Cav1.3 channel α1D protein is overexpressed and modulates androgen receptor transactivation in prostate cancers

doi:10.1016/j.urolonc.2013.05.011

Urologic Oncology: Seminars and Original Investigations

Volume 32, Issue 5, July 2014, Pages 524-536

https://doi.org/10.1016/j.urolonc.2013.05.011 Get rights and content

Abstract

Widespread use of L-type calcium channel blockers for treating hypertension has led to multiple epidemiologic studies to assess the risk of prostate cancer incidence. These studies revealed a reverse correlation between the likelihood of prostate cancer risk and the use of L-type calcium channel blockers among men without family history but the mechanism was not clear. In this study, we examined the expression profiles of multiple L-type calcium channel genes in prostate cancers and determined their functional roles in androgen receptor (AR) transactivation and cell growth. By reanalyzing the ONCOMINE database, we found that L-type calcium channel CACNA1D gene expression levels in cancer tissues were significantly higher than noncancer tissues in 14 of 15 published complementary deoxyribonucleic acid microarray data sets, of which 9 data sets showed an increase of 2- to 17-folds. Quantitative polymerase chain reaction and immunostaining experiments revealed that CACNA1D gene and its coding protein α_1D were highly expressed in prostate cancers, especially in castration-resistant diseases, compared with benign prostate tissues. Consistent with the notion of CACNA1D as an ERG-regulated gene, CACNA1D gene expression levels were significantly higher in prostate cancers with TMPRSS2-ERG gene fusion compared with the cases without this gene fusion. Blocking L-type channel’s function or knocking down CACNA1D gene expression significantly suppressed androgen-stimulated Ca²⁺ influx, AR transactivation, and cell growth in prostate cancer cells. Taken together, these data suggest that CACNA1D gene overexpression is associated with prostate cancer progression and might play an important role in Ca²⁺ influx, AR activation, and cell growth in prostate cancer cells.

Introduction

Calcium signaling is a common mechanism involved in the majority of cellular functions, and Ca²⁺ homeostasis is tightly modulated by multiple channel mechanisms in all excitable and many nonexcitable cells [1], [2], [3], [4]. Functional machineries for calcium influx and intracellular calcium release exist in prostate cancer cells, and maintenance of intracellular Ca²⁺ pool is required for cell growth, whereas blocking Ca²⁺ influx reduces cell growth and invasion as well as prostate-specific antigen (PSA) secretion [5], [6], [7], [8]. It has been shown that androgens increase intracellular calcium level that could be suppressed by antiandrogens and L-type calcium channel blocker verapamil [9], [10]. However, excessive elevation of intracellular calcium level was associated with reduced androgen receptor (AR) expression in prostate cancer LNCaP cells [11], and blocking L-type calcium channeL-mediated Ca²⁺ influx suppressed castration-induced apoptotic cell death in prostate epithelial cells [12], [13]. These early studies indicate that Ca²⁺ influx is involved in cell growth, invasion, and death of prostate cancer cells.

L-type calcium channels consist of multiple membrane proteins, of which the α₁ proteins are the major functional unit for calcium influx. There are 4 L-type α₁ proteins: α_1C, α_1D, α_1F, and α_1S. The α₁ proteins are associated with auxiliary subunits (β_1–4, α₂δ_1–4, and γ_1–8) [1], [2]. Although these calcium channel proteins are mostly expressed in electrically excitable cells, α_1D was also found in nonexcitable cells, including AR-positive prostate cancer VCaP and LAPC-4 cells [14].

Owing to wide use of L-type calcium channel blockers for treating hypertension since 1990s, multiple epidemiologic surveys were conducted to assess the risk of prostate cancer incidence in blocker users [15], [16], [17], [18], [19]. Although multiple reports found no excessive risk of prostate cancer incidence, interestingly, 2 reports showed a reverse correlation between the likelihood of prostate cancer risk and the use of calcium channel blockers among men without family history [18], [19]. These studies indicate a potential role of L-type calcium channels in prostate cancer development.

