Purpose: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-a (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics.Experimental Design: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. Results:We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity.Conclusions: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival.

No abstract

17β-estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra-nuclear localized receptor triggers the membrane-dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2-acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)-based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high-content method to detect all aspects of E2:ERα signaling (nuclear and extra-nuclear receptor activity, ERα levels, E2-induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96-well plate format. To demonstrate its robustness, we also challenged 4OH-tamoxifen resistant (Tam-Res) BC cells with a library of anti-cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high-content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti-ERα and anti-proliferative use for MTX in Tam-Res BCs.

17β-estradiol (E2) regulates diverse physiological effects, including cell proliferation, by binding to estrogen receptor α (ERα). ERα is both a transcription factor that drives E2-sensitive gene expression and an extra-nuclear localized receptor that triggers the activation of diverse kinase cascades. While E2 triggers cell proliferation, it also induces ERα degradation in a typical hormone-dependent feedback loop. Although ERα breakdown proceeds through the 26S proteasome, a role for lysosomes and for some endocytic proteins in controlling ERα degradation has been reported. Here, we studied the role of the endocytic protein dynamin II in E2-dependent ERα signaling and degradation. The results indicate that dynamin II siRNA-mediated knock-down partially prevents E2-induced ERα degradation through the inhibition of an autophagy-based pathway and impairs E2-induced cell proliferation signaling. Altogether, these data demonstrate that dynamin II is required for the E2:ERα signaling of physiological functions and uncovers a role for autophagy in the control of ERα turnover.