TUMOR VIRUSES AND ONCOGENES

1. TUMOR VIRUSESAND ONCOGENES Mohammed El-Khateeb DSVL-4 April7th2015

2. Types of Cancers • Leukemias (derived from lymphoid cells) • Carcinomas (derived from epithelial or endothelial cells) • Sarcomas (derived from connective tissue cells)

3. Human Cancer Viruses • Contributing factor in at least 15% of human cancers worldwide caused by viruses • Major cause of liver & cervical cancer

4. Characteristics of Cancer Cells • Undergo unregulated growth • Become immortal (active growth when they should be quiescent) • Have increased nutrient uptake • In tissue culture become anchorage independent. • The cell cycle in cancer cells becomes active • Growth signaling pathways activated (oncogenes – RNA tumor viruses) • Pathways to prevent cell proliferation are disrupted (tumor suppressors – DNA tumor viruses)

5. Transformed cells forming foci (clusters) due to loss of cell-contact inhibition

6. Cancer • Activated oncogenes transform normal cells into cancerous cells. • Transformed cells have increased growth, loss of contact inhibition, tumor specific transplant and T antigens. • The genetic material of oncogenic viruses becomes integrated into the host cell's DNA.

7. Clonality of Tumors • Tumors induced by acutely transforming viruses arepolyclonalin nature, that is, many cells lose growth controlas a result of infection with an oncogene-bearing virus • Tumors caused by slow transforming viruses aremonoclonalor possiblyoligoclonalin nature, they occur asa result of an outgrowth of a single rare cell with virusintegrated into a specific site near or within a proto-oncogene • The clonality of a tumor thus provides clues to themechanisms of tumor induction

8. The hallmarks of cancer comprise six biological Capabilities acquired during the multistep development of human tumors. • Sustaining proliferative signaling • Evading growth suppressors, • Resisting cell death, • Enabling replicative immortality, • Inducing angiogenesis, • Activating invasion and metastasis.

9. HPV SV-40 BK, JC HBV EBV KSHV HTLV-1 Hepatitis C virus Taxonomy of Tumor Viruses • DNA viruses:PapovavirusesHepadnaviruses HerpesvirusesAdenoviruses Poxviruses • RNA viruses: Retroviruses Flaviviruses

10. Tumor Viruses For most viruses: Replication Lysis Progeny virions Lytic Life Cycle Genome all viral proteins

11. Virus Cell Integration (usually) Transformation Tumor Viruses Latent Life Cycle Some virus-specific proteins expressed - No mature virus Viral structural proteins are not expressed Sometimes latency may terminate – cell must be infected by complete virus Changes in the properties of host cell - TRANSFORMATION

12. Tumor Viruses • Transformation: • Loss of growth control • Reduced adhesion • Motility • Invasion • Ability to form tumors - viral genes interfere with control of cell replication • Transformed cells frequently exhibit chromosomal aberrations

13. Oncogenic Viruses • Oncogenic DNA Viruses • Adenoviridae • Heresviridae • Poxviridae • Papovaviridae • Hepadnaviridae • Oncogenic RNAviruses • Retroviridae • Viral RNA is transcribed to DNA which can integrate into host DNA • HTLV 1 • HTLV 2

14. Human Viruses and Associated Malignancies • HPV 16, 18, 31, 33, 45 Cervical Carcinoma • Hepatitis B&C viruses Hepatocellular Carcinoma • HTLV1 Adult T cell Leukemia • Epstein-Barr virus (HHV-4) Burkitt’s Lymphoma • Hodgkin’s Disease • PTLD • Nasopharyngeal Carcinoma • Gastric Carcinoma? • Kaposi sarcoma-associated Kaposi’s Sarcoma herpesvirus (KSHV, HHV-8)

