1. FARUK MOHAMMED FARUK MOHAMMED δ, ψ F.mohammed2@wlv.ac.uk; elfarukpath11@yahoo.com F.mohammed2@wlv.ac.uk δ- THE UNIVERSITY OF WOLVERHAMPTON, SCHOOL OF APPLIED SCIENCES, DEPARTMENT OF BIOMEDICAL SCIENCE & PHYSIOLOGY, ENGLAND-UK Ψ- AHMADU BELLO UNIVERSITY, FACULTY OF MEDICINE, DEPARTMENT OF HISTOPATHOLGY, ZARIA-NIGERIA ©Faruk Mohammed ROLE OF HUMAN ENDOGENOUS RETROVIRUSES IN THE PATHOLOGY OF BREAST, PROSTATE AND TESTICULAR CANCERS: A REVIEW A PAPER PRESENTED AT THE BIOMEDICAL SCIENCE CONGRESS 2013. INTERNATIONAL CONVENTION CENTRE BIRMINGHAM, ENGLAND-UK

2. Human Endogenous Retroviruses (HERVs) and Malignancies HERVs are exogenous retrovirus-like infections, that are 8-10% in constituent of the entire human genome. Breast and Prostate cancers are the most common diagnosed type of malignancies, whereas testicular cancer is one of the most common cancer in men aged 15–44 years. Cancer incidence may rise from 127 million in 2008 to 222 million by 2030.

4. TYPE OF HERV AUTHORSMETHODSSAMPLES USEDCOMMENTS HERV-K (1) Ono et al., 1987SB (4) and NB (5) T47D cell line Progesterone stimulates HERV-K. Hormonal level in vivo stimulate HERV-K ? HERV-K Ejthadi et al., 2005 RT-PCR (2) and SB MCF-7, MCF-7, and T47D breast cancer cell lines HERV-K over-expressed HERV-K Contreras-Galindo et al., 2008 RT-PCR, WB (6) IM (7) EM (8) 18 subjectsHERV-K over-expressed HERV-K Golan et al., 2008 RT-PCR, WB, NB, and IF (9) 195 FFPE (3) breast tissues, T47D, MDA-MB- 231, and BT549 cell line HERV-K may be prognostic marker for breast cancer. HERV-K Zhao et al., 2011 RT-PCR and IM 305 FFPE breast malignant tissues HERV-K related to lymph node metastasis, staging, tumour size and decreased survival rate of patients. HERV-K Wang-Johanning et al., 2011 WB, ELISA, IM and FC (10) MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1 breast cancer cell line, MCF-10A and MCF-10AT cell lines (non-malignant) HERV-K can be targeted with Monoclonal antibodies. HERV-K Wang-Johanning et al., 2012 IHC, ELISA, WB 110 FFPE breast tissues (119 malignant and 76 non- malignant tissues) HERV-K over-expression results to T-cell responses stimulated by dentritic cell. HERV-T, F, and E Frank et al., 2008QRT-PCR46 breast tissues (23 malignant) There is evidence of HERV-T, HERV-E, HERV-F, and many of the class II families including HML-6 in breast cancers. TABLE II: HERVs Implicated in Breast cancer and the identification methods employed (1) HERV-K- Human Endogenous Retrovirus K. (2) Real time polymerases chain reaction. (3) Formalin fixed paraffin embedded. (4) Southern blot (5) Northern blot. (6) Western blot. (7) Immunohistochemistry. (8) Electron microscopy (9) Immunofluorescence. (10) Flow cytometry

