Impairment of Retinoic Acid Receptor-Ceramide Signaling in Retinoic Acid-Resistant Breast Cancer Cells. Implications for Retinoid Differentiation Therapy of Breast Cancer

Despite promising preclinical animal studies, retinoic acid (RA), the bioactive derivative of vitamin A, has given disappointing results in clinical trials of human breast cancer because of the hurdle of RA resistance. RA resistance seems to have an heterogeneous molecular basis. One form of RA resistance in breast cancer has been traced to epigenetic silencing of RA receptors (RARs). In the breast cancer cell line T47D, RA treatment (1 mmol/L, 72 h) induced impaired cell growth and apoptosis concomitant with a 2.3-fold increase of endogenous ceramide, a sphingolipid with a recognized role as proapoptotic second messenger. In contrast, in the breast cancer cell line MDA-MB-231 the same treatment failed to induce both growth arrest and accumulation of proapoptotic ceramide. The two cell lines differ in their RAR profiles in that T47D cells are RAR-a positive and RAR-b inducible whereas MDA-MB-231 cells are RAR-a and RAR-b negative. Thus we hypothesized that ceramide accumulation may be mediated by RARs. To test this hypothesis we engineered T47D cells to stably express an RAR-a dominant-negative (DN) construct. DN T47D cells present an RA-resistant phenotype and fail to accumulate ceramide in response to RA treatment. In addition, by inhibiting RAR-a in T47D cells with RAR-a antagonists, we observed a failure to accumulate endogenous ceramide. Altogether these data indicate that RA-induced ceramide in T47D cells is mediated by RAR-a signaling. The synthetic retinoid fenretinide, 4-HPR, whose action is largely independent of RARs, was able to induce cell death and a concomitant ceramide increase in the MDA-MB-231 cell line, thus indicating that the biochemical machinery required for endogenous ceramide production is intact. In conclusion, we show that RA resistance associated with a lack of functional RARs in breast cancer cells can be overcome by using synthetic retinoids such as 4-HPR.