-cell, cell-matrix (less adhesive) 4. not sensitive to contact inhibition 5,6. secrete proteases for invasion, growth factors for angiogenesis 7. don’t differentiate normally, stay undifferentiated 8, 9. not undergo apoptosis; even after DNA damage 10. unlimited DNA replication; (over) express telomerase

Cancer cells have lost normal control – 10 properties Cancer cells lack Density-dependent inhibition - Continue growing to high densities Normal cells proliferate to finite cell density, (availability of growth factors). cease proliferating, arrest in G0 Cell5e-Fig-18-07-0.jpg Fig. 18.7

The Development and Causes of Cancer 2. Cancer cells reduced requirements for growth factors contributes to unregulated proliferation. Can stimulate own proliferation (autocrine stimulation). Fig. 18.8

The Development and Causes of Cancer 3. Cancer cells are less regulated by cell-cell, cell- matrix interactions: Reduced expression of adhesion molecules contributes to invasion, metastasis Loss of E-cadherin (main adhesion molecule), aids development of carcinomas (epithelial cancers). 4. Cancer cells lack Contact inhibition Normal fibroblasts migrate until contact neighbor cell, stop, adhere. Tumor cells move after contact with neighbor cells, migrate over adjacent cells, grow in disordered, multilayered patterns. Fig. 18.9

The Development and Causes of Cancer 5,6. Cancer cells secrete: Proteases to digest extracellular matrix Growth factors for angiogenesis Permits invasion of normal tissues Digest collagen allows penetration of basal laminae, invasion of connective tissue. New blood vessels needed after tumor about 106 cells; penetrate capillaries

The Development and Causes of Cancer 7. Cancer cells don’t differentiate normally. Most fully differentiated cells cease cell division. Ex. Leukemias: Blood cells from hematopoietic stem cells in bone marrow. Leukemic cells don’t undergo terminal differentiation; arrest at early stages, retain capacity for proliferation Fig. 18.10

The Development and Causes of Cancer 8.9. Cancer cells fail to undergo programmed cell death or apoptosis: Longer life span than normal; not require growth factors Lack of apoptosis after DNA damage increases resistance of cancer cells to irradiation, chemotherapeutic drugs, (which damage DNA) 10. Cancer cells overexpress telomerase: Capacity for unlimited DNA replication Telomerase required to maintain ends of chromosomes after replication Fig. 6.16 telomerase

The Development and Causes of Cancer Ex. Assay for cell transformation in vitro: Study of tumor induction by radiation, chemicals, or viruses Detect conversion of normal cells to tumor cells in culture (altered growth properties) Focus assay (1958) recognizes group of transformed cells morphologically distinct “focus” versus normal cells on dish. Fig. 18.11 Focus: RSV and chicken fibroblasts

18.2 Tumor viruses Tumor Viruses directly cause cancer in humans or animals Critical role in research - models for cellular, molecular study Small genomes allowed identification of viral genes responsible for cancer induction.

Hepatitis B and C viruses Tumor Viruses Hepatitis B and C viruses Principal causes of liver cancer. Viruses infect liver cells, long-term chronic infections, associated with high risk of liver cancer. HBV is DNA virus HCV is RNA virus

Simian virus 40 (SV40, monkey) (and polyomavirus, mice) Tumor Viruses Simian virus 40 (SV40, monkey) (and polyomavirus, mice) not cause human cancer, important model small genome sizes Replicates in permissive host Transforms non-permissive host (inactivates Rb) Early region encodes proteins (small and large T antigens) Proteins stimulate host cell gene expression, DNA synthesis. Figs. 18.12,13

Papillomaviruses are small DNA viruses. Tumor Viruses Papillomaviruses are small DNA viruses. About 100 different types infect epithelial cells. Some cause benign tumors (warts); others cause malignant carcinomas, particularly cervical cancer Transformation by expression of early genes, E6,E7: E7 binds Rb; E6 stimulates degradation of p53. Fig. 18.14

Tumor Viruses Adenoviruses large family of DNA viruses not associated with human cancer, important models. Adenoviruses lytic in cells of their natural hosts, can induce transformation in nonpermissive cells. Adenoviruses potential gene therapy vector