15. TRANSFORMATION Both DNA and RNA tumor viruses can transform cells Integration occurs (usually) Similar mechanisms VIRAL TRANSFORMATION The changes in the biological functions of a cell that result from REGULATION of the cell’s metabolism by viral genes and that confer on the infected cell certain properties characteristic of NEOPLASIA These changes often result from the integration of the viral genome into the host cell DNA

16. Two Major Classes of Tumor Viruses DNA Tumor Viruses DNA viral genome DNA-dependentDNA polymerase(Host or viral) Host RNA polymerase Viral mRNA Viral protein

17. Proto-oncogenes  oncogenes: • Proto-oncogenes • Proto-oncgenes are genes that possess normal gene products and stimulate normal cell development. • Oncogenes • Oncogenes arise from mutant proto-oncogenes. • Oncogenes are more active than normal or active at inappropriate times and stimulate unregulated cell proliferation. • Some tumor viruses that infect cells possess oncogenes: • RNA tumor viruses = possess viral oncogenes (derived form cellular proto-oncogenes) capable of transforming cells to a cancerous state. • DNA tumor viruses = another class of tumor viruses; do not carry oncogenes, but induce cancer by activity of viral gene products on the cell (no transformation per se).

18. Definitions Oncogene:“Gain of function” An altered gene whose product can act in a dominant fashion to help make a cell cancerous. Usually, an oncogene is a mutant form of a normal gene (a “proto-oncogene”) involved in the control of cell growth or division. Tumor Suppressor gene: “Loss of function” A gene whose normal activity prevents formation of a cancer. Loss of this function by mutation enhance the likelihood that a cell can become cancerous (a normal process to maintain control of cell division is lost).

19. DNA tumor viruses target tumor suppressors Virus Gene Product Cellular target Adenovirus SV40 Polyomavirus Papillomavirus E1AE1B Rb p53 Large T antigen Large T antigen Middle T antigen Rb, p53 Rb Src, PI3K E7 E6 E5 Rb PDGF receptor

20. DNA Virus Transforming Activities via Cellular Homologues • EBV LMP1 mimics CD40 (tumor necrosis factor receptor) • E5 gene of bovine papillomavirus is molecular mimic of growth factor (activates PDGF receptor signaling cascade) • Polyomavirus middle T: src signaling pathway • HHV 8: Encodes viral D cyclin, vIL-6

21. Human Papillomaviruses • Virus: 55 nm diameter • Genome: ds DNA, circular, 8 kbp • Highly tropic for epithelial cells of the skin and mucous membrane. • Viral replication is strictly associated with the differentiated keratinocytes. • Papillomaviruses causes warts, including skin warts, plantar warts, flat warts, genital condylomas and laryngeal papillomas.

22. Human Papillomaviruses • HPVs are accepted as the cause of anogenital cancers including cervical cancer. • Cervical cancer is caused most commonly by HPV-16 and -18 (high risk types) and less commonly by types 31, 33, 35 and 45. Types 6 and 11 are considered low risk types causing benign tumors. • Integrated copies of viral DNA are present in cancer cells. HPV DNA is episomal in non cancerous cells or pre-malignant lesions.

24. Progression of Cervical Carinogenesis

25. Herpesviruses • Large viruses (100 – 200 nm diameter), enveloped. • Linear ds DNA genome (124 – 235 kpb). • Causes acute infections followed by latency. • EBV causes acute infectious mononucleosis when it infects B lymphocytes of susceptible humans. EBV can immortalize such lymphocytes. • Pthogenesis of EBV IN: ImmunocompetentImmunocompremised • Burkitt’s lymphomaPost-transplantlymphoproliferative diseases (PTLD) • Nasopharyngeal carcinoma • Hodgkin’s diseaseHodgkin lymphoma Non-Hodgkin’s lymphoblastoid malignancies

26. Herpesviruses • EBV encodes a viral oncoproteins (LMP1) that mimics an activated growth factor receptor that is essential for transformation of B lymphocytes. EB nuclear antigens (EBNAs) are also essential for immortalization of B cells • Most of these tumors show chromosomal translocations between the c-myc gene and immunoglobulin loci, leading to the constitutive activation of myc expression • Human herpes virus 8 (HHV-8) is associated with Kaposi’s sarcoma.

