Oncogenesis

Molecular Perspectives In Oncogenesis Marigold D.R. Majarucon-Ferrolino, MD , FPCP, FPSMO Department of Biochemistry & Nutrition Our Lady of Fatima University College of Medicine

INTRODUCTION CANCER Cancer is an overgrowth of cells bearing cumulative genetic injuries that confer growth advantage over the normal cells [ Nowell’s Law ] Cancer cells can be characterized as antisocial, fairly autonomous units that appear to be indifferent to the constraints and regulatory signals imposed on normal cells [ Robbin’s ]

CANCER CELLS AND NORMAL CELLS CANCER CELLS NORMAL CELLS Loss of contact inhibition Increase in growth factor secretion Increase in oncogene expression Loss of tumor suppressor genes Oncogene expression is rare Intermittent or co-ordinated growth factor secretion Presence of tumor suppressor genes Normal cell Few mitoses Frequent mitoses Nucleus Blood vessel Abnormal heterogeneous cells

CHARACTERISTICS OF CANCER Clonality Autonomy Anaplasia Metastasis

CHARACTERISTICS OF CANCER Clonality Cancer is a genetic disease at the cellular level. Genetic mutations play a critical role in pathogenesis of cancer. Consequences of genetic instability: Phenotypic heterogeneity Tumor progression Proto-oncogenes and oncogenes Dominant and recessive mutations

Cancer Genetics Tumors arise as clones from a single cell. At the cellular level, cancer is a genetic disease. The development of the malignant clone is due to mutations in DNA due to: Random replication errors Exposure to carcinogens Faulty DNA repair process

Evidence that Mutations Cause Cancer Recurring sites of chromosome change are observed in cancers at sites of genes involved in cellular growth control. Most carcinogens are mutagens. Defects in DNA repair systems increase the possibility of cancer.

Cancer cells are able to proliferate despite regulatory influences. Unrestricted proliferation results in tumor formation. Mechanisms: Growth factor secretion Increased number of cell receptors Independent activation of key biochemical process Proliferation depends on the cell cycle. CHARACTERISTICS OF CANCER Autonomy

AUTONOMY Brought about by mutations in the cell’s genetic apparatus Most common in tissues with rapid turnover, especially: - those exposed to environmental agents - those whose proliferation is hormone-dependent Proliferation is dependent on the cell regeneration cycle

The Cell Cycle G1/S checkpoint G2/M checkpoint DEATH DIFFERENTIATION DNA content = 2n Mitosis M S DNA synthesis G 2 G 1 G 0 DNA content = 4n

CYCLIN, CDK,CDKI: PHOSPHORYLATION

Cell Cycle Regulation Process assures that cell accurately duplicates its contents. Important checkpoints are present at G1 and G2 and are regulated by protein kinases called cyclins (cdk). Checkpoints determine whether the cell proceeds to next phase of the cycle.

G2/M Checkpoint Regulated by the cyclin B/cdc2 (mitosis promoting factor or MPF). Activity of this cyclin with its substrate results in: Chromosome condensation Nuclear membrane breakdown Spindle formation

G1/S Checkpoint Area most often disrupted in cancer. Mechanism of regulation is complex and involves the phosphorylation of the Rb gene. This results in: Activation of several genes needed for S phase progression. Promotes differentiation through association with transcription factors.

Cyclin Regulators Regulated by cdk inhibitors (cdki). May be induced by growth inhibitors and inhibited by positive growth factors. Genetic alterations in cdki occur with high frequency in some cancers.

Cyclin Regulators p 21 : inhibits cell cycle progression and permits DNA repair to take place. P53 : “the guardian of the genome” In the presence of DNA damage, influences transcription to either: Halt cell cycle progression to facilitate DNA repair. In cases of severe DNA damage, activates apoptosis. Mutations in p53 are the most common genetic alterations found in human cancer.

CHARACTERISTICS OF CANCER: Anaplasia Loss of differentiated function resulting to bizarre-looking cells Large nuclei, prominent nucleoli, increased chromatin Increased and/or abnormal mitosis Aneuploidy Partial or complete loss of normal architecture

Invasion and Metastasis The defining characteristic of a malignancy. Invasion: active translocation of neoplastic cells across tissue barriers. Critical pathologic point: local invasion and neovascularization. These events may occur before clinical detection.

