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.

Estrogen receptor α expressing breast cancers (BC) are classically treated with endocrine therapy. Prolonged endocrine therapy often results in a metastatic disease (MBC), for which a standardized effective therapy is still lacking. Thus, new drugs are required for primary and metastatic BC treatment. Here, we report that the Food and Drug Administration (FDA)-approved drugs, ouabain and digoxin, induce ERα degradation and prevent proliferation in cells modeling primary and metastatic BC. Ouabain and digoxin activate the cellular proteasome, instigating ERα degradation, which causes the inhibition of 17β-estradiol signaling, induces the cell cycle blockade in the G2 phase, and triggers apoptosis. Remarkably, these effects are independent of the inhibition of the Na/K pump. The antiproliferative effects of ouabain and digoxin occur also in diverse cancer models (i.e., tumor spheroids and xenografts). Additionally, gene profiling analysis reveals that these drugs downregulate the expression of genes related to endocrine therapy resistance. Therefore, ouabain and digoxin behave as ‘anti-estrogen’-like drugs, and are appealing candidates for the treatment of primary and metastatic 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.

Most cases of breast cancer (BC) are estrogen receptor α-positive (ERα+) at diagnosis. The presence of ERα drives the therapeutic approach for this disease, which often consists of endocrine therapy (ET). 4OH-Tamoxifen and faslodex (i.e., fulvestrant - ICI182,780) are two ETs that render tumor cells insensitive to 17β-estradiol (E2)-dependent proliferative stimuli and prevent BC progression. However, ET has limitations and serious failures in different tissues and organs. Thus, there is an urgent need to identify novel drugs to fight BC in the clinic. Re-positioning of old drugs for new clinical purposes is an attractive alternative for drug discovery. For this analysis, we focused on the modulation of intracellular ERα levels in BC cells as target for the screening of about 900 Food and Drug Administration (FDA) approved compounds that would hinder E2:ERα signaling and inhibit BC cell proliferation. We found that carfilzomib induces ERα degradation and prevents E2 signaling and cell proliferation in two ERα+ BC cell lines. Remarkably, the analysis of carfilzomib effects on a cell model system with an acquired resistance to 4OH-tamoxifen revealed that this drug has an antiproliferative effect superior to faslodex in BC cells. Therefore, our results identify carfilzomib as a drug preventing E2:ERα signaling and cell proliferation in BC cells and suggest its possible re-position for the treatment of ERα+ BC as well as for those diseases that have acquired resistance to 4OH-tamoxifen.

Kinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels.17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor.Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signaling, the knowledge of real-time ERα

No abstract