27. EPSTEIN-BARR VIRUS • EBV has a very limited host range and tissue tropism defined by the limited cellular expression of its receptor (CD21). • This receptor is expressed on • B lymphocytes • Epithelial cells of the oro – and nasopharynx • Diseases • Infectious Mononucleosis • African Burkitt’s Lymphoma • Nasopharyngeal Carcinoma • EBV-induced lymphoproliferative disease

28. African (endemic) Burkitt’s lymphoma • Poorly differentiated monoclonal B-cell lymphoma • The tumor cells contain chromosomal translocations that moves the C-myc oncogene to a very active promoter. (Immunoglobulin gene promoter) • African (endemic) Burkitt’s lymphoma • Poorly differentiated monoclonal B-cell lymphoma • Affecting the Jaw and face • Endemic to children of malarial regions of Africa. • The tumor cells contain chromosomal translocations that moves the C-myc oncogene to a very active promoter. (Immunoglobulin gene promoter)

29. * In Burkitt's lymphoma, the c-myc on chromosome 8 is brought to a site on chromosome 14 close to the gene for immunoglobulin heavy chains. It seems that the proto-oncogene may thus be brought under the control of the Ig promotor, which is presumably very active in B lymphocytes.

30. THE LATENT CYCLE EBV in saliva B cells proliferation T cells activation Liver Lymph node Spleen Epithelial cells of oropharynx Shedding in saliva Atypical lymphocytes Heterophile antibodies swelling Pharyngitis

31. THE LATENT CYCLE EB nuclear antigen 1 (EBNA) EBNA-2 Viral promoter (ori P) Monoclonal antibodies Heterophile antibodies B cellimmortalization • Antibodies to EBNA persist for life. • Antibodies to viral capsid antigen (VCA)appear during active • disease. • CD8+ T cells are activated against EBNA proteins • Destroy infected B cells • Atypical lymphocytes T cell immunodeficiencies B cell lymphoma

32. EBV Latency Genes • Non-antigenic:EBNA1 (Epstein Barr Nuclear Antigen 1) - episomal replication and segregation function • Antigenic • EBNA2 • EBNA3A, 3B, 3C • EBNA-LP • LMP1 (Latent Membrane Protein 1) • LMP2A • 4 different types of latency • True Latency - no viral gene expression • EBNA1 only - EBNA1 (non-antigenic) • Default - EBNA1, LMP1, and LMP2 (moderately antigenic) • Growth - EBNA1, LMP1, LMP2, EBNA2, EBNA-LP, EBNA3A, 3B, 3C (highly antigenic)

34. Kaposi’s sarcoma Kaposi’s Sarcoma Herpes Virus - HHV-8 • Hematologic malignancies • Primary effusion lymphoma • MulticentricCastleman's disease (MCD) – a rare lymphoproliferative disorder (AIDS) • MCD-related immunoblastic/plasmablastic lymphoma • Various atypical lymphoproliferative disorders

35. Hepatitis B virus • Epidemiology: • Vast public health problem • 10% of population in underdeveloped countries are chronic carriers • Long latency • Strong correlation between HBV andhepatocellular carcinoma • China: 500,000 - 1 million new cases of hepatocellular carcinoma per year • Taiwan: Relative risk of getting HCC is 217 x risk of non-carriers

36. HBV DNA genome RNA polymerase II RNA Provirus Reverse transcriptase DNA genome Hepatitis B Virus Host enzyme Viral enzyme

37. Adenoviruses • Highly oncogenic in animals • Only part of virus integrated • Always the same part • Early functions • E1A region: 2 T antigens • E1B region: 1 T antigen • E1A and E1B = Oncogenes