ATTRIBUTES OF CANCER Metastasis Two basic steps: Destruction of the BM Attachment to the laminin of distant BM Genes up-regulated among good metastasizers: EDGF receptor Basic Fibroblast Growth Factor Type IV Collagenase  -Cathepsin (under-expressed) Cathepsin B (a lamininase) Heparanase

Angiogenesis Process of new blood vessel formation. Clinical importance: Tumor vessel number correlates positively with risk and degree of dissemination. Several cytokines that stimulate endothelial cell proliferation also stimulate proliferation of malignant cells.

Triad of Invasion Adhesion with the basement membrane. Local proteolysis Mobility and ability to translocate through vents in body’s structural barriers.

MOLECULAR CARCINOGENESIS Mutation the molecular hallmark of cancer Gene Families in Cancer Development 1 - Oncogenes 2 - Tumor Suppressor genes 3 - Mutator genes

Cancer Genes Proto-oncogenes – normally promote normal cell growth; mutations convert them to oncogenes. Tumor suppressor genes – normally restrain cell growth; loss of function results in unregulated growth. Mutator or DNA repair genes – when faulty, result in an accumulated rate of mutations.

ONCOGENE FAMILY + oncogenes Oncogenes promote cell proliferation dominant & highly conserved types : viral oncogenes [ v-oncs ] cellular oncogenes [ c-oncs ] Proto-oncogene  “Mutation”  Oncogene

ONCOGENE FAMILY Classification of Oncogenes A. Secreted Growth Factors B. Cell Surface Receptors C. Intracellular Transducers D. DNA-binding Nuclear Proteins E. Regulators of the Cell Cycle Components of signal transduction pathways c-sis, hst erb B, fms, ret, trk, fes, fms c-src, c-abl, mst, ras myc, jun, fos bcl, bax, bad

ONCOGENE FAMILY Mechanisms of Oncogene Activation 1. Point Mutation H-ras [codon 12] Normal C G C  Gly Bladder ca C T C  Val H-ras GTP Perpetual cell division 2. Gene Amplification Double minutes HSRs Normal copy Multiple copies

ONCOGENE FAMILY Mechanisms of Oncogene Activation 3. Gene Translocation Ex. Burkitt’s Lymphoma

ONCOGENE FAMILY Mechanisms of Oncogene Activation 3. Gene Translocation Ex. Chronic Myelogenous Leukemia [CML]

ONCOGENE FAMILY Mechanisms of Oncogene Activation 4. Viral Gene Integration promoter Viral promoter

TUMOR SUPPRESSOR GENE FAMILY TS Genes inhibit growth and multiplication of mutated cells prevent neoplastic transformation recessive & highly conserved Classification of TS genes A. Cell Adhesion Molecules B. Regulators of the Cell Cycle APC, DCC RB1, Tp53

TUMOR SUPPRESSOR GENE FAMILY KNUDSON’S Two-Hit Hypothesis 1st Hit : TS mutation or Inherited mutation 2nd Hit : gross chromosomal loss

TUMOR SUPPRESSOR GENE FAMILY Retinoblastoma gene [ RB1 gene] rare form of childhood malignancy forms: hereditary & sporadic pRb 105-KDa nuclear protein inhibits E2F [prevents G 1  S transition] inhibited by: phosphorylation viral oncoproteins [E1A, HPV E7]

TUMOR SUPPRESSOR GENE FAMILY Tp53 gene location : 17p13.1 product : p53 protein [53 KDa] function : induces DNA repair or apoptosis mutation : point mutation > deletion results to: loss of function & extended lifespan of p53 Clinical conditions : carcinomas, Li Fraumeni Syndrome p53 inhibited by: E1B, HPV E6, mdm2