5. TYPE OF HERVAUTHORMETHODSSAMPLES USEDCOMMENTS XMRV (1) Urisman et al., 2006 M and RT-PCR (3), FISH (4), IM (5), H and E (6), and cytokeratin IHC (7) 86 prostatic malignant tumour tissues XMRV Over-expressed XMRV Fischer et al., 2008 RT-PCR, allele-specific PCR 175 FFPE prostate tissues (105) malignant and 70 non- malignant tissues cell line No XMRV Expression XMRV Schlaberg et al., 2009 qPCR (2) IHC, TEM (8), WB (9) 334 FFPE prostate malignant tissues4, 293T cell line XMRV over-expressed XMRV Hohn, et al., 2009 Nested RT-PCR, IM, EM, and ELISA 589 FFPE prostate malignant tissues, 293T cell line No XMRV over-expression probably due to geographical reasons. XMRV Knouf et al., 2009 SB 22Rv1 and HTX cell line XMRV over-expressed XMRV Aloia et al., 2010 qPCR, WB, IM 1048 FFPE(3) prostate tissues, 22Rv1 cell line, DU145 and PC3 cell lines No XMRV over-expression. XMRV Arnold et al., 2010 RT-PCR, FISH, TaqMan genotyping assay 40 prostate tissues, and peripheral blood DNA XMRV over-expressed XMRV Martinez-Fierro, et al., 2010 RT-PCR, FISH, TaqMan genotyping assay 130 FFPE prostate tissues (55 Ca. and 75 controls), peripheral blood DNA No XMRV over-expression. XMRV Yang, et al., 2010 PCR, SNPG (10) CWR22 and 22Rv1 cell lines No XMRV over-expression. XMRV presence in 22Rv1 cells, a lab. Contaminant. XMRV Switzer et al., 2011 PCR, WB 162 FFPE prostate malignant tissues No XMRV over-expression XMRV Lee et al., 2012 qRT-PCR, RTPCR, PCR, FISH, and serological, VMA (11), and CMI (12) 40 FFPE prostate malignant tissues, 39 plasma samples. No XMRV over-expression. HERV -K Ishida et al., 2008 RT-PCRH and E, IF, FC, and ELISA 293T and COS7 cells lines There is HERV-K overexpressed in Prostate ovarian cancers, and leukemia TABLE I: HERVs Implicated in Prostate Cancer and the Identification Methods employed (1) XMRV- Xenotropic murine leukemia virus-related virus. (2) qPCR- quantitative polymerases chain reaction. (3) Microarrays and RT-PCR, (4) Florescence In situ hybridisation. (5) Immunofluorescence (6) Haematoxylin and Eosin. (7) Immunohistochemistry. (8) Transmission Electron Microscopy. (9) Western blot. (10) Single nucleotide polymorphism genotyping. (11) ViroChip Microarray Analysis (12) Chemiluminescent Microparticle Immunoassay.

6. TABLE III: HERVs Implicated in Testicular Cancer and the Identification Methods Employed TYPE OF HERVAUTHORMETHODSSAMPLES USEDCOMMENTS HERV-K (1) Roelofs et al., 1998 RT-PCR (2), and H & E (4) FFPE testicular cancer tissues HERV-K overexpressed.. HERV-K Goedert et al., 1999 Immunofluorescence 136 serum sample (52 prostate cancer patients and 84 control patients HERV-K10 is overexpressed in testicular malignancy which are not related to HIV. HERV-W (3) Gimenez, et al., 2010 RT-PCR and SB (5) RNA samples biological samples and DNA pair sample of human testicular malignant and normal sample There is evidence of overexpression of HERV-W in testicular cancer. HERV-K Kaufmann et al., 2010 Western blotting 293T cell lines HERV-K overexpressed in testicular cancer. HERV-K Rec protein may have unblock oncogenic transcription factors. HERV-HLower, et al., 1993 PCRProstate cancer tissues. HERV-H and HERV-K are overexpressed in human teratocarcinoma cell lines which produce retroviral particles (1) Human Endogenous Retrovirus-K. (2) Real time polymerases chain reaction. (3) Human Endogenous Retroviruses W(4) Haematoxylin and eosin stain (5) Southern blot.

7. CLASS I (GAMMARETROVIRUS) CLASS II (BETARETROVIRUS ) Class III (SPUMARETROVIRUS) Group 1: HERV-HF HERV-H, HERV-F Group 2: HERV-RW HERV-W HERV-R HERV-P Group 3: HERV-ER1 HERV-E HERV-R Group 4 : HERV-T HERV-T (S71, CRTK1, CRTK6) Group 5: HERV-IP HERV-I (RTVL-I) HERV-IP-T47D Group 6: HERV-FRD ERV-FRD Group 1: HML-1 HERV-K (HML-1.1) Group 2: HML-2 HERV-K10 HERV-K-HTDV Group 3: HML-3 HERV-K (HML3.1) Group 4: HML-4 HERV-K-T47D Group 5: HML-5 HERV-K-NMWV2 Group 6: HML-6 HERV-K (HML-6p) Group 7: HML-7 HERV-K-NMWV7 Group 8: HML-8 HERV-K-NMWV3 Group 9: HML-9 HERV-K-NMWV9 Group 10: HML-10 HERV-KC4 HERV-L HERV-S (HERV18) OTHERS: HRES-1 AVAILABLE HERVs CLASSIFICATION FROM LITERATURES

9. FIGURE 5: Phylogenetic Tree of Human Endogenous Retroviruses (HERVs) showing the three classes of HERVs in contradictory some some commonly used classical classifications (Class I [Gammaretroviruses], Class II [Betaretroviruses] and Class III [Spumaretroviruses]), and as relate to some major cancers. The Phylogenetic analysis was performed using different nucleotide sequence and analysing the sequence in ClustalX2 Version 2.1.0.0 software from the University of Wolverhampton SAS IT Teaching Suite A, MA143A, England-UK. NB: Load Sequence>Align Sequence>Output format>Bootstrap N-J trees at 2000>OK. ©Faruk Mohammed ©Fruk ohammed