To understand whether L-type calcium channels are associated with prostate cancer development and progression, we conducted a comprehensive analysis of calcium channel gene expression profiles in prostate cancers using complementary deoxyribonucleic acid (cDNA) microarray data, quantitative polymerase chain reaction (PCR) methods, as well as immunohistochemistry assays. Our analyses revealed that L-type calcium channel Ca_v1.3 α₁ subunit CACNA1D gene is highly expressed in prostate cancers at both the messenger ribonucleic acid (mRNA) and protein levels, and its expression level is significantly increased in TRMPSS2-ERG gene fusion cases and castration-resistant cases. Functional analyses demonstrated that Ca_v1.3 α_1D protein is involved in androgen-stimulated Ca²⁺ influx, AR transactivation, and cell growth. These results suggest that L-type channel protein α_1D might play an important role in prostate cancer development and progression.

Section snippets

Cell lines, plasmid construct, small interfering RNA (siRNA), antibodies, and channel blockers

A set of 8 human prostate cell lines, including benign prostate epithelial cells RWPE-1 and BPH-1, as well as prostate cancer cells LAPC-4, LNCaP, C4-2, 22RV1, PC-3, and DU145 were used in this study and described previously in our publications [20], [21]. Mammalian expression construct pcDNA6/V5-His-Ca_v1.3e (plasmid 26576), harboring the rat CACNA1D gene cDNA [22], was obtained from Addgene (Cambridge, MA). siRNAs for the negative control, CACNA1D and CACNA1C genes as well as Ca_v1.3 α_1D and

L-type calcium channel CACNA1D gene is overexpressed in prostate cancers

Previous epidemiologic surveys showed a reduced prostate cancer incidence in L-type calcium channel blocker users compared with the general population [18], [19], indicating a potential involvement of L-type channels in prostate cancer development. To understand whether L-type calcium channels are involved in prostate cancers, we took the advantage of published cDNA microarray data sets available at the ONCOMINE database and analyzed the expression profiles of L-type calcium channel α₁ subunit

Discussion

In this study, we found that L-type calcium channel Ca_v1.3 α₁ subunit CACNA1D gene at the mRNA level and its coding protein α_1D is overexpressed in most prostate cancers, especially in castration-resistant cases. We also found that primary prostate cancers with TMPRSS2-ERG fusion showed a significantly higher CACNA1D gene expression in comparison with the negative cases. Further analysis determined that Ca_v1.3 channel is involved in androgen-induced Ca²⁺ influx and that blocking Ca_v1.3 function

Conclusions

L-type calcium channel Cα_v1.3 α₁ subunit CACNA1D gene is highly expressed in prostate cancers compared with case-matched benign prostate tissues, especially in CRPC as well as in cases with TMPRSS2-ERG gene fusion. Functional Cα_v1.3 channel is required for androgen-stimulated Ca²⁺ influx, AR-mediated gene expression, and cell growth in prostate cancer cells. Taken together, it is plausible that CACNA1D gene overexpression might be involved in prostate cancer development and progression.

