In 2006, upon completion of the Human Genome Project, it was discovered that nearly 8% of the human genome is made up of viral DNA. These viral remnants are composed of ancient germline infections known as human endogenous retroviruses (HERVs) which are passed on to future generations in a Mendelian fashion1–4. Although these viral elements were previously thought to be ‘junk DNA’ or DNA with no functions in the body, research has slowly been emerging over the years that show that these viral sequences play a key role in many cancers including breast cancer.
Structure of the Human Endogenous Retrovirus (HERV)
The HERV-K group of endogenous retroviruses consists of 11 subtypes each of which resulted from a separate germline infection during evolution. HML-2, a subgroup of the HERV-K endogenous retroviruses is the most recently integrated provirus and maintains a complete open reading frame for its viral polyproteins5,6. There are two main types of HERV-K (HML-2) present in humans: Type 1 and Type 2 which differ from each other due to a 292 base-pair deletion and thus give rise to different accessory proteins Np9 in type I and Rec in Type II(Fig 1). HERV-K expression has been reported in many different solid tumours including ovarian, melanoma, breast and prostate7–14. Our lab has also demonstrated that HERV-K expression is predictive of a prostate cancer diagnosis15.
Figure 1: Structure of HERV-K provirus.
HERV-K and Cancer
HERV-K was first successfully cloned in the 1980s by Ono et al who also showed that human breast cancer cell lines stimulated with oestrogen and progesterone showed elevated levels of HERV-K mRNA16. This work was refined upon by our collaborator Wang-Johanning and colleagues who quantified HERV-K envelope transcripts and spliced transcripts in breast tumours showing elevated levels of expression compared to healthy controls14. We then demonstrated that elevated HERV-K expression in breast tumours is associated with increased risk of lymph node metastasis and poor outcome in two separate US cohorts and a cohort of Chinese breast cancer patients17,18. Wang-Johanning et al also demonstrated that HERV-K serum mRNA and anti-Rec titres are predictive of early stage breast cancer and HERV-K gag serum mRNA tend to be higher in breast cancer patients with a primary tumour who later developed metastasis3. Recently, this group has also shown that downregulation of HEV-K env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth.1
HERV-K induced immunomodulation in Cancer
HERV proteins possess immunogenic properties as evidenced by HML-2 antibodies being present in patients with melanoma, breast and ovarian cancer8,3. HERV-K 18, Env protein is upregulated in response to the Epstein Barr Virus and can induce T-Cell responses19.
Another interesting property of the Env protein of HERV-K is immunosuppression via the protein’s transmembrane domain that could aid tumour progression in cancer19. Morozov et al identified an immunosuppressive HERV-K env protein that regulates cytokine expression and suppresses immune cell proliferation in vitro20.
Nitric oxide synthase 2 (NOS2) is a predictor of poor outcome in estrogen receptor-negative breast cancer and it co-occurs with HML-2 Env expression in breast cancer21. It is possible that Env mediates an inflammatory response via its activation of the nitic oxide signalling pathway.
HERV-K and breast cancer
Understanding the consequences of HERV-K activation in breast cancer is of critical importance for understanding the emergence of breast neoplasms. HERV-K may induce tumorigenesis via the pathway we have postulated (fig.2). Data has come into light that suggests that the spliced accessory proteins of HERV-K, Np9 and Rec could be oncogenic22,23. Rec, the accessory protein developed from the splicing event of type II proviruses, is a shuttle protein and has functional homology to the Rev protein of HIV-1. Np9 (from type I proviruses) is spliced from an alternative splice donor site and shares only 14aa with Rec. Np9 has been shown to act as a molecular switch for co-activating the ERK/Akt pathway in human leukaemia and its expression is significantly higher in leukaemia patients compared to normal donors22. Both Rec and Np9 bind to the promyelocytic leukaemia zinc finger protein (PLZF) which is a transcriptional repressor of C-Myc, leading to its de-repression19. Rec leads to similar effects by binding to testicular zinc finger protein (TZFP), a repressor of the androgen receptor. Another piece of evidence that points towards the oncogenic potential of these two accessory proteins of HERV-K is that mice transgenic for Rec are prone to the development of seminomas.
Although it is now understood that HERVs play a significant role in inducing chronic diseases including cancer, most of our knowledge to date is limited to the functionality of the Env protein. My project aims to elucidate the expression of each protein produced by the HERV-K provirus and its implications in the different subtypes of breast cancer. What makes this a unique and relevant study is our previous findings that HERV-K is expressed in 66% of primary breast tumours irrespective of estrogen receptor status and is associated with increased risk of lymph node metastasis and poor outcome1718.
