The Role of Mdm2 in Estrogen-Mediated Breast Cancer Cell Proliferation

Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the presence of estrogen increased p21 and the percent of cells in the G1 phase. Interestingly, knockdown of p53 had no effect on the estrogen-mediated cell proliferation. Estrogen also up-regulated the Mdm2 protein levels in cells exposed to the DNA damaging agent, etoposide, and the Mdm2 inhibitor, Nutlin-3. In turn, estrogen inhibited etoposide- and Nutlin-3-induced transcription of puma, a pro-apoptotic p53 target gene, without changing the p53 protein levels or p53 recruitment to the chromatin. The decrease in puma gene transcription correlated with a decrease in Puma protein and an increase in Bcl-2 protein, an anti-apoptotic estrogen receptor target. Overall, our findings suggest that estrogen signals to an Mdm2-mediated pathway to provoke cell proliferation and that this pathway is associated with inhibition of the G1 checkpoint

Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway

The Cancer Genome Atlas (TCGA) data indicate that high MDM2 expression correlates with all subtypes of breast cancer. Overexpression of MDM2 drives breast oncogenesis in the presence of wild-type or mutant p53 (mtp53). Importantly, estrogen-receptor positive (ER+) breast cancers overexpress MDM2 and estrogen mediates this expression. We previously demonstrated that this estrogen-MDM2 axis activates the proliferation of breast cancer cell lines T47D (mtp53 L194F) and MCF7 (wild-type p53) in a manner independent of increased degradation of wildtype p53 (ie, p53-independently). Herein we present data supporting the role of the estrogen-MDM2 axis in regulating cell proliferation and mammary tissue architecture of MCF7 and T47D cells in a p53-independent manner. Inducible shRNA mediated MDM2 knockdown inhibited colony formation in soft agar, decreased mass size and induced lumen formation in matrigel and also significantly reduced mitosis as seen by decreased phospho-histone H3 positive cells. The knockdown of MDM2 in both cell lines decreased Rb phosphorylation and the level of E2F1 protein. This signaling was through the estrogen receptor because fulvestrant (a selective estrogen receptor degrader) decreased MDM2 protein levels and decreased phosphorylation of Rb. Taken together these data indicate that in some ER+ breast cancers the estrogen- MDM2-Rb-E2F1 axis is a central hub for estrogen-mediated p53-independent signal transduction. This is the first indication that estrogen signaling utilizes the estrogen- MDM2 axis to provoke phosphorylation of Rb and increase E2F1 while promoting abnormal mammary architecture