TUMOR SUPPRESSOR GENE FAMILY p53 protein

MUTATOR GENE FAMILY Mutator Genes involved in ensuring the fidelity of replication function : checks for & corrects mismatched pairs mutation  inefficient repair & replication leading increased propensity of oncogenes and tumor suppressor genes to undergo mutation first described in E coli [ Mut-HSL system ] Fischel, et al = Human homologs leads to the formation of Microsatellite Instability [MIN+]

In summary ….. ONCOGENES TS GENES MUTATOR GENES

Re-cap of Molecular Carcinogenesis Proto-oncogene Gain-of-function TS gene Loss-of-function Mutator gene Loss-of-function CANCER

CARCINOGENS Occupation related causes Lifestyle related causes Tobacco Diet Sexual practices Multifactorial causes Viral carcinogens Chemical carcinogens Ionizing radiation

Occupational Risk Factors Etiology Arsenic Asbestos Benzene Benzedine Chromium cpds Radiation (mining) Mustard gas Polycyclic hydrocarbons Vinyl Chloride Site of Malignancy Lung, skin, liver Mesothelium, lung Leukemia Bladder Lung Numerous locations Lung Lung, skin Angiosarcoma of liver

Lifestyle Risk Factors Tobacco-related: Lung cancer Pancreatic cancer Bladder cancer Renal cancer Cervical cancer

Diet-Related Risk Factors Nitrates Salt Low vitamins A, C, E Low consumption of yellow-green vegetables Gastric Cancer Esophageal Cancer

Diet-Related Risk Factors High fat Low fiber Low calcium High fried foods Colon Cancer Pancreatic Cancer Prostate Cancer Breast Cancer Uterine Cancer Mycotoxins Liver Cancer

Sexual Practices Risk Factors Cervical Cancer Sexual promiscuity Multiple partners Unsafe Sex Human Papillomavirus

Multifactorial Factors Tobacco + Alcohol Oral Cavity Cancer Esophageal Cancer Tobacco + Asbestos Tobacco + mining Tobacco + uranium + radium Respiratory Tract Cancer Lung Cancer

CARCINOGEN METABOLISM Three Main Categories: I. Chemical Carcinogens II. Physical Carcinogens III. Viral Agents Carcinogens Mutations Cancer Environmental factors ?

CHEMICAL CARCINOGENESIS Stages: Initiation - primary exposure Promotion - transformation Progression - Cancer growth Frank Cancer

CHEMICAL CARCINOGENESIS Initiation normal cells are exposed to a carcinogen not enough to cause malignant transformation requires one round of cell division normal cells are exposed to a carcinogen 1. Direct-acting carcinogens 2. Indirect-acting carcinogens procarcinogen Cytochrome P450 Ultimate carcinogen

CHEMICAL CARCINOGENESIS Promotion initiated cells are exposed to promoters promoters are not carcinogens ! properties of promoters reversible dose-dependent time-dependent Types of Carcinogens …... 1. Direct carcinogens 2. Procarcinogens  Ultimate carcinogens

CHEMICAL CARCINOGENESIS Direct-acting Carcinogens cyclophosphamide chlorambucil busulfan melphalan Procarcinogens PAHs Aromatic amines & Azo dyes Aflatoxin B1 Nitrosamine & Amides Asbestos Vinyl chloride Chromium, nickel, other metals Arsenic Promoters saccharine & cyclamates Estrogen Diesthystilbestrol [DES]

Physical Carcinogenesis Radiation-induced mutation in the host cell Transmits irreversible changes in gene expression to cell progeny

Sources of Potentially Carcinogenic Radiation Sunlight Artificial sources of UV light X-rays Radio-chemicals Nuclear fission

PHYSICAL CARCINOGENESIS Ultraviolet Rays UV-A = 320 - 400 nm UV-B = 280 - 320 nm UV-C = 200 - 280 nm

PHYSICAL CARCINOGENESIS Ultraviolet Rays UV-C  filtered by ozone UV-B Inhibition of cell division inactivation of enzymes induction of mutations cell death at high doses Squamous cell cancer Basal cell cancer Melanocarcinoma

PHYSICAL CARCINOGENESIS Ionizing Radiation includes electromagnetic rays & particulate matter mechanism:  free radicals & mutations pathology: leukemias > thyroid ca > lung & breast ca resistant tissues: bone, skin and the GIT PRE -IRRADIATION POST -IRRADIATION

Viral Carcinogenesis Viral carcinogens are classified into RNA and DNA viruses. Most RNA oncogenic viruses belong to the family of retroviruses that contain reverse transcriptase mediates transfer of viral RNA into virus specific DNA.