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Hypospadias is a congenital genitourinary malformation, with the etiology remaining complex and poorly understood. Despite several genes have been identified to be associated with the risk of hypospadias, current understanding of the susceptibility loci for hypospadias yet remained largely improved. The CACNA1D gene encodes calcium voltage-gated channel subunit alpha 1d and may be involved in androgen signaling. However, the genetic susceptibility of CACNA1D associated with hypospadias has yet been addressed.
To evaluate the association between CACNA1D polymorphisms and the susceptibility to hypospadias.
In this study, we accessed the association between two potential regulatory SNPs (rs3774491 and rs898415) within CACNA1D and hypospadias in a cohort of southern Chinese population which comprised of 740 cases and 948 healthy individuals. Both SNP and haplotypic associations were evaluated. Bioinformatic analysis of the regulatory abilities of the CACNA1D SNPs were carried out by utilizing public ChIP-seq and DNase-seq data. The expression of Cacna1d in mouse external genitalia and testis was evaluated by qPCR.
We found that the allele C in rs3774491 and allele G in rs898415 were significantly associated with an increased risk of hypospadias, especially for proximal hypospadias. Further model-based genotypic analyses showed that these association were prominent in additive model and recessive models. Bioinformatic analyses indicated that both SNPs were colocalized with DNase and multiple histone marker across multiple tissues, suggesting the regulatory potentials for these variants. Cacna1d is detectable in both testis and external genitalia of mouse, but the expression level was more prominent in testis than that in external genitalia, suggesting tissue-specific differences in its expression.
Our findings provide evidence for CACNA1D as a novel predisposing gene for hypospadias, shedding new light on the genetic basis of malformation of urinary tract. Further investigations are warranted to elucidate the functional implication of CACNA1D underlying the development of hypospadias.
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CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer
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Citation Excerpt :
In addition, it is a known target of the common somatic alternation present in fifty percent of PCa patients, TMPRSS2:ERG [13]. To date, the single PCa study exploring its protein form, CaV1.3, found it increased Cai2+ following androgen stimulation of androgen receptor (AR) positive PCa cell lines [14]. Channel inhibition with calcium channel blockers (CCB), lead to reduced cell proliferation [14].
Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Ca_i²⁺) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Ca_i²⁺ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.
The interactive effect of amlodipine and chemotherapeutic agents in lung cancer cells
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Chemoresistance is one of the major challenges in lung cancer treatment. The identification of new chemotherapeutic therapies with increased efficacy and minimal toxicity over those currently in use is a key area of current research. Calcium channel blockers are emerging as anticancer agents in lung and other cancers. This study evaluates the ability of amlodipine to sensitize non-small cell lung cancer (NSCLC) cells to the first-line chemotherapeutics used in lung cancer treatment. Cell viability in the presence of carboplatin, gemcitabine, pemetrexed, or paclitaxel alone or combined with amlodipine was evaluated in A549 and H1299 NSCLC cell lines using the colorimetric MTT cell proliferation assay. Nonlinear regression curve fit analyses were used to calculate half-maximal inhibitory concentrations (IC₅₀). Sensitization was estimated by the ratio of the IC₅₀ of the monotherapy to the IC₅₀ of the combined therapy that included that drug (R-value). We found that amlodipine significantly reduced cell viability in a dose-dependent manner, with IC₅₀ values of 23 and 25.66 μM in A549 and H1299 NSCLC cells, respectively. In addition, amlodipine combined with carboplatin, gemcitabine, pemetrexed, or paclitaxel had a significant sensitization effect on A549 and H1299 NSCLC cells compared to their single-drug treatments (R > 1.0). Our findings confirm the ability of amlodipine to sensitize NSCLC cells to first-line chemotherapy treatments.
Association Between the Overall Risk of Prostate Cancer and Use of Calcium Channel Blockers: A Systematic Review and Meta-analysis
2020, Clinical Therapeutics
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Thus, the anti–T-type CCBs may inhibit the growth of cancer cells.4 One study suggested that CCB use significantly reduced the risk of PCa with a fusion gene (OR, 0.38; 95% CI, 0.19–0.78) of the androgen-regulated gene TMPRSS2 and the oncogene ERG (TMPRSS2:ERG or T2E).22,30,31 Therefore, TMPRSS2:ERG or T2E might be a reversal factor in the relationship between CCB use and PCa risk.
Although calcium channel blockers (CCBs) are now commonly prescribed to treat hypertension as a first-line drug therapy, their impact on prostate cancer (PCa) is unclear. This systematic review and meta-analysis was conducted to determine the association between CCB use and the overall risk of PCa.
PubMed, EMBASE, and Cochrane were searched up to December 26, 2019, stratified according to statistical method of outcome [odd ratios (ORs), relative ratios (RRs), hazard ratios (HRs)] and cumulative duration of CCB use. The quality assessment of included studies was evaluated by using the Newcastle–Ottawa Scale. Fixed effects models were used to study the association between CCB use and the risk of PCa. Between-study heterogeneity was quantified by using Cochran's Q-statistic and I² statistics. Sensitivity analysis was performed by excluding the studies one by one, and publication bias was analyzed by using funnel plots.
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CCB use had a tendency to increase the overall risk of PCa, and cumulative duration of CCB use might also be positively correlated with the overall risk of PCa.
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Ca^2 + is a ubiquitous and dynamic second messenger molecule that is induced by many factors including receptor activation, environmental factors, and voltage, leading to pleiotropic effects on cell function including changes in migration, metabolism and transcription. As such, it is not surprising that aberrant regulation of Ca^2 + signals can lead to pathological phenotypes, including cancer progression. However, given the highly context-specific nature of Ca^2 +-dependent changes in cell function, delineation of its role in cancer has been a challenge. Herein, we discuss the distinct roles of Ca^2 + signaling within and between each type of cancer, including consideration of the potential of therapeutic strategies targeting these signaling pathways.