Tumor Viruses Herpesviruses among the most complex viruses, enveloped DNA genomes 100 to 200 kb: HSV-1, HSV-2 cause cold sores, genital sores Varicella zoster virus (VZV) causes chicken pox, shingles Kaposi’s sarcoma-associated herpesvirus causes Kaposi’s sarcoma (common with AIDS) Epstein-Barr virus cause mononucleosis (transient) and Burkitt’s lymphoma

Tumor Viruses Retroviruses (RNA genome, DNA intermediate) cause cancer in animals, including humans. HTLV-I causes adult T-cell leukemia Human immunodeficiency virus (HIV) causes AIDS. HIV not cause cancer directly, but infects & destroys T cells; AIDS patients malignancies (lymphomas, Kaposi’s sarcoma) Most retroviruses only 3 genes (gag, pol, and env): for virus replication, not transformation; rarely induce tumors Other retroviruses have specific extra genes induce cell transformation, oncogenes (carcinogens Fig. 18.15

Fig 18.16 Cell transformation by RSV and ALV 18.3 Oncogenes - genes that transform cells: Rous sarcoma virus (RSV) Prototype highly oncogenic retrovirus 1st oncogene identified by comparison of RSV to ALV (avian leukosis virus does not induce tumors) ** Cellular oncogenes are involved in development of non-virus-induced cancers. Cell5e-Fig-18-16-0.jpg Fig. 18.16

RSV mutants revealed gene responsible for tumors Fig 18.17 The RSV genome RSV mutants revealed gene responsible for tumors RSV causes sarcomas: oncogene is src. (not in ALV) Src was 1st protein-tyrosine kinase identified. * other oncogenic retroviruses have key proteins of cell signaling path: ras, raf, myc, erbB, fos, jun, Cell5e-Fig-18-17-0.jpg Fig. 18.17

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Fig 18.18 Isolation of Abelson leukemia virus Viral oncogenes are derived from genes of host cell: Key expt: isolation of oncogenic retrovirus Abelson leukemia virus from mice injected with a nontransforming MuLV virus. One mouse developed lymphoma from which a new, highly oncogenic virus was isolated: Virus contained an oncogene (abl) Abl is related to normal cell gene Normal gene called proto-oncogene Cell5e-Fig-18-18-0.jpg Fig. 18.18

*18.3 Proto-oncogenes: Oncogenes Normal-cell genes from which oncogenes originated Often proteins of signal transduction pathways that control cell proliferation (e.g., src, ras, and raf ) Retroviral oncogenes differ from proto-oncogenes: transcribed under control of viral promoter, enhancer expressed at much higher levels, or in inappropriate cells. point mutations lead to unregulated activity (ex. Ras) can differ in structure and function from normal proteins: Fig. 18.19 Raf oncogene: loss of regulatory domain makes oncogene protein unregulated

Detection of human tumor oncogene by gene transfer Evidence cellular oncogenes in human tumors (gene transfer experiments 1981) DNA from human bladder carcinoma induced transformation of mouse cells in culture, indicating tumor had oncogene Many other examples later: ras, raf, c-myc erbB, cdk4, PDGFR Cell5e-Fig-18-20-0.jpg Fig. 18.20

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Fig 18.21 Point mutations in ras oncogenes First human oncogene: homolog of rasH oncogene of Harvey sarcoma virus. 3 members of ras gene family (rasH, rasK, rasN) are oncogenes most frequent in human tumors. ras oncogenes have point mutations critical sites: Mutations maintain Ras proteins constitutively in active GTP-bound conformation Cell5e-Fig-18-21-0.jpg Fig. 18.21 mutation in RasH of bladder cancers

*Cancer cells have abnormal chromosomes: Oncogenes *Cancer cells have abnormal chromosomes: translocations, duplications, deletions. Creates oncogenes from proto-oncogenes: altered promoters, gene fusions, amplification of normal gene Ex Burkitt’s lymphomas: translocations involve genes encoding immunoglobulins. c-myc transcription factor Expressed abnormally from immunoglobulin promoter Tumors of B cells Fig. 18.22

Translocations rearrange coding sequences → abnormal gene products. Oncogenes Translocations rearrange coding sequences → abnormal gene products. Ex: Translocation of c-abl proto-oncogene causes chronic myeloid leukemia (CML); fusion protein has constitutive Abl tyr-kinase activity Fig. 18.23 Bcr-Abl