38. RNA TUMOR VIRUSES

39. RNA Tumor Viruses Viral RNA genome Reverse transcriptase (Virus-encoded) Viral DNA genome (integrated) DNA-dependent RNA polymerase (Host RNA pol II) Viral genomic RNA Splicing (Host splicing enzymes) messenger RNA viral protein Virus IMPORTANT Important: Use HOSTRNA polymerase to make its genome An enzyme that normallymakes mRNA

40. Retrovirus Lifecycle Simple retrovirus • LTR-gag-pol-env-LTR

41. Retroviruses • RNA tumor viruses “create” oncogenes by acquiring, modifying, deregulating cellular genes (proto-oncogenes) • v-onc not essential viral gene & unrelated to strategy of viral replication • Replication of RNA viruses is not cytocidal nor is it required for tumorigenesis

42. Mechanisms of cell transformation by retroviruses 1) Retroviral transduction of oncogene (transducing retrovirus) 2) Oncogene activation by retroviral insertion (cis-acting / nontransducing retrovirus) 3) Oncogenesis mediated by essential retrovirus proteins (trans-activating / nontransducing long-latency retrovirus)

43. v-ONC c-ONC Transducing retroviruses • Viral acquisition of cellular proto-oncogene with capacity to transform if deregulated, usually replacing viral coding sequences (exception is RSV=src oncogene) • Overexpression versus structural change in v-onc mos vs src • Becomes replication defective, secondary to the loss of viral coding information; requires helper virus Host DNA cell

44. Acquired Genes Are Components of Signaling Networks • External signal molecules or growth factors (receptor ligands) (sis) • Cellular receptors (erbB, fms, kit) • Second messengers in signaling cascade (kinases: src, abl, fgr, yes; mos raf) • Transcription factors (jun, fos, myc, myb, ets, rel)

45. Outcome of Retroviral Transduction • “Single hit” carcinogenesis (one event) • Polyclonal: tumor growth initiated in every infected cell • Tumors form within days • Characteristic of animal retroviruses

46. Mechanisms of cell transformation by retroviruses 1) Retroviral transduction of oncogene (transducing retrovirus) 2) Oncogene activation by retroviral insertion (cis-acting / nontransducing retrovirus) 3) Oncogenesis mediated by essential retrovirus proteins (trans-activating / nontransducing long-latency retrovirus)

47. Outcome of Oncogene Activation by Retrovirus Insertion • Cell transformation rare event because insertion near potential oncogenes is infrequent • Monoclonal tumors: proviral sequences integrated at same chromosomal site • Tumors induced more slowly (months) since tumor derived from single cell

48. Human T cell Leukemia Virus type I (HTLV-I) • Associated with 2 fatal human diseases • Adult T cell leukemia (ATL) • clonal malignancy of infected mature CD4+ T cells • Tropical spastic paraparesis/HTLV-1 associated myelopathy • neurodegenerative disease • Endemic in parts of Japan, South America, Africa, and the Caribbean • With an estimated 10-20 million people infected worldwide • Asymptomatic in majority of individuals with approximately 2-5% of HTLV-I carriers developing disease 20-40yrs post infection. • The long clinical latency and low percentage of individuals who develop leukemia suggest that T-cell transformation occurs after a series of cellular alterations and mutations. • Infects primarily CD4+ T cells.

49. HTLV 1 Transmission • Extended close contact (cell-associated virus) • Sexual (60% male to female versus 1% female to male transmission) • Blood products (screening of blood supply since 1988) • Mother to child (breast feeding: 20% children with seropositive mothers acquire virus)

50. HTLV-I and ATL • 1980 Gallo isolated type C retrovirus (HTLV1) from patient with “cutaneous T cell lymphoma” • The provirus is present in all cases ATL • Integration occurs at the same site in all cells derived from an ATL tumor (monoclonal). • Integration site varies in different patients • Integration does not occur at a preferred chromosomal site (no cis-activation of oncogenes).