ERα negative breast cancer cells restore response to endocrine therapy by combination treatment with both HDAC inhibitor and DNMT inhibitor

Estrogen receptor α (ERα) mediates the growth stimulation of estrogen in breast cancer cells and is a useful predictive factor for response to endocrine therapy. It is reported that ERα was induced in ERα negative breast cancer cells by both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2′-deoxycytidine (AZA) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, whether the breast cancer cells with induced ERα restore response to endocrine therapy requires to be further researched.

Reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to explore the change in the mRNA of ERα, PR and pS2 in the ERα negative breast cancer cells MDA-MB-435 treated with two chemicals (AZA + TSA). Water-soluble tetrazolium salt-8 (WST-8) method was used to study the proliferation rate of the breast cancer cells. Flow cytometer (FCW) was used to analyze the distribution of cell cycle of these breast cancer cells. Some xenograft models in nude mice were used to further study the results we found in vitro.

In this study we observed that the mRNA of ERα, PR and pS2 in the ERα negative breast cancer cells MDA-MB-435 was re-expressed by treatment with AZA + TSA. The proliferation assay analysis showed AZA + TSA suppressed the proliferation of MDA-MB-435 cells, which were further suppressed by addition of 4-OH Tamoxifen (4-OHT). On the contrary, the proliferation of cells treated with 4-OHT alone showed no difference compared with the vehicle control. Cell cycle analysis showed AZA + TSA treated cells showed S phase arrest, which was partially attenuated by addition of estradiol (E2); furthermore, the effect of E2 on stimulation of cell cycle could be reversed by 4-OHT in the treated cells with induced ERα. In vivo experiment xenograft volume of MDA-MB-435 cells treated with AZA + TSA was smaller than that of the control (P < 0.01), and the xenograft of AZA + TSA treated cells was further suppressed by ovarectomy (P < 0.01).

Our data indicate that DNMT1 inhibitor AZA and HDAC inhibitor TSA play important roles in restoring sensitivity of the ERα negative breast cancer cells to endocrine therapy in vitro and in vivo.