*Proto-oncogene proteins have normal roles in Oncogenes *Proto-oncogene proteins have normal roles in growth factor-stimulated signal transduction pathways. Ex. ERK pathway oncogenes: polypeptide growth factors, growth factor receptors (ErbB), intracellular signaling proteins (Ras, Raf, MEK, ERK), transcription factors (fos), transcription targets (cyclin D1). Fig. 18.24 ERK path See also Fig. 15.34

Mechanism of Tel/PDGFR oncogene activation Many oncogenes encode growth factor receptors, mostly protein-tyrosine kinases. Ex. Receptor for PDGF converted to oncogene by chromosome translocation, replacement of amino terminus by transcription factor Tel. Fusion protein Tel sequences dimerize in absence of PDGF, → activate oncogene tyr protein kinase. Cell5e-Fig-18-25-0.jpg Fig. 18.25

Mutant fos or jun always activate Cyclin D1 proto-oncogene, Oncogenes Oncogenes can encode transcription factors normally induced by growth factors EX. Transcription of fos proto-oncogene is induced by phosphorylation of Elk-1 by ERK (Fig. 18.24). Fos and Jun dimerize to form AP-1 transcription factor, activates transcription of cyclin D1 Mutant fos or jun always activate Cyclin D1 proto-oncogene, can become oncogene (CCND1) by chromosome translocation or gene amplification Fig. 18.26

Oncogenes Oncogenic activity of transcription factor can result from inhibition of differentiation. Ex. Mutated form of retinoic acid receptor (PML/RARa) oncoprotein in acute promyelocytic leukemia (APL) Mutated receptors interfere with action of normal homologs, block cell differentiation, maintain leukemic state. Treatment with retinoic acid induces differentiation, blocks cell proliferation. Fig. 18.28

Tumor Suppressor Genes *18.4 Tumor suppressor genes normally act to inhibit cell proliferation and tumor development. In many tumors, both genes are lost or inactivated, contributes to abnormal proliferation of tumor cells Tumor suppression first noticed during somatic cell hybridization experiments in 1969; hybrids did not cause tumor, suggesting genes in normal cell suppressed tumors. Fig. 18.30

Fig 18.31 Inheritance of retinoblastoma First tumor suppressor gene found was retinoblastoma, inherited childhood eye tumor. About 50% of children of affected parent develop retinoblastoma: inherited as dominant autosomal susceptibility to tumor development: Need somatic mutation of other copy photo Cell5e-Fig-18-31-0.jpg Fig. 18.31; retinoblastoma; Purple, affected people

Tumor Suppressor Genes Retinoblastoma requires loss of both functional copies of tumor susceptibility gene (Rb gene); People inherit one mutated gene, later other mutates Fig. 18.32

Mutations of Rb during retinoblastoma development Noninherited retinoblastoma is very rare: needs two independent somatic mutations in cell. Cell5e-Fig-18-32-3.jpg Fig. 18.32

Tumor Suppressor Genes Rb is tumor suppressor: Deletions of chromosome 13q14 in some retinoblastomas suggested loss (rather than activation) of Rb gene led to tumor development Mutations of Rb contribute to many human cancers Oncogene proteins of some DNA tumor viruses, including SV40, adenoviruses, and human papillomaviruses, bind to Rb and inhibit it. . Fig. 18.33,34

Mutations ruining tumor suppressor genes: common molecular alterations in human tumors Cell5e-Table-18-05-0.jpg

Tumor Suppressor Genes Mutations ruining tumor suppressor genes are common molecular alterations in tumors p53 - second tumor repressor gene identified. inactivated in many cancers, leukemias, lymphomas, sarcomas, brain tumors, carcinomas. Mutations of p53 in about 50% of all cancers. Proteins encoded by most tumor suppressor genes inhibit cell proliferation or survival. Tumor suppressor proteins inhibit regulatory pathways stimulated by products of oncogenes.