The connection between HERVs and the human genome is a complex one, coming about due to millions of years of co-evolution. Even though there is now established evidence connecting HERVs to cancer and other diseases, the field is still limited by a lack of patient studies and functional studies of the impact of the different proteins produced by the virus. Our hypothesis points towards the inflammatory pathway being activated by HERV-K leading to tumour progression. Thus, the findings of this project will point towards a mechanistic role of HERV-K in breast cancer which is lacking thus far.
- Nelson, P. N. et al. Demystified . . . Human endogenous retroviruses. 11–18 (2003).
- Downey, R. F. et al. Human endogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice? Int. J. Cancer 137, 1249–1257 (2015).
- Wang-Johanning, F. et al. Human endogenous retrovirus type K antibodies and mRNA as serum biomarkers of early-stage breast cancer. Int. J. Cancer 134, 587–595 (2013).
- Hohn, O., Hanke, K. & Bannert, N. HERV-K(HML-2), the Best Preserved Family of HERVs: Endogenization, Expression, and Implications in Health and Disease. Front. Oncol. 3, 246 (2013).
- Subramanian, R. P., Wildschutte, J. H., Russo, C. & Coffin, J. M. Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. Retrovirology 8, 90 (2011).
- Young, G. R., Stoye, J. P. & Kassiotis, G. Are human endogenous retroviruses pathogenic? An approach to testing the hypothesis. BioEssays 35, 794–803 (2013).
- Iramaneerat, K., Rattanatunyong, P., Khemapech, N., Triratanachat, S. & Mutirangura, A. HERV-K Hypomethylation in Ovarian Clear Cell Carcinoma Is Associated With a Poor Prognosis and Platinum Resistance. Int. J. Gynecol. Cancer 21, 51–57 (2011).
- Wang-Johanning, F. et al. Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer. Int. J. Cancer 120, 81–90 (2007).
- Serafino, A. et al. The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation. Exp. Cell Res. 315, 849–862 (2009).
- Büscher, K. et al. Expression of Human Endogenous Retrovirus K in Melanomas and Melanoma Cell Lines. Cancer Res. 65, 4172–4180 (2005).
- Reiche, J., Pauli, G. & Ellerbrok, H. Differential expression of human endogenous retrovirus K transcripts in primary human melanocytes and melanoma cell lines after UV irradiation. Melanoma Res. 20, 1 (2010).
- Li, M. et al. Downregulation of Human Endogenous Retrovirus Type K (HERV-K) Viral env RNA in Pancreatic Cancer Cells Decreases Cell Proliferation and Tumor Growth. Clin. Cancer Res. 23, 5892–5911 (2017).
- Golan, M. et al. Human Endogenous Retrovirus (HERV-K) Reverse Transcriptase as a Breast Cancer Prognostic Marker 1,2,3. Neoplasia 10, 521–533 (2008).
- Wang-Johanning, F. et al. Quantitation of HERV-K env gene expression and splicing in human breast cancer. Oncogene 22, 1528–1535 (2003).
- Wallace, T. A. et al. Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers. Carcinogenesis 35, 2074–2083 (2014).
- Ono, M., Yasunaga, T., Miyata, T. & Ushikubo, H. Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. J. Virol. 60, 589–98 (1986).
- Wang-Johanning, F. et al. Immunotherapeutic potential of anti-human endogenous retrovirus-k envelope protein antibodies in targeting breast tumors. J. Natl. Cancer Inst. 104, 189–210 (2012).
- Zhao, J. et al. Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer. Genes Cancer 2, 914–22 (2011).
- Sutkowski, N., Conrad, B., Thorley-Lawson, D. A. & Huber, B. T. Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen. Immunity 15, 579–89 (2001).
- Morozov, V. A., Dao Thi, V. L. & Denner, J. The Transmembrane Protein of the Human Endogenous Retrovirus – K (HERV-K) Modulates Cytokine Release and Gene Expression. PLoS One 8, (2013).
- Burke, A. J., Sullivan, F. J., Giles, F. J. & Glynn, S. A. The yin and yang of nitric oxide in cancer progression. Carcinogenesis 34, 503–512 (2013).
- Chen, T. et al. The viral oncogene Np9 acts as a critical molecular switch for co-activating β-catenin, ERK, Akt and Notch1 and promoting the growth of human leukemia stem/progenitor cells. Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund, U.K 1–10 (2013). doi:10.1038/leu.2013.8
- Hanke, K. et al. Staufen-1 interacts with the human endogenous retrovirus family HERV-K(HML-2) rec and gag proteins and increases virion production. J. Virol. 87, 11019–30 (2013).