TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.

only GFP constructs did not vary in LNCaP and PC3 cells. However, we observed that the transfection efficiency of GFP-tagged TRPM8 in PC3 cells was higher than in LNCaP. PC3 cells showed massive cell death after 48 h of transfection with GFP-TRPM8.

Transfection
All transfection experiments were performed with FuGene HD transfection reagent (Roche, Indianapolis, IN). Briefly, plasmid was mixed with FuGene HD reagent (1:3 ratio) in 200-500 µl of serum free-medium and left for 30 min at RT. The complex was then added to the 6-well or 100 mm plate containing 0.5 mL or 3 mL of serum-free medium, and around 1 g plasmid per mL of medium was used. After 8 h of transfection, complete medium containing 10 % FBS was added, and incubated for 64 h. Control cells were processed in the same way as treated cells and were incubated with either equal volumes of FuGENE HD or transfected with vector alone.

Immunocytochemistry and immunohistochemistry
For immunocytochemical analysis, treated cells were seeded onto 4-well chamber slides, fixed, permeabilized with ice-cold methanol and blocked for 1 h using 3% BSA in PBS. Cells were incubated with primary antibody and Alexa Fluor conjugated secondary antibody for 60 min at room temperature. Before mounting, the slides were washed with PBS and incubated for 5 min with a 1:100 dilution of 4'-6-Diamidino-2-phenylindole (DAPI) for nuclear staining. For immunohistochemical analysis, prostate tissue array (PR208) sections were deparaffinized in xylene, rehydrated in graded ethanol solutions. Antigen retrieval was carried out with 10 mM citrate buffer (pH 6) at boiling temperature for 60 min and permeabilization in 0.1% Triton-X-100. The tissue sections were incubated with primary antibodies for DHT/Testosterone, TRPM8 and androgen receptor (AR) for overnight, incubated with donkey Alexa Fluor conjugated secondary antibodies. The images were captured using confocal microscopy (Olympus BX61 Fluoview, Minneapolis, MN) at 40X magnification.

Chromatin immunoprecipitation (ChIP) assay
Chromatin was sheared by sonication method. The anti-DHT/testosterone or control IgG antibody was used to immunoprecipitate the chromatin processed from LNCaP, PC3 and HEK/TRPM8 control and testosterone-induced cells for overnight at 4 0 C followed by DNA purification and elution. The ChIP-enriched DNA was subjected to end filling using End-It™ For the analysis of DHT/testosterone binding to TRPM8 promoter, the ChIP DNA immunoprecipitated by anti-DHT/testosterone and anti-IgG antibody was used as template for ChIP specific primers which were designed to cover the Regions 1-10 (R1, R2, R3, R4, R5, R6, R8, R9 and R10) of TRPM8 gene promoter (ref|NW_004929306.1) and were used to perform quantitative RT-PCR following standard protocol. The coefficient of androgen/AR interaction with different regions (R1-R10) in the TRPM8 promoter was quantified and represented graphically. The ChIP specific R1-R10 primer sequences are listed in Supplementary table 1.

Real-Time PCR (RT-PCR) analysis
Total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA). 1 g of RNA was subjected to reverse transcription using the Superscript III reverse transcriptase cDNA synthesis kit (Roche Applied Science, Indianapolis, IN). Reverse transcription reaction was carried out in a thermal cycler for 30 minutes at 55°C followed by 5 minutes at 85°C. Real-time PCR analysis was then performed in triplicate using the SYBR Green PCR Master Mix (Applied Biosystems).
The following PCR conditions were used: 95°C for 10 minutes, followed by 40 cycles at 95°C/10 sec and 60°C/30 sec. The fold enrichment of androgen interaction with different regions (R1-R10) in the trpm8 promoter was quantified and was normalized to ChIP DNA immunoprecipitated by anti-IgG antibodies.

Reverse-transcription PCR.
Total RNA was isolated from control and treated cells using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the standard protocol. Using 1 g of RNA as a template, first-strand cDNA was synthesized using Superscript III reverse transcriptase (Roche Applied Science, Indianapolis, IN). PCR analysis using 100 ng of first-strand cDNA was completed using specific primers (Supplementary Table 2). GAPDH was used as an internal control. The PCR reaction was performed using standard conditions in an AB Applied Biosystems Thermocycler (model 9700). PCR amplified products were electrophoresed on 1.5% agarose gel.

LC/MS
LC-MS/MS spectra was generated following standard protocol (see SMethods) at University of Florida. Briefly, the enzymatically digested samples were injected onto a capillary trap (LC Packings PepMap) and de-salted for 5 min with a flow rate of 3 µl / min of 0.1% v/v acetic acid. potential and ion spray voltage was set to 225 V and 2400 V, respectively. The informationdependent acquisition (IDA) mode of operation was employed in which a survey scan from m/z 400-1800 was acquired followed by collision-induced dissociation (CID) of the four most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 seconds, respectively. The obtained spectra was analysed by using scaffold (™) software version 3_00_07 (Portland, OR). The cut off for these putative identified proteins were set to 99 % identity.