Tumor Suppressor Genes Tumor suppressor proteins inhibit pathways stimulated by products of oncogenes. Ex. Products of Rb and INK4 (p16) tumor suppressor genes regulate cell cycle at restriction point in G1 Point affected by cyclin D1/ Cdk4 (which can be oncogenes). Fig. 18.36

Tumor Suppressor Genes Ex. p53 gene product regulates both cell cycle progression and apoptosis (programmed cell death) DNA damage induces p53, activates cell cycle inhibitory gene p21, transcription of proapoptotic genes Cells lacking p53 do not cycle arrest, not do apoptosis after DNA damaging agents p53 mutated in many cancers (Fig. 16.20 p21 inhibits Cdk2/cycE) Fig. 18.37

Tumor Suppressor Genes Ex. BRCA1 and BRCA2 genes (responsible for some inherited breast and ovarian cancers) function as stability genes that maintain integrity of genome: Checkpoints in cell cycle progression, Repair of DNA double-strand breaks Mutations inactivating these genes leads to a high frequency of mutations in oncogenes or tumor suppressor genes.

Tumor Suppressor Genes MicroRNAs (miRNAs) also regulate gene expression post-transcriptionally, inhibit translation and/or induce mRNA degradation, contribute to regulation of about 1/3 of protein-coding genes. Expression of miRNAs is lower in tumors, suggesting they are tumor suppressors. Example: let-7 targets oncogene c-myc [Other miRNAs may be oncogenes] Fig. 18.38

Tumor Suppressor Genes Development of cancer is multistep process: Accumulated damage in multiple genes → increased proliferation, survival, invasiveness, metastatic potential. Large-scale genome sequencing detects frequency mutations 100 colorectal cancers: each tumor ~ 15 mutations in genes thought to be involved in cancer development: Oncogenes rasK and PI3K ; Tumor suppressor genes APC and p53. Breast cancers ~ 14 mutations per tumor, p53 tumor suppressor and PI3K oncogene Fig. 18.39

Molecular Approaches to Cancer Treatment Molecular defects being deciphered-> new approaches to prevention and treatment 1. prevent development of cancer 2. detect early premalignant, before metastasis 3. identify susceptible individuals (gene tests) 4. molecular diagnosis of oncogenes, tumor suppressor genes in patient 5. drugs specifically targeted to mutant proteins

Molecular Approaches to Cancer Treatment Early detection of cancer: Cured by localized treatment, before metastasis. Ex. Early stages of colon cancer (adenomas) usually curable by minor surgical procedures. Fig. 18.40

Molecular Approaches to Cancer Treatment Identify individuals with inherited susceptibilities to cancer development, diagnose tumors. Mutations in tumor suppressor genes (p53, Rb), oncogenes (myc and cdk4), stability genes (BRCA1 and BRCA2) detected with genetic testing. Molecular analysis of oncogenes, tumor suppressor genes used in diagnosis of tumor (Bcr-Abl, ErbB-2) Molecular markers monitor course of disease during treatment (loss of PML/RAR with treatment).

Molecular Approaches to Cancer Treatment Treat with drugs that inhibit angiogenesis: Blocks proliferation of endothelial cells, less toxic to normal cells. Most cancer drugs damage DNA or inhibit DNA replication, are toxic to normal cells Especially cells continually replaced by division of stem cells (hematopoietic cells, epithelial cells of gastrointestinal tract, hair follicle cells)

New drugs targeted specifically against oncogenes recall proto-oncogenes important roles in normal cells. Acute promyelocytic leukemia (APL):. Gene that encodes retinoic acid receptor (RARa) fused with PML to form PML/RARa oncogene. Treatment with retinoic acid differentiates cells, remission of leukemia in most patients. Breast cancer: Herceptin - monoclonal antibody against ErbB-2 oncogene protein, which is over-expressed in 25–30% of breast cancers (amplification of erbB-2 gene).

Molecular Approaches to Cancer Treatment Small molecule inhibitors of oncogene proteins, protein kinases: Imatinib or Gleevec, specific inhibitor of Bcr/Abl protein kinase, blocks proliferation of chronic myeloid leukemia cells (CML). Imatinib inhibits PDGF receptor and Kit protein-tyrosine kinases: Kit oncogene in most gastrointestinal stromal tumors. Imatinib active against tumors with PDGF receptor activated as oncogene Catalytic domain abl plus imatinib

Molecular Approaches to Cancer Treatment gefitinib and erlotinib, (small molecule inhibitors of EGF receptor), active against some lung cancers. Responsive lung cancers had mutations for constitutive activation EGF receptor tyrosine kinase. Fig. 18.41