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Published OnlineFirst December 20, 2013; DOI: 10.1158/1541-7786.MCR-13-0485 Molecular Cancer Cell Death and Survival Research T-Type Ca2þ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through Activation of p38-MAPK in Colon Cancer Cells Barbara Dziegielewska1, David L. Brautigan2,3, James M. Larner1,3, and Jaroslaw Dziegielewski1,3 Abstract þ Epithelial tumor cells express T-type Ca2 channels, which are thought to promote cell proliferation. This study þ investigated the cellular response to T-type Ca2 channel inhibition either by small-molecule antagonists or by þ RNAi-mediated knockdown. Selective T-type Ca2 channel antagonists caused growth inhibition and apoptosis À À more effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116 mutant p53 / cells. These antagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at specific target þ sequences. The knockdown of a single T-type Ca2 channel subunit (CACNA1G) reduced cell growth and À À induced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53 / cells. Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdown þ when p53wt was stably expressed. Upon T-type Ca2 channel inhibition, p38-MAPK promoted phosphorylation at Ser392 of p53wt. Cells treated with the inhibitor SB203580 or specific RNAi targeting p38-MAPKa/b þ (MAPK14/MAPK11) showed resistance to T-type Ca2 channel inhibition. Finally, the decreased sensitivity to channel inhibition was associated with decreased accumulation of p53 and decreased expression of p53 target genes, p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA). Implications: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumor þ therapies that target T-type Ca2 channels. Mol Cancer Res; 12(3); 348–58. Ó2013 AACR. Introduction with activity in vivo in combination with chemo- (9) or þ þ 2 Intracellular Ca2 regulates many cellular processes, radiotherapy (10). Aberrant expression of T-type Ca including cell cycle, proliferation, transcription, exocytosis, channels in cancer cells is thought to promote cell survival, hormone release, cell motility, and apoptosis (1, 2). Voltage proliferation, and motility (3, 11); however, the molecular 2þ 2þ mechanisms for these effects are poorly understood. gated Ca channels facilitate transient Ca influx from þ Signaling through voltage gated Ca2 channels involves the environment into the cytoplasm and appear mostly in þ Ca2 -binding proteins such as calmodulin (CaM) and excitable tissues, but also are unusually expressed in cancer þ þ 2 – cells. Low-voltage activated Ca2 channels, termed T-type activation of its binding partner Ca /calmodulin depen- 2þ dent kinase II (CaMKII), which in turn regulates T-type Ca channels, recently gained attention in cancer therapy, 2þ because their inhibition decreased proliferation of glioblas- Ca channels activity (12, 13). Consequently, proteins toma cells (3, 4), breast adenocarcinoma cells (5, 6), mel- downstream from activated CaMKII, such as mitogen-acti- anoma cells (7), and esophageal carcinoma cells (8). In vated protein 3 kinase 5 (MAP3K5, also known as apoptosis þ – addition, an antagonist selective for T-type Ca2 channels, signal regulating kinase 1 or ASK-1) or prosurvival protein kinase B (PKB also known as AKT) are affected by changes in mibefradil, has been proposed recently as a sensitizing agent þ intracellular Ca2 concentration (14–16). Increased CaM- KII activity regulates gene expression directly through phos- Authors' Affiliations: Departments of 1Radiation Oncology, 2Microbiol- phorylation of transcription factors, such as cyclic-AMP ogy, Immunology, and Cancer Biology, Center for Cell Signaling, University response element binding protein (CREB; reviewed in of Virginia School of Medicine; and 3Cancer Center, University of Virginia, Charlottesville, Virginia ref. 17) or indirectly involving p53 activation (18, 19). For example, CREB transcription factor could be responsible for Note: Supplementary data for this article are available at Molecular Cancer radiation resistance through regulation of DNA repair genes Research Online (http://mcr.aacrjournals.org/). þ (20), whereas intracellular Ca2 and CaMKII could regulate Corresponding Author: Jaroslaw Dziegielewski, Department of Radiation fi fl Oncology, University of Virginia School of Medicine, P.O. Box 800383, ef cient p53 activation upon 5- uorouracil treatment that Charlottesville, VA 22908. Phone: 434-982-0076; Fax: 434-243-9789; involves activated p38-MAPK (19). E-mail: [email protected] Under physiologic conditions the levels of p53 protein doi: 10.1158/1541-7786.MCR-13-0485 and its activity are low, but exposing cells to stress results Ó2013 American