Targetable T-type Calcium Channels Drive Glioblastoma.

Glioblastoma (GBM) stem-like cells (GSC) promote tumor initiation, progression, and therapeutic resistance. Here, we show how GSCs can be targeted by the FDA-approved drug mibefradil, which inhibits the T-type calcium channel Cav3.2. This calcium channel was highly expressed in human GBM specimens and enriched in GSCs. Analyses of the The Cancer Genome Atlas and REMBRANDT databases confirmed upregulation of Cav3.2 in a subset of tumors and showed that overexpression associated with worse prognosis. Mibefradil treatment or RNAi-mediated attenuation of Cav3.2 was sufficient to inhibit the growth, survival, and stemness of GSCs and also sensitized them to temozolomide chemotherapy. Proteomic and transcriptomic analyses revealed that Cav3.2 inhibition altered cancer signaling pathways and gene transcription. Cav3.2 inhibition suppressed GSC growth in part by inhibiting prosurvival AKT/mTOR pathways and stimulating proapoptotic survivin and BAX pathways. Furthermore, Cav3.2 inhibition decreased expression of oncogenes (PDGFA, PDGFB, and TGFB1) and increased expression of tumor suppressor genes (TNFRSF14 and HSD17B14). Oral administration of mibefradil inhibited growth of GSC-derived GBM murine xenografts, prolonged host survival, and sensitized tumors to temozolomide treatment. Our results offer a comprehensive characterization of Cav3.2 in GBM tumors and GSCs and provide a preclinical proof of concept for repurposing mibefradil as a mechanism-based treatment strategy for GBM. Cancer Res; 77(13); 3479-90. ©2017 AACR.

T-Type Ca2+ Channel Inhibition Sensitizes Ovarian Cancer to Carboplatin.

Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca(2+)) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca(2+) channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca(2+) channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer.

T-type calcium channels blockers as new tools in cancer therapies.

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca(2+) channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca(2+) channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca(2+) channels could be attractive molecular targets for anticancer therapy.

A model for the regulation of T-type Ca(2+) channels in proliferation: roles in stem cells and cancer.

Ca(2+) influx at critical points in the cell cycle is required for proliferation. This requirement is so ubiquitous that its occurrence is often treated as background noise. Yet without it, cells stop dividing, suggesting an obvious and potentially effective way to treat cancer. To control proliferation by controlling Ca(2+) influx requires that the mechanism be elucidated, but this field of study has been filled with controversy and devoid of therapeutic utility. In this study, the authors present a model for the regulation of Ca(2+) influx at the G1/S restriction point in cancer and stem cells that is simple, cohesive and, we believe, reasonably complete. The model illustrates the essential role of T-type Ca(2+) channels in mediating influx and points clearly to the therapeutic strategies that have recently entered clinical trials.

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells.

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca(2+) channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca(2+) channels, as inhibition of L-type Ca(2+) channels did not induce similar effects. Our results implicate T-type Ca(2+) channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

The pharmacology and regulation of T type calcium channels: new opportunities for unique therapeutics for cancer.

T type calcium channels are implicated in a fascinating array of physiological and pathophysiological phenomena, as reviewed extensively in this issue of Cell Calcium. It is also evident from this timely collection of papers that knowledge about T channels is nascent. This creates the exciting potential to explore an uncharted territory for new insights into important physiological processes and associated diseases. Equally exciting and perhaps more important is the opportunity to exploit the developing understanding of T channels to design new therapeutic approaches to diseases that are now intractable to varying degrees. This review will focus on the controversy surrounding the proposed novel functions for and means of regulation of T type Ca2+ channels, particularly with regard to the potential role of pharmacologic modulation of T type Ca2+ channels in cytostatic cancer chemotherapy. In addition, we will concentrate on recent studies of the molecular pharmacology and physiology of T type channels, since these areas (molecular pharmacology [T.N. Heady, J.C. Gomora, T.L. Macdonald, E. Perez-Reyes, Molecular pharmacology of T-type Ca2+ channels, Jpn. J. Pharmacol. 85 (2001) 339-350.]; physiology [E. Perez-Reyes, Molecular physiology of low-voltage-activated t-type calcium channels, Physiol. Rev. 83 (2003) 117-161.]) have been very well-reviewed and the reader is referred to these authoritative sources.

Investigation into the structure-activity relationship of novel concentration dependent, dual action T-type calcium channel agonists/antagonists.

This paper describes the synthesis and biological evaluation of a series of straight chain analogs of a compound (1) that was previously synthesized in our research program. These compounds, which are T-type calcium channel antagonists, exhibits potent anti-proliferative activity against a variety of cancer cells. A structure-activity relationship of these analogs against a variety of cancer cells has provided insight into a logical pharmacophore for this series of compounds. Furthermore, this series of compounds has presented itself as a set of novel, concentration dependent, dual action agonists/antagonists for the T-type calcium channel.

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells.

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.

Design, synthesis, and biological evaluation of novel T-Type calcium channel antagonists.

This paper describes the synthesis of several novel T-type calcium channel antagonists that inhibit calcium influx into the cell, which in turn regulates unknown aspects of the cell cycle pathway that are responsible for cellular proliferation. A library of compounds was synthesized and a brief structure activity relationship will be described. From these studies we have identified a compound (1) that displays anti-proliferative activity in the low micromolar range across a variety of cancer cell lines.

THE INFLUENCE OF ENVIRONMENTAL LIGHTING ON THE GROWTH AND PROMETAMORPHIC DEVELOPMENT OF LARVAL RANA PIPIENS.

This study was designed to investigate whether the larval development of an anuran amphibian could be modified by raising the animals in continuous light or darkness instead of under conditions of diurnal illumination, and to quantify the effects of these treatments at various intervals during this period of development. Larvae of the frog, Rana pipiens, were raised through metamorphosis under conditions of constant light, constant darkness, or diurnal lighting. As measured by stages of development, body weight, tail length and body length at 20-day intervals, no significant differences in growth rate or metamorphic change were observed until near the middle of the prometamorphic period, which began at approximately the 50th day of development. After midmetamorphosis, a significant acceleration in the measured parameters was seen for the animals raised in conditions of constant light in comparison with those in constant darkness. Those with diurnal lighting were intermediate. These results suggested that light, or its absence, can respectively stimulate or retard amphibian metamorphosis in late larval stages after the hypothalamo-hypophyseal-thyroid axis has matured. Neither continuous light nor continuous darkness during larval development prevented the transformation from tadpole to frog.