Estrogen receptor-a (ERa) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERa protein by targeting receptor for destruction by the 26S proteasome. However, ERa is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulsechase analysis was developed using a tet-inducible ERa expression system to determine the rate of ERa protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERa half-life is shortened from 3 to 1 h. However, ERa half-life increases over time, achieving a half-life of w6 h in 72 h of estrogen treatment. Analysis of ERa half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERa and showed that the half-life of the phosphomimetic ERa mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERa protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.

Estrogen receptor-alpha (ERα) is a major therapeutic target of hormonal therapies in breast cancer and its expression in tumors is predictive of clinical response. Protein levels of ERα are tightly controlled by the 26S proteasome, yet how the clinical proteasome inhibitor, bortezomib, impacts ERα regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERα protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERα levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERα mRNA due to direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERα gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related to occupancy of ERα and RNA PolII on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERα in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERα pathway.

Breast cancer cell growth and therapeutic response are manipulated extrinsically by microenvironment signals. Despite recognition of the importance of the microenvironment in a variety of tumor processes, predictive measures that incorporate the activity of the surrounding cellular environment are lacking. In contrast, tumor cell biomarkers are well established in the clinic. Expression of Estrogen Receptor-alpha (ERα) is the primary defining feature of hormonally responsive tumors and is the molecular target of therapy in the most commonly diagnosed molecular subtype of breast cancer. While a number of soluble factors have been implicated in ERα activation, the complexity of signaling between the cellular microenvironment and the cancer cell implies multivariate control. The cumulative impact of the microenvironment signaling, which we define as microenvironmental activity, is more difficult to predict based on the sum of its parts. Here we tested the impact of an array of microenvironments on ERα signaling utilizing a microfluidic co-culture model. Quantitative immunofluorescence was employed to assess changes in ERα protein levels, combined with gene expression and phosphorylation status, as measures of activation. Analysis of microenvironment-induced growth under the same conditions revealed a previously undescribed correlation between growth and ERα protein down-regulation. These data suggest an expanded utility for the tumor biomarker ERα, in which the combination of dynamic regulation of ERα protein and growth in a breast cancer biosensor cell become a read-out of the microenvironmental activity.

Phosphodiesterase 4D (PDE4D) has recently been implicated as a proliferation-promoting factor in prostate cancer and is over-expressed in human prostate carcinoma. However, the effects of PDE4D inhibition using pharmacological inhibitors have not been examined in prostate cancer. These studies examined the effects of selective PDE4D inhibitors, NVP-ABE171 and cilomilast, as anti-prostate cancer therapies in both in vitro and in vivo models. The effects of PDE4D inhibitors on pathways that are critical in prostate cancer and/or downstream of cyclic AMP (cAMP) were examined. Both NVP-ABE171 and cilomilast decreased cell growth. In vitro, PDE4D inhibitors lead to decreased signaling of the sonic hedgehog (SHH), Androgen Receptor (AR), and MAPK pathways, but growth inhibition was best correlated to the sonic hedgehog pathway. PDE4D inhibition also reduced proliferation of epithelial cells induced by paracrine signaling from co-cultured stromal cells that had activated hedgehog signaling. In addition, PDE4D inhibitors decreased the weight of the prostate in wild-type mice. Prostate cancer xenografts grown in nude mice that were treated with cilomilast or NVP-ABE171 had decreased wet weight and increased apoptosis compared to vehicle treated controls. These studies suggest the pharmacological inhibition of PDE4D using small molecule inhibitors is an effective option for prostate cancer therapy. Implications PDE4D inhibitors decrease the growth of prostate cancer cells in vivo and in vitro, and PDE4D inhibition has therapeutic potential in prostate cancer.