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¹: This work was partially supported by grants from DoD PCRP program (W81XWH-09-1-0455) and KUMC Valk Foundation to Dr Benyi Li, and grants from China Natural Science Foundation to Dr Benyi Li (NSFC #81172427) and Dr Jun Yang (NSFC #81101927). This project was also supported by the “Chutian Scholar” program funded by Hubei Province of China dedicated to China Three Gorges University.

²: These authors contributed equally to this work.

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Translational studies in urologic oncologyCav1.3 channel α1D protein is overexpressed and modulates androgen receptor transactivation in prostate cancers1

Abstract

Introduction

Section snippets

Cell lines, plasmid construct, small interfering RNA (siRNA), antibodies, and channel blockers

L-type calcium channel CACNA1D gene is overexpressed in prostate cancers

Discussion

Conclusions

Cell Calcium

J Biol Chem

Biochem Biophys Res Commun

Am J Hypertens

Ann Epidemiol

J Biol Chem

Am J Pathol

Cancer Cell

Cancer Cell

Cancer Cell

J Biol Chem

Biochem Pharmacol

Biochem Pharmacol

Trends Pharmacol Sci

J Biol Chem

Voltage-gated calcium channels and disease

Biofactors

The ss subunit of voltage-gated Ca2+ channels

Physiol Rev

Calcium in tumour metastasis: new roles for known actors

Nat Rev Cancer

Permeation and gating mechanisms in store-operated CRAC channels

Front Biosci

Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines

Int J Cancer

Inhibition by anticonvulsants of prostate-specific antigen and interleukin-6 secretion by human prostate cancer cells

Anticancer Res

Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations

Biol Reprod

Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP

Endocrinology

Role of calcium in the programmed death of rat prostatic glandular cells

Prostate

Calcium channel antagonists delay regression of androgen-dependent tissues and suppress gene activity associated with cell death

Prostate

1{alpha},25-Dihydroxyvitamin D3 inhibits growth of VCaP prostate cancer cells despite inducing the growth-promoting TMPRSS2:ERG gene fusion

Endocrinology

Cancer risk in users of calcium channel blockers

Hypertension

Cancer risk of hypertensive patients taking calcium antagonists

J Hypertens

Inverse association between prostate cancer and the use of calcium channel blockers

Cancer Epidemiol Biomarkers Prev

Small-interfering RNA-induced androgen receptor silencing leads to apoptotic cell death in prostate cancer

Mol Cancer Ther

Phosphoinositide 3-OH kinase p85alpha and p110beta are essential for androgen receptor transactivation and tumor progression in prostate cancers

Oncogene

Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines

J Neurosci

Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer

Science

Translational studies in urologic oncology
Ca_v1.3 channel α_1D protein is overexpressed and modulates androgen receptor transactivation in prostate cancers¹

The ss subunit of voltage-gated Ca²⁺ channels