Ubiquitination assay
For the ubiquitination assay, whole cell lysates (500 μg protein/sample) were incubated with UbiCapture-Q Matrix (VWR International) by gentle agitation at 4°C overnight to pull down all ubiquitinated proteins according to the manufacturer's instructions. After washing three times, captured proteins were eluted with 2× SDS-PAGE loading buffer and analyzed by western blotting using anti-TRPM8 antibody.

Biotinylation assay
Biotinylation assays were performed using Pierce Cell Surface Protein Isolation kit (Thermo Scientific). Three 10 cm 2 dishes of 80%-90% confluent LNCaP cells were harvested. Cells were washed twice with PBS and incubated with 0.25 mg/ml of sulfo-NHS-SS-biotin in PBS for 30 min. After addition of 300 μl of quenching solution, cells were gently scrapped and centrifuged at 1000× g for 3 min, washed with TBS and centrifuged again. Cells were then lysed with 500 μl of lysis buffer (Thermo Scientific) supplemented with 1% PMSF and 1% protease inhibitors (ProteCEASE). After sonication on ice (five 1 s bursts), cells were incubated on shaker for 30 min at 4°C. After centrifugation at 10,000 × g for 2 min at 4°C, supernatants were transferred to a new tube. Neutravidin beads (Pierce) were prepared as described by Thermo Scientific and incubated with supernatant on a rotating shaker for 60 min at RT. After centrifugation at 1000 × g for 1 min, beads were washed three times with 400 μl of wash buffer.
Elution was performed with 120 μl of sample buffer containing DTT by heating for 4 min at 95°C. Samples were then processed for Western blot analysis and visualized by using an anti-TRPM8 antibody.

Cell cycle analysis by fluorescence-activated cell sorting (FACS)
The 8

TUNEL assay
The TUNEL assay was done using in situ cell death detection kit (Roche) in accordance with the manufacturer's protocol. In brief, control and treated LNCaP cells were fixed in 4-well chambered slides with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Next, cells were incubated with a TUNEL reaction mixture for 60 min at 37°C in a humidified incubator.
The slides were washed 3 times with phosphate-buffered saline (PBS), and the incorporated biotin-dUTP was detected under a fluorescence microscope (Olympus BX61 Fluoview, Minneapolis, MN) at 40X magnification.

Clonogenic assay
The single cell suspension of LNCaP and PC3 control and treated cells were prepared following imaging system mounted on an Zeiss-AXIO observed D1 microscope, equipped with a DeltaRAM excitationlight source or with a Ratiomaster 5 imaging system (PhotonTechnology International) equipped with a Cool-snap HQ2(Roper) camera.

Preparation of the TRPM8 Protein from Human Embryonic Kidney (HEK-293) Cells
HEK-293 cells stably expressing TRPM8 were grown to 70-80% confluence, washed, and collected with PBS. Cells were harvested and resuspended in NCB buffer, containing 500 mM NaCl, 50 mM NaH2PO4, 20 mM Hepes, 10% glycerol, pH 7.5, with the addition of 1mM of protease inhibitor PMSF, 5 mM β-mercaptoethanol. Then the cells were lysed by the freezethawing method and centrifuged at low speed to remove cell debris and DNA. The supernatant was further centrifuged at 40,000 x g for 2.5 h, and the pellet was resuspended in NCB buffer with the addition of a protease inhibitor mixture (Roche Applied Science), 0.1% Nonidet P-40 (Roche Applied Science), and 0.5% dodecylmaltoside (Calbiochem). The suspension was incubated overnight at 4 °C on a shaker with gentle agitation and then centrifuged for 1 h at 40,000 x g. Further, the TRPM8 protein was purified by immunoprecipitation with anti-Myc-IgG conjugated to A/G protein magnetic beads (Thermo Scientific Pierce), following the procedure provided by the manufacturer. All steps of the purification were performed at 4 °C.
For the planar lipid bilayer experiments, the protein was eluted with Myc-peptide (50 µg / ml).

p53 transcription factor assay
The p53 activation in nuclear extracts of PC cells was monitored using the ELISA-based TransAM™ p53 kit (TransAM p53, Active Motif, CA) according to the manufacturer's protocol.
2g of total nuclear protein were loaded onto a 96-well plate coated with an immobilized oligonucleotide containing a p53 consensus binding site. Anti-p53 and anti-rabbit HRP were used to quantify the amount of bound p53 protein. The HRP signal was developed by a substrate provided by the manufacturer, and samples were analyzed with an ELISA reader at 450 nm. The OD value for negative control was deducted from all the test samples.

Statistical Analysis
Statistical analyses were performed using Origin 8.0 software (Microcal Software Inc., Northampton, MA). Statistical significance was calculated using one-way ANOVA combined with Fisher's LSD test and data were presented as mean ± SEM. Statistical differences are presented at * P <0.05, * * P <0.01 and * * * P <0.001.