Li Tang, MD, PhD Roswell Park Cancer Institute Buffalo, NY, USA Li.Tang@roswellpark

1. Retroviruses and Novel Drugs, 2015 Associations between insertional polymorphisms of human endogenous retrovirus and breast cancer risk in African American and European American women Li Tang, MD, PhD Roswell Park Cancer Institute Buffalo, NY, USA Li.Tang@roswellpark.org June 2015

2. Outlines • Human endogenous retrovirus (HERV) and insertional polymorphism • HERV K and breast cancer • Research questions and Study design • Research findings • Summary

3. HERVs • Are sequences within the genome that closely resemble infectious retroviruses. • remnants of ancient germ line infections by exogenous retrovirus • comprise up to 8% of the human genome • Contribute to cancer development and progression. • effect of insertional mutagenesis on oncogene activation and/or tumor suppressor gene disruption • role of HERV-encoded proteins on tumorigenesis and immunosurveillance

4. HERV-K family and Breast cancer • The most biologically active class of HERVs, with the ability to • encode functional retrovirus proteins • produce retrovirus-like particles • High genetic homology to mouse mammary tumor virus (MMTV) • Identification of HERV-K viral transcripts and proteins in breast cancer cell lines, tissues, and plasma of breast cancer patients

5. Cancer-specific activation of HERV-K • expression level in breast tissue: Cancer tissue > adjacent normal tissue > normal tissue • expression level in plasma: Breast cancer patients > healthy individuals Activation of HERV-K in the early stage of breast tumorigenesis

6. HERV-K activation and hormones • in breast cancer cell lines: Increase of HERV-K transcription and translation by estradiol and progesterone • in MMTV-integrated mouse model: Transcription of HERV-K during pregnancy and lactation Increase of tumorigenic transformation by multiple pregnancies Hormone exposure-related risk factors may interact with HERV-K to contribute to breast cancer

7. HERV-K113 and -K115 • Coding competence: -K113: has complete full-length open reading frames for all of the viral proteins; is the only one so far to show capacity of producing intact viral particles. -K115: differs from –K113 in one single base-pair deletion which introduces a frameshift mutation and results in inability of producing some of the viral proteins.

8. HERV-K113 and -K115 • Insertional polymorphism: Full-length insertions exist only in a proportion of human population and show clear racial disparity Herrera et al. J Hum Genet 2006

9. Breast cancer in European American (EA) and African American (AA) women • Compared to EA women, AA women are more likely to be diagnosed at a younger age and have more aggressive tumors • Compared to EA women, AA women tend to experience menarche and pregnancy at early age and have more children • Similarities in breast cancer characteristics between sub-Saharan African and AA women supports a role of ancestry in racial disparities in breast cancer

10. Hypothesis: The prevalence of HERV-K113 and -K115 insertional polymorphism differs between AA and EA women, and that this disparity together with hormone-related factors is associated with differences in breast cancer characteristics between AA and EA women.

11. Research Questions Q1. Whether insertional polymorphism of HERV-K113 and K115 is associated with breast cancer risk in EA and AA women. Q2. Whether insertional polymorphism of HERV-K113 and K115 is associated with early-onset and aggressive characteristics of breast cancer in EA and AA women. Q3. Whether hormone-related factors modify the effect of insertional polymorphism of HERV-K113 and -K115 on breast cancer risk and aggressiveness.

12. Study Design Study population: The study is nested in The Women’s Circle of Health Study (WCHS), an ongoing multi-center case-control study of breast cancer in AA and EA women. Participants are enrolled in metropolitan New York City and eastern New Jersey area from AA and EA women between the ages of 20 to 64 years with newly diagnosed breast cancer for cases and for controls without prior history of cancer other than non-melanoma skin cancer. Cases and controls are matched on age, race, and county of residence.

13. Study Design A total of 1242 cases (608 AA and 634 EA) and 1422 controls (783 AA and 639 EA) were included in the analysis. Analytic population: Data and Specimens: • Breast cancer risk information collected by interviewer- administrated questionnaires at enrollment; 2. Tumor characteristic information obtained from pathology report and/or cancer registry; • DNA samples extracted from blood and/or saliva samples which are collected at enrollment.

14. Insertional Polymorphism Assay PCR followed by fragment analysis: Integration site a gag pro pol env 5´ 3´ LTR LTR c b LTR: Long Terminal Repeat

15. Integration site Insertional Polymorphism Assay a gag pro pol env 5´ 3´ LTR LTR c b c a b No insertion Heterozygote (1 insertion) Homozygote (2 insertion) LTR only

16. Analytic Methods: Insertional polymorphism assay: PCR and fragment analysis Population structure analysis: A panel of 128 single nucleotide polymorphisms (SNPs) were used as ancestry informative markers (AIMs) to correct for population stratification Statistical method: Logistic regression model was used to calculate Odd Ratio (OR) and 95% Confidence Interval (95% CI) adjusting for age at diagnosis and proportion of European ancestry

17. Characteristics of study population

18. Distribution of HERV-113 and -115 K113 K115 K113/K115/LTR 100% 80% 51.5% 60% Distribution of HERV insertion (%) 23.3% 40% 20% 0 L 1 2 2+ 0 L 1 2 2+ AA EA AA EA AA EA P <0.0001 P <0.0001 P <0.0001 0, no insertion; 1, one copy; 2, two copy; 2+, more than 2 insertion; L, LTR insertion only 0 0 0 0 1 1 1 1 2 2 2 2

19. Relationship between HERV Insertion and European Ancestry

20. HERV Insertion and Breast Cancer Risk

21. HERV Insertion and Breast Cancer Risk

22. Interaction between K113 Insertion and Hormonal-related Factors among All

23. Summary of Results 1. A two-fold higher prevalence of HERV-K113 and -K115 was observed in AA women compared to EA women. 2. The prevalence of HERV-K113 and –K115 was inversely correlated with proportions of European Ancestry. 3. HERV-K113 insertion was significantly associated with reduced risk of breast cancer in both AA and EA women, while K113 is the only HERV identified so far with a capacity to produce viral particles in cells. 4. Hormone exposure may modulate the association of HERV-K113 insertion with breast cancer risk.

24. Conclusion Anti-HERV-K specific antibody has been shown to inhibit breast cancer growth in animal models. Polymorphic insertion of HERV-K113 may interact with host immune system and offer some protection against breast cancer. Future Directions Comparisons of Immune activity among patients with or without HERV-K113 insertion. Association studies of HERV-K113 insertion and breast cancer survival. Development of anti-HERV-K antibody based prevention strategy in high risk women.

25. Acknowledgements Laboratory Assay: Steven R. Gregory Chris Borrelli Genomics core Facility The WCHS Study: Warren Davis Gregory L. Ciupak Elisa V. Bandera Christine B. Ambrosone Statistical Support: David Tritchler at UB Gary R. Zirpoli Song Yao Immunology: Yasmin Thanavala The study is supported by grant 5R03CA156645, K07 CA148888, R01CA100598, and P01151135 from National Cancer Institute (NCI).

26. Thank You!