Origin of oncogenes? Oncogenes and Proto-oncogenes. Jekyll and Hyde. Oncogene hypothesis. Retroviral oncogenes and cell proto-oncogenes

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1 Oncogenes and Proto-oncogenes Jekyll and Hyde A double edged sword Origin of oncogenes? Oncogene hypothesis Retroviral oncogenes and cell proto-oncogenes (v-onc) (c-onc) The role of c-onc in cancer How many c-onc in human genome?

2 Tumor viruses and cancer The viral origin of the majority of all malignant tumors..have now been documented beyond any reasonable doubt. It..would be rather difficult to assume a fundamentally different etiology for human cancer. Ludwik Gross 1970 Endogenous retroviruses and cancer

3 Epidemiological observation of cancer is inconsistent with viral etiology for human cancer Two schools of thought from virologists firmly believed in viral etiology: Can reactivation of endogenous viruses cause cancer? Ans: Could be in some cases in animals, but no evidence in human Can reactivation of integrated viral oncogene(s) cause cancer? Ans: No such evidence in human cancer Murine versus Human endogenous viruses Murine: 4-5% of the genome *Some endogenous viruses are infectious. *Continue to be infected in recent evolution Human: 1% of the genome No infectious endogenous viruses have been found so far. Pattern of human endogenous retroviral sequences are fixed, and no evidence of recent infection in evolution.

4 Origin of Endogenous viruses Murine Human Figure 4.1 The Biology of Cancer ( Garland Science 2007) Figure 4.1a (part 1 of 2) The Biology of Cancer ( Garland Science 2007)

5 Figure 4.1a (part 2 of 2) The Biology of Cancer ( Garland Science 2007) Figure 4.1b The Biology of Cancer ( Garland Science 2007)

6 Figure 4.1c The Biology of Cancer ( Garland Science 2007) Gene transfer technology and cancer 1972 Calcium phosphate DNA transfection method Cooper and Temin RSV transformed cells ---> DNA ---> NIH 3T3 cells --- > transformation MC (carcinogen) ---> C3H 10T1/2 cells transformation---> isolate DNA ---> transfect NIH 3T3 cells ---> transformation

7 DNA transfection using chemically transformed cells and Subsequent tumor formation in mouse using the cells Figure 4.2 The Biology of Cancer ( Garland Science 2007) Figure 4.2 (part 1 of 2) The Biology of Cancer ( Garland Science 2007)

8 Figure 4.2 (part 2 of 2) The Biology of Cancer ( Garland Science 2007) T24 bladder carcinoma cells DNA transfected NIH 3T3 A transformed focus Magnification of the focus Surrounding Normal cells Figure 4.3 The Biology of Cancer ( Garland Science 2007)

9 How many oncogenes are needed to transform cells? One hit, two hits or multiple hits phenomenon? * Only 0.1% of a genome set was transfected to induce transformation. If one hit: probability : yes If two hits: probability : no Any other way to show the hitness? (Ans: serial dilution kinetics) Human cancer genes = viral oncogenes?

10 DNA blotting and hybridization Mid 1970 s: Southern blot Figure 4.4 The Biology of Cancer ( Garland Science 2007)

11 Figure 4.4 (part 1 of 4) The Biology of Cancer ( Garland Science 2007) Figure 4.4 (part 2 of 4) The Biology of Cancer ( Garland Science 2007)

12 Figure 4.4 (part 3 of 4) The Biology of Cancer ( Garland Science 2007) Figure 4.4 (part 4 of 4) The Biology of Cancer ( Garland Science 2007)

13 Southern blot: DNA Northern blot: RNA Western blot: protein Eastern blot:? (carbohydrates) T24 DNA transformed NIH 3T3 lines Southern blot H-ras probe Figure 4.5 The Biology of Cancer ( Garland Science 2007)

14 Amplification of proto-oncogenes in cancer Myc: promyelocytic leukemia line HL60 N-Myc: neuroblastoma ErbB (EGFR): stomach, lung, brain, and breast cancers ErbB2 (HER2): 25 to 30% breast cancer Table 4.1 The Biology of Cancer ( Garland Science 2007)

15 Human breast carcinoma with amplified ErbB2/Neu Southern blot using ErbB2 probe Figure 4.6a The Biology of Cancer ( Garland Science 2007) Kaplan-Meier plot Figure 4.6b The Biology of Cancer ( Garland Science 2007)

16 Amplified ErbB2 gene Elevated ErbB2 transcription Elevated ErbB2 translation Figure 4.6c The Biology of Cancer ( Garland Science 2007) Gene cluster analysis: 160 genes on 17q 25% Figure 4.7 The Biology of Cancer ( Garland Science 2007)

17 Activation of proto-oncogenes by point mutation(s) Early 1980 s: Cloning and sequence analysis of bladder and colon cancer genes: H-Ras; K-Ras Codon 12, 61 and 13 (less frequent) Sequential transfection with human cancer cells DNA Figure 4.8 The Biology of Cancer ( Garland Science 2007)

18 Localization of an activating oncogene in human cancer DNA Figure 4.9 The Biology of Cancer ( Garland Science 2007) H-ras oncogene activation: codon 12 Gly Val Figure 4.10 The Biology of Cancer ( Garland Science 2007)

19 Table 4.2 The Biology of Cancer ( Garland Science 2007) Homogeneously Staining regions (HSRs) and Double minutes (DMs) HSRs: tandem gene amplification in the chromosome DMs: amplified genes broken off from chromosome