Association for Cancer Research. in p53 induction and activation (21). Activated p53 348 Mol Cancer Res; 12(3) March 2014 Downloaded from mcr.aacrjournals.org on October 6, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst December 20, 2013; DOI: 10.1158/1541-7786.MCR-13-0485 p53 and p38-MAPK Response to T-Type Ca2þ Channels Inhibition accumulates as a tetramer in the cell nucleus and acts as a blockers as chemotherapeutic agents that can induce cancer transcription factor, mediating expression of cell cycle-reg- cell death. ulating, senescence-inducing, and proapoptotic genes (22). Previously, mibefradil was shown to decrease proliferation of Materials and Methods esophageal carcinoma cells by increasing transcription of the Cell culture and drug treatment cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 Colon carcinoma HCT116 p53wt and p53-deleted (8). This suggested to us that cellular responses to T-type 2þ mutant were described previously (35) and were maintained Ca channels might be p53 dependent. in McCoy 5A medium (Life Technologies) supplemented In general, p53 is phosphorylated by protein kinases of with 10% FBS (Life Technologies). CaCo2 colon adeno- the phosphatidylinositol 3-kinase-related kinase family carcinoma cell line was purchased from American Type that are activated by DNA damage, such as ataxia telan- Culture Collection and cultured in Minimum Essential giectasia mutated (ATM), ataxia telangiectasia mutated Medium (MEM) supplemented with L-glutamine, nones- and Rad3 related (ATR), and DNA-dependent protein sential amino acids, and 20% FBS (Life Technologies). The kinase (DNAPK). Phosphorylation releases p53 from its cell lines were not authenticated. Mibefradil and TTL1177 interactions with MDM2 ubiquitin ligase, which stabilizes were generously provided by Tau Therapeutics LLC. fi p53, and allows for its speci c DNA binding (23, 24). NNC55–0396 hydrate was from Sigma-Aldrich. The Following DNA damage induced by ionizing radiation or p38-MAPK inhibitor SB203580 was from Santa Cruz UV radiation, at least 17 Ser/Thr residues are phosphor- Biotechnology. All inhibitors were prepared in dimethyl fi ylated within p53. Signi cant redundancies are observed sulfoxide (DMSO; 10 mmol/L stock) and diluted in media as a single residue can be subject to phosphorylation by before the use. multiple protein kinases, for example, Ser15-p53 is report- edly phosphorylated by ATM, ATR, DNAPK, p38- Drug-induced growth inhibition MAPK, and ERK (extracellular signal–regulated kinase) HCT116 cells were seeded in 96-well plates at 1,000 cells kinases (reviewed in ref. 21). Interestingly, some of the per well. Twenty-four hours later, cells were treated in protein kinases that interact and phosphorylate p53 þ triplicates with different concentrations of T-type Ca2 belong to the calmodulin-dependent kinase super family channels inhibitors (mibefradil or TTL1177). After contin- (which includes Chk1, Chk2, and death associated protein uous exposure to drug for 4 days, cells were fixed with kinase 1 and 3; ref. 25), and these may phosphorylate p53 2þ trichloroacetic acid (10%) and stained with sulforhodamine in response to pathologic Ca signaling. 2þ B solution (0.1%; ref. 36). Percentage of growth inhibition Cross-talk is apparent between T-type Ca channel signaling and mitogen-activated protein kinase (MAPK) was determined after background subtraction based on the comparison of the absorbance signal of drug-treated cells pathways with consequences for p53. Mobilization of 2þ 2þ versus the control. Ca through T-type Ca channels (Cav 3.1; CACNA1G) produces a transient decrease in activity of Raf/MEK/ERK (26). Although ERK tends to induce prosurvival pathways, Plasmid DNA and siRNA transfection and growth stress-activated MAPKs, such as p38-MAPK and JNK (c- inhibition jun– – NH2 kinase), are linked to induction of apoptosis (27, CaCo2 cells were transfected with pcDNA3.1 p53wt or 28). In particular, upon UV-induced DNA damage or pcDNA3.1 empty vector plasmids [kind gift from Dr. A. osmotic shock, p38-MAPK phosphorylates p53 at Ser46 Dutta, University of Virginia (UVA), Charlottesville, VA] (29) or Ser33 (30), thus stabilizing p53. Moreover, UV- using lipofectamine LTX and plus reagent (Life Technolo- induced DNA damage activates p38-MAPK to activate gies) according to the manufacturer's instructions. Colonies casein kinase 2 (CK2), which in turn also phosphorylates were selected for 8 weeks in media containing selection p53 at Ser392, increasing p53 transcriptional activity (31– antibiotic geneticin G418 (600 mg/mL; Life Technologies). In RNAi experiments the cells were transfected with 25 33). Thus, p38-MAPK supports increased p53 levels and