BT

Increasing evidence points to the role of endogenous retroviruses (ERVs) in driving cancer cell proliferation. The purpose of this study was to explore the ...

As expected, DTG treatment reduced the motility of BT-20 cells, so did HERV-K knockdown (Figures 7B, 7D, 7E). A transwell assay revealed reduced invasiveness of BT-20 cells through the basement membrane extract in the presence of DTG, an effect similar to that of HERV-K knockdown (Figures 7C, 7F, 6G). Surprisingly, the inhibitory effect of DTG on MMTV pol observed in vitro became insignificant in vivo and the drug significantly enhanced env expression in mouse tumor tissues, accompanied by enhanced metastasis to the lungs. These results highlight the different effects of the same drug in cell cultures and in an animal model. In the five human breast and prostate cancer cell lines tested (MDA-MB-453, SKBR3, BT-20, LNCaP, and T47D), the antiproliferative effect of DTG was strongly correlated with the expression of HERV-K in the cell lines. Since there are fundamental differences between human and mouse ERVs and between human and mouse immune systems, using xenograft models to study the effect of DTG on HERVs and human breast cancers in immunocompromised mice will eliminate the immunity factor and may yield different results from what we observed using MMTV-related murine cancer in immunocompetent mice. Quantitative RT-PCR revealed that DTG inhibited both the pol and env genes of HERV-K in multiple cell lines including the breast cancer cell lines MDA-MB-453, BT-20, and T47D, as well as the LNCaP prostate cancer cell line (Figure 1A). The pol gene of HERV-K was more significantly inhibited than the env gene. An anti-Gag antibody revealed reduced expression of the 30-kDa Gag product in the knockdown cell lines (Figure 4A). The knockdown cell lines proliferated slower and became more resistant to DTG. Figures 4B, 4C show the proliferation and survival of one of the knockdown cell lines. The correlation coefficient between ED50 of DTG and the expression of HERV-K pol was 0.99, and that between ED50 and the expression of HERV-K env was 0.76, with the former being statistically significant (Figures 2A, 2B). Figure 2C shows the dosage-dependent antiproliferative effect of DTG on 4T1 cells. DTG also inhibited the expression of the pol gene, but not the env gene, of MMTV in the murine 4T1 mammary cancer cell line (Figures 1B, 1C). Moreover, DTG treatment of BT-20 cells reduced the quantity of HERV-K genes in genomic DNA (Figure 1D), while the quantity of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene in genomic DNA was not affected. Primers of Pgk1, YWHAZ, and GAPDH were used at 100 nM. Primers for HERV-K and MMTV genes were used at 400 nM. HERV-K genes and the human YWHAZ gene were amplified at 95°C 15”, 57°C 25”, and 72°C 45”, while MMTV genes and the mouse Pgk1 gene were amplified at 95°C 15”, 58°C 25”, and 72°C 45”. BT-20 cells were pre-treated with 50 μM DTG or DMSO for 48 hours and seeded in quadruplet wells on a 24-well plate in serum-free EMEM. DTG-treated cells continued to receive the same concentration of drug during wound healing. BT-20 breast tumor cell line (kindly provided by Dr. Jiayuh Lin of the University of Maryland) was maintained in Eagle's minimum essential medium (EMEM) with 10% fetal bovine serum (FBS) and 5% CO2. T47D breast cancer cell line (kindly provided by Dr. Joseph Brewer of Edward Via College of Osteopathic Medicine) and 4T1 murine mammary cancer cell line (American type culture collection, ATCC, Manassas, Virginia, USA) were maintained in RPMI 1640 with 10% FBS and 5% CO2. MBA-MD-453 breast cancer cell line (kindly provided by Dr. Jiayuh Lin, University of Maryland) was maintained in Leibovitz's L-15 medium in atmospheric air.