20 N-myc in Neuroblastoma FISH analysis HSRs DMs Figure 4.11a The Biology of Cancer ( Garland Science 2007) Kaplan-Meier plot Figure 4.11b The Biology of Cancer ( Garland Science 2007)

21 Table 4.3 The Biology of Cancer ( Garland Science 2007) Gene translocation C-myc translocation: Burkitt s lymphoma Mimicking promoter insertion activation of c-myc transcription in chicken leukemia

22 Figure 4.12 The Biology of Cancer ( Garland Science 2007) Figure 4.13a The Biology of Cancer ( Garland Science 2007)

23 Chromosomal translocation Figure 4.13b The Biology of Cancer ( Garland Science 2007) C-Myc translocation: transcriptional activation Ig heavy chain : 75% Ig lambda chain: 16% Ig kapa chain: 9%

24 What is the role of EBV in Burkitt s lymphoma? Hypotheses: Promoted immune response of B-cell gene rearrangement myc translocation Chronic infection viral reactivation chromosomal instability What is the role of malaria in Burkitt s lymphoma? Hypothesis: Malaria CIDR-1 EBV reactivation

25 C-myc in cancer Promoter insertion Gene amplification Chromosomal translocation Table 4.4 The Biology of Cancer ( Garland Science 2007)

26 Why gene translocation often seen in leukemia? CML Philadelphia chromosome Figure 4.15a The Biology of Cancer ( Garland Science 2007)

27 Chromos 9 Chromos 22 Figure 4.15b The Biology of Cancer ( Garland Science 2007) Table 4.5 The Biology of Cancer ( Garland Science 2007)

28 Activation of cellular tyrosine kinases Non-receptor tyrosine kinases: ex Src Receptor tyrosine kinases: ex EGFR Src Protein N unique SH3 SH2 PTK/SH1 regulatory C membrane binding myristoylation site K295 ATP binding Y416 (+) Y527 ( )

29 Figure 4.14 The Biology of Cancer ( Garland Science 2007) Autoinhibition of Cytoplasmic PTK and PTP

30 PTK Receptors & Oncogenes Kinase src fps fes yes fgr abl fyn syn lyn hck lck tkl. etc EGFR neu v-erbb sea eph ltk. etc InsR IGF-1R met ros trk PDGFRs v-ros v-fms Flk CSF1R Flt kit ret Tie1 Tie2 flt FGFR (3 loops) flg ( ) bek ( ) Location and Function of Retroviral Oncogene Products PDGF sis Receptor PTKs: erbb2, fms, (ros), (kit) Kinase GTP binding: K-ras, H-ras Serine/Threonine Kinases: mos, rel, mil/raf Kinase Intracellular Non-receptor PTKs: src, fps/fes, yes, fgr, abl Extracellular DNA binding; Transcription Factors: myc, fos, rel, ski, myb, jun, ets Nucleus

31 Products and Functions of Various Oncogenes Growth factors: ex.sis of Simian sarcoma virus (SSV) Growth factor receptors: receptor protein tyrosine kinases (RPTKs); ex. v- erbb of Avian erythroblostosis virus (AEV); fms of SM-FeSV; ros of ASV UR2 Cytoplasmic PTKs: ex.src of RSV; fps/fes of FSV & FeSV GTP-binding protein: ex.ras of Ki-MuSV and Ha-MuSV Serine/threonine Protein Kinases: ex.mos of Mo-MuSV; raf of MSV; mhl/mil of MH2 Nuclear proteins (transcription factors): ex.myc of MC29; myb of AMV; rel of REV-T; jun of ASV17. Signaling Adaptors: crk of ATV110 Lipid and protein kinase (PI3 Kinase) : Avian sarcoma virus Activation of Proto-oncogenes A) Point mutations single mutation:ex. c-ras codons 12 or 61 mutation resulting in enhanced binding to GTP and persisted activation of ras; ex. c-neu conversion is due to oncogenic neu conversion is due to a single mutation in the transmembrane domain; ex. c-src can be activated by a single point mutation. multiple point mutations: most of the oncogenes contain several point mutations. B) Deletions Almost invariably retroviral oncogenes suffer 5' and 3' truncations comparing to their proto-oncogenes. The 5' truncated gene is fused in-frame to one of the viral gene, most often the 5' gag gene, resulting in the generation of a fusion protein. The 3' region of the proto-oncogene is also frequently fused to the viral sequence. Those mutations result in constitutive activation of the proto-oncogenes. Aside from the terminal truncations, internal deletions can also activate a proto-oncogene in a fashion equivalent to those of point mutations. C) Amplification Several types of human tumors have been found to contain amplified proto-oncogenes. Examples include myc amplification in carcinomas of lung, breast, cervix and erbb/erb2 amplification in squamous cell carcinoma and astrocytoma. Although it is not clear whether the amplified genes also harbor mutations and either or both is responsible for the oncogenesis, it has been demonstrated experimentally for several protooncogenes that mere overexpression of them can lead to cell transformation. D) Gene translocation and rearrangement This type of gene aberration not only can lead to structural mutation, but could also result in deregulated expression. The best known examples are the c-abl translocation leading to formation of Philadelphia chromosome in chronic myelogenous leukemia (CML) and the c-myc translocation in Burkitt's and certain T- and B-cell lymphomas. The resulting rearranged genes could still code for the original proteins, or in other cases, code for novel fusion proteins.

32 Table 4.6 The Biology of Cancer ( Garland Science 2007)