10. HUMAN ENDOGENOUS RETROVIRUSES INVOLVED IN CANCERS CLASS I (GAMMARETROVIRUS) CLASS II (BETARETROVIRUS ) Class III (SPUMARETROVIRUS) Group 1: HERV-F HERV-H Group 2: HERV-S-71-Related ERV9 (HERV. pHE.1) MSRV Group 3: HERV-K1.1 HERV-E ∞ HERV R (ERV-3) GROUP 4: HERV-I RTLV-H HERV-S HRES-1 (HERV-18) GROUP 1: HERV-K (HML-7) GROUP 2: HERV-KC4 GROUP 3: HERV-K-18 HERV-K4 HERV-K11 GROUP 4: ∞∂ HERV-K8 HERV-K-5 HERV-K (gag, pol, and env) HERV-10 HERV-K (HML-2) HERV-K108 Group 5: HERV-K12 HERV-K7 HERV-K6 Group 6: HERV-L HERV-K1.3 HERV-P-T47D Group 7: HERV-K113 HERV-K (HML-1.1) XMRV Group 1: HERV-T HERV-P HERV FRD (ERV-FRD) GROUP 2: HERV-K-HTDV HERV-W ¥ BREAST CANCER ¥ TESTICLUAR CANCER ∂ PROSTATE CANCER ∞

11. HERV-W Figure A: Putative conserved domain of HERV-W protein showing mapped query sequence alignment, heptad repeat 1 and 2 (HR1 and HR2), and immunosuppressive region and other important component located around sequence length of 350-425 amino acids (aa). The 17 amino acids long immunosuppressive region is present in most retroviral envelope proteins. Synthetic peptides derived from this immunosuppressive region inhibit immune function in vitro and in vivo. B): These regions are also highlighted in red colour within the query sequence matches. C): Backbone structure of HERV-W showing ligand domain, hydrogen bonding locations and the location of the different amino acid contained. Homology and BLAST analysis using the NCBI website revealed that HERV-W refers to 52.9kDa protein encoded by mRNA gene with an open reading frame encoded by 538 amino acids (aa). This corresponds to accession number NP_055405 located on q21.2 region of human chromosome 7 which encodes the Homo sapiens member of HERV. This gene is part of HERV provirus that has inactivating mutations in the gag and pol genes. HERV-W gene's protein produce is expressed in the placental syncytiotrophoblast towards fusion of the cytotrophoblast cells for placenta syncytial layer formation. Putative Conserved Domain of HERV-W with Query Sequence Matches and Structure. A B C

12. Figure B: Query sequence matches of HERV-K envelop protein from 1 to 699 amino acids. The amino acids highlighted in red signify the HERV-K proteins topological domain. Areas underlined in black lines signifies the specific hit of HERV-K envelop protein located around amino acid sequence length of 180-300. Homology searches and BLAST analysis using the NCBI website and other necessary tools revealed that HERV-K env refers to a 13 kDa protein encoded by mRNA gene. This has an apparent retrained introns (non-coding region) and an open reading frame encoded by 699 amino acids (aa). This corresponds to accession number Q69384 located on p22.1 region of human chromosome 7 which encodes the Homo sapiens member of human endogenous retrovirus (HERV). Figure A: Putative conserved domain of HERV-K env protein showing mapped query sequence alignment of HERV-Kenv-2, heptad repeat 1 and 2 (HR1 and HR2), homotrimer interface, HIV-1-like_HR1-HR2, ebola_HIV-1-like-HR1-HR2. The specific hit of HERV-K envelop protein is located around sequence length of 180-300. The retro-transcribing virus envelop protein originate from HERV-K-env glycoproteins. Putative Conserved Domain of HERV-K with Query Sequence Matches HERV-K A B

13. CONCLUSIONS The study of HERVs in today's medical research may be a better surrogate towards eradicating cancer. The possibility that due to our strategies, other HERVs were missed cannot be excluded.

14. Future Research If we have got adequate financial support from the govt. & general public, we should be able to contribute our quota towards cancer vaccine development. Perhaps, we may take into consideration cancer samples, as regard to geographical location of patients (UK, US and Sub- Saharan Africa for e.g.).

15. We sincerely wish to acknowledge Professors P.N Nelson, Wang-Johanning, P. Hooley, Y. Iliyasu, S. Smith and J. Idoko, for their kind contribution and motivation. Acknowledgements

16. THANK YOU