Viral Oncogenes RETROVIRUS Oncogene Viral RNA Viral DNA NUCLEUS DNA Oncogene REVERSE TRANSCRIPTASE INSERTION TRANSCRIPTION Oncogene Viral RNA CELL MEMBRANE CYTOPLASM Viral genome RNA messenger TRANSCRIPTION Oncogene protein

Viruses Associated With The Development Of Human Neoplasia VIRUSES NEOPLASMS DNA VIRUSES Human papilloma virus Cervical Ca, warts, ano- genital carcinoma Herpes simplex virus II Cervical carcinoma Epstein-Barr virus NPCa, African Burkitt’s Human Herpes virus 8 Kaposi’s sarcoma Hepatitis B virus Hepatocellular Ca Herpes simplex virus 6 Certain B cell (HBLV) lymphomas

VIRUSES NEOPLASMS RNA VIRUSES Human T-cell leukemia virus I Some T-cell leukemia, lymphoma Human T-cell leukemia virus II Some cases of hairy cell leukemia Human immunodeficiency virus I Lymphoma; Kaposi’s sarcoma Viruses Associated With The Development Of Human Neoplasia

VIRAL AGENTS: DNA viruses Human Papillomavirus [HPV types 16, 18, 31, 33 & 35] Interruption of the E1/E2 ORF E2 is not expressed Over-expression of E6 & E7

VIRAL AGENTS: DNA viruses Epstein-Barr Virus [EBV] in Burkitt’s, B-cell & Hodgkin’s lymphomas + NP ca tropism: CD21+ cells [e.g., B cells, epithelial cells] mechanism: viral entry  episomal existence  latency  (+) LMP-1 , EBNA-1 , EBNA-2  immortalization Hepatitis B virus [HBV] induction of chronic hepatocyte injury  (+) HBx HBx activates protein kinase c for transformation

VIRAL AGENTS: R NA viruses Human T-cell Leukemia Virus [HTLV] a retrovirus tropism: CD4+ cells mechanism: Tax protein  transcription  c-fos, c-sis, IL-1 and IL-2 Viral replication T cell proliferation

Principal Pathways of Malignancy 1. Proliferation 2. Cell-Cycle Progression 3. DNA Repair 4. Immortalization 5. Apoptosis 6. Angiogenesis 7. Metastasis and Invasion

PROLIFERATION (Growth Factor Signaling Pathway) Uncontrolled and uncoordinated proliferation Uncontrolled growth stimulated by: Increased secretion of Growth Factors (PDGF,EGF,FGF,VEGF,NGF) Increased Growth Factor receptors Independent activation of certain enzyme and protein production pathways

PROLIFERATION (Growth Factor Signaling Pathway) Receptor Tyrosine kinase Pathway (RTK)- Main pathway RTK ligands: NGF PDGF FGF EGF Functions of RTK: promotion of cell survival regulation of cell proliferation and differentiation modulation of cellular metabolism

PROLIFERATION (Growth Factor Signaling Pathway) RTK SIGNALING PATHWAYS Ras-MAP Kinase Pathway- most prominent PI3 kinase Pathway Phospholipase C Pathway

PROLIFERATION (Growth Factor Signaling Pathway) Therapeutic implications Blocking of GF mitogenic signaling is achieved by: Preventing binding of GF to receptor or receptor dimerization with specific agent Preventing receptor activation with small molecule inhibitors Blocking cytoplasmic proteins downstream of the activated receptor pathway

Cell Cycle Regulation Process assures that cell accurately duplicates its contents. Important checkpoints are present at G1 and G2 and are regulated by proteins Cyclins and Cyclin-dependent Kinases (CDKs). Checkpoints determine whether the cell proceeds to next phase of the cycle.

Cyclins and Cyclin-dependent Kinases (CDKs) CYCLINS – activate protein kinases CDKs – protein enzymes which selectively phosphorylate specific serine/threonine residues in their substrates Dimeric complex with catalytic subunit (CDK 1-9) regulatory subunit (Cyclin A-H,T)

G2/M Checkpoint Regulated by the cyclin B/cdc2 (mitosis promoting factor or MPF). Regulated mainly by intracellular signal (Completion of DNA Synthesis) MPF is activated by dephosphorylation by cdc25 Cyclin B is degraded by Anaphase Promoting Complex (APC) Role of G2/M checkpoint: to prevent mitosis when DNA is damaged and not yet repaired

G1/S Checkpoint Area most often disrupted in cancer. Mechanism of regulation is complex and involves the phosphorylation of the Rb gene. Regulated by extracellular signals (e.g. GF) “ R” point (restriction)- point late in G1 beyond which cell cycle progression becomes independent from external GF Regulated mainly by CDK4/cyclin D

Cyclin Regulators- CDK Inhibitors CDK inhibitors – inhibit the activity of CDK-cyclin complex Two Groups: 1) INK4 family – p15 16 18 19 2) CIP-KIP family – p21 p27 Actions: P15- change response to anti-mitogenic agents P16- inhibits CDK4/cyclin D P19- induces p53 stabilization P21-induces cell cycle arrest via activation by p53 P27- inhibits CDK2/cyclin E

Cyclin Regulators p 21 : activated by p53 inhibiting cell cycle progression and permitting DNA repair to take place. P53 : “the guardian of the genome” In the presence of DNA damage, influences transcription to either: Halt cell cycle progression to facilitate DNA repair. In cases of severe DNA damage, activates apoptosis. Mutations in p53 are the most common genetic alterations found in human cancer.

Clinical Significance Oncogenic alterations in cell cycle regulators: Loss of p53 and pRB function as tumor suppressors Increased expression of Cyclin D1(Mantle Cell Lymphoma) CDK4 amplification in sarcomas, glioma Mutations in p16-binding domain of CDK4(Familial Melanoma) Inactivation of INK4 Alterations in Cyclin D1,p16 Decreased levels of p27 (Breast Ca) Over expression of cdc25

Therapeutic Implications Approaches using Inhibitors of CDKs as therapeutic agents Small molecules Protein therapy Antisense Gene therapy Most cytotoxic agents block the cell cycle in the S/G2/M phases

DNA REPAIR PATHWAYS Cancer as “Malady of Genes” Defects in the maintenance of genome stability Repair Mechanisms: Mismatch excision repair Base excision repair Nucleotide excision repair Double strand base repair

DNA REPAIR PATHWAYS Clinical Significance HNPCC – mutations in genes involved in DNA repair pathways (MSH1 MSH2) Somatic defects in repeated DNA elements leading to Microsatellite instability (MSI) Inactivation of TGF- β (tumor suppressor) Inactivation of BAX gene

Telomeres and Telomerase Telomeres - specialized structures at chromosome ends generated and maintained by telomerase Telomerase - ribonucleoprotein enzyme which preserves the integrity of telomeres * key component in immortalization of cancer cells Telomere length- represents a molecular clock that determines the life span of the cell

Telomeres and Telomerase Clinical Significance Most normal adult tissues have NO telomerase activity Telomerase activity is present in 90% of tumors Therapeutic Implication hTERT - protein identified to be catalytic subunit of telomerase limiting component of telomerase activity can be a target for small molecule inhibitor

APOPTOSIS APOPTOSIS – programmed cell death Important in: Steady-state kinetics of normal tissues Focal deletion of cells during normal embryonic development Seen after chemotherapy and radiation * Balance between proliferation and apoptosis is critical in determining growth or regression

Components of Apoptotic Pathway 1) CASPASES (Cysteine-containing aspartate-specific proteases) Initiator Caspases – activated in response to cell death signal Executioner or Effector Caspases- progress the death signal activating cascade resulting to DNA fragmentation and cell death Caspase prodomains – DED CARD Death ligands – TNF- α , Fas , TRAIL Survival Signals – NF κβ

Components of Apoptotic Pathway 2) CYTOCHROME C – component of mitochondria released in response to apoptotic signals 3) BCL-2 Family of Proteins- located upstream in the pathway Provides pivotal decisional checkpoint in the fate of the cell after a death stimulus Contains BH1-BH4 domains necessary for interaction Anti-apoptotic – BCL-2 BCL-xL Pro-apoptotic – BAX BAD BAK BID

APOPTOTIC PATHWAYS 1) FAS-mediated apoptosis FAS – cell surface receptor of TNF family which binds to FAS-L Eliminates unwanted activated T cells Pathway for cytotoxic-mediated signaling 2) P53-mediated apoptosis important after chemotherapy and radiation Induction of BAX and downregulation of BCL-2 Induced expression of FAS and DR5

Clinical Significance Over expression of BCL-2 as a prognostic indicator Mutations of BAX in GI Ca and leukemias P53 provides a link between cell proliferation and apoptosis Cell survival signals: NF κβ BCL-2 P53 mutations confer chemoresistance

Therapeutic Implications Antisense oligonucleotide against BCL-2 in the treatment of lymphoma BCL-2 antisense as chemosensitizing agent in solid tumors TRAIL ( TNF-related apoptosis inducing ligand) to induce apoptosis

ANGIOGENESIS Formation of new blood vessels from existing vascular bed Carried out by endothelial cells (EC) and extra cellular matrix (ECM) Regulated by angiogenic factors (inducers and inhibitors) * A tumor is unable to grow larger than 1 mm3 w/o developing a new blood supply

Components of Angiogenesis ENDOTHELIAL CELLS Fenestrated Increased cell adhesion molecules ( E-selectin) Increased integrins αγβ 3 essential for viability during growth Activated ECs release: bFGF PDGF IGF-1

Components of Angiogenesis 2) INDUCERS OF ANGIOGENESIS VEGF – main inducer TGF- β TNF- α low concentration - inducer high concentration - inhibitor PDGF/thymidine phosphorylase TGF- α EGF IL-8

Components of Angiogenesis 3) CELL ADHESION MOLECULES (CAM) Mediate cell-cell adhesion processes Selectins IG Supergene family- ICAM VCAM Cadherins Integrins- vitronectin receptor 4) PROTEASES Degrade ECM to provide suitable environment for EC migration thru adjacent stroma Ex: Metalloproteinases (MMP)

Components of Angiogenesis ANGIOGENESIS INHIBITORS Interferon TSP-1 Angiostatin Endostatin Vasostatin CLINICAL SIGNIFICANCE : Tumor angiogenesis switch is triggered as a result of shift in the balance of stimulators to inhibitors

Therapeutic Implications Metalloproteinase inhibitors (MMPI) – block the degradation of basement membrane Inhibitors of endothelial function- thalidomide, TNP 470,endostatin Anti-angiogenic factors – tyrosine kinase inhibitors of VEGF bFGF PDGF Interferon – angiogenic inhibitor COX-2 inhibitor – thromboxane A2 as critical intermediary of angiogenesis

Triad of Invasion Adhesion with the basement membrane. Local proteolysis Mobility and ability to translocate through rents in body’s structural barriers.

ADHESION De-regulated function of CAM (E-cadherin) Changes in catenin expression leads to loss of cadherin function Integrin over expression in naturally occurring cancers Downregulation of integrin in more advanced stages of cancer Upregulation of ICAM-1 which enhances extravasation Adhesion molecules on EC: E-selectin,VCAM ICAM

LOCAL PROTEOLYSIS Degradation of basement membrane to traverse barriers Carried out by: Serine proteases -uPA elastase plasmin cathepsin G Cysteine proteases- cathepsin B L Aspartate proteases – cathepsin D Matrix metalloproteinases- gelatinases interstitial collagenases stromelysins matrilysins

MOTILITY Tumor cells can move randomly or directionally toward attractants Modulators of motility GF, hyaluronases, components of ECM, tumor-secreted factors, host-derived factors THERAPEUTIC IMPLICATIONS : MMPI and monoclonal antibodies against integrin

Oncogenesis

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