- Email: [email protected]
Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells
Accepted Manuscript Title: Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells Authors: Bhakti R. Pathak, Ananya A. Breed, Priyanka Deshmukh, Smita D. Mahale PII: DOI: Reference:
S0960-0760(18)30108-0 https://doi.org/10.1016/j.jsbmb.2018.02.012 SBMB 5123
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
9-6-2017 9-2-2018 20-2-2018
Please cite this article as: Pathak BR, Breed AA, Deshmukh P, Mahale SD, Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells, Journal of Steroid Biochemistry and Molecular Biology (2010), https://doi.org/10.1016/j.jsbmb.2018.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells Bhakti R Pathak1#, Ananya A Breed1, Priyanka Deshmukh and Smita D Mahale
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Division of Structural Biology, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Mumbai, India. 1
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Equal contribution by both authors Correspondence to:
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Dr. Bhakti R Pathak
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Division of Structural Biology,
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National Institute for Research in Reproductive Health (ICMR)
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Jehangir Merwanji Street, Parel, Mumbai-400012, India.
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Phone: 91-22-24192014, Fax: 91-22-24139412
Highlights
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Email: [email protected], [email protected]
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Three overlapping fragments of human CRISP-3 promoter were cloned and characterized for promoter activity. Stable clones of LNCaP and PC3 cell line carrying 1133 bp CRISP-3 promoter –reporter construct were generated and high reporter activity was observed in LNCaP cells which endogenously express CRISP-3 Endogenous CRISP-3 expression was responsive to DHT in LNCaP CRISP-3 promoter showed histone H3 acetylation in LNCaP but not in PC3 cells AR occupied CRISP-3 promoter in LNCaP cells and was associated with acetylated histone H3
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Abstract Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer
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(CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1
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cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency
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of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide
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molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP
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PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is
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epigenetically regulated by AR.
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Keywords: - CRISP3; Androgen receptor; DHT; Prostate cancer
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1. Introduction Prostate cancer is the most commonly diagnosed cancer among men in Western countries. CRISP3 (Cysteine Rich Secretory Protein-3) is upregulated in prostate cancer [1, 2] and appears to be a promising prognostic marker for prostate cancer. Overexpression of CRISP3
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along with loss of PTEN has been shown to define a subgroup of prostate cancer which shows recurrence and worst prognosis [3, 4]. Association of overexpression of CRISP3 with disease recurrence is probably due to its role in prostate cancer invasion [5]. CRISP3 upregulation occurs right from the early stages of prostate tumorigenesis that is high-grade intraepithelial neoplasia up to the most advanced stage of castration resistant prostate cancer (CRPC) [6, 7]. As proliferation of prostate cancer cells depends on androgen signaling, androgen deprivation
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therapy (ADT) is the first line of therapy for prostate cancer [8]. Eventually 10-20% of the patients who undergo ADT, show castration-resistant prostate cancer (CRPC) with proliferation
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of tumor cells at the castrate levels of circulating testosterone and increase in PSA levels [9].
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Androgen receptor (AR) still remains a key player in progression and maintenance of CRPC
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where it shows hyperactivation either due to mutation/s, overexpression or alterations in interacting proteins [10, 11]. Whether AR directly regulates expression of CRISP3 during
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prostate cancer development and progression still remains unexplored.
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CRISP3 was originally described as an androgen-dependent protein in mouse salivary gland [12] and lacrimal glands [13]. In mice, males expressed high CRISP3 mRNA in the
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salivary glands than females and the levels significantly reduced post castration. Both mouse and human CRISP3 genes have shown presence of androgen response elements (ARE) in the
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promoter suggesting possible regulation by AR [14, 15]. Stimulation of human submandibular gland cells with dehydroepiandrosterone (DHEA) has been shown to increase CRISP3 mRNA [15]. Though there is no direct evidence for CRISP3 expression to be regulated by AR in human
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prostate cancer cells, a study by Bjartell et al. [6] which looked at levels of PSA and CRISP3 in the sera of prostate cancer patients undergoing orchiectomy suggested that CRISP3 expression by the prostate cancer cells may not be as strongly regulated by androgens as PSA. On the other hand, CRISP3 overexpression was shown to be frequently associated with TMPRSS-ERG fusion positive prostate tumors and CRISP3 was shown as a direct target of the ERG transcription
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factor [16]. Using prostate cancer cell line as a model, we have earlier shown that overexpression of CRISP3 in prostate tumor may maintain higher PSA expression [5]. In order to study the regulation of CRISP3 expression in prostate cancer cells, different overlapping fragments of CRISP3 promoter were cloned in a luciferase reporter vector and their
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activity was assessed in LNCaP and PC3 cells. Luciferase activity was evaluated upon transient as well as stable transfections and in the presence or absence of DHT. Effect of DHT on expression of endogenous CRISP3 was also investigated in androgen-dependent LNCaP cells. CRISP3 promoter was found to be epigenetically regulated which was demonstrated by ChIP in LNCaP and PC3 cells.
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2. Materials and Methods
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2.1 Reagents and chemicals
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pGL3 basic luciferase reporter vector (E1751), pRL-TK (Renilla luciferase reporter vector,
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E2241), firefly luciferase assay system and Renilla luciferase assay system (E2810) were purchased from Promega Corp (Madison, WI). X-tremeGENE HP DNA transfection reagent
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(06366236001) was purchased from Roche diagnostics (Mannheim, Germany). The antibodies used for this study included anti-CRISP3 antibody (AF2397, R&D Systems, Minneapolis, MN,
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USA), anti-PAP antibody (P905470A, US Biological, Swampscott, MA), HRP-conjugated rabbit anti-goat (Santacruz Biotech, CA, USA) and rabbit anti-mouse secondary antibodies (Dako,
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Agilent technologies, Santa Clara, CA, USA), ChIP grade anti-Histone H3 (06-755, Merck Millipore, Bangalore, India) and anti-acetyl-Histone H3 (06-599, Millipore). ECL Plus Western
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Blotting substrate (32132) was obtained from Pierce (Rockford, IL, USA). The reagents used for ChIP assay were as follows: Formaldehyde (12765, Qualigens, Thermo Fisher scientific, India),
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Glycine (24755, Thermo Fisher scientific), Salmon sperm DNA Protein A agarose beads (16157, Millipore), Proteinase K (03115836001, Roche diagnostics), Glycogen (G0885, Sigma, St. Louis, MO, USA) and Phenol/chloroform (77618, Sigma). Trizol, DNase I, oligo dT primer, and Superscript III reverse transcriptase were purchased from Life technologies (Invitrogen, Carlsbad, CA, USA). RPMI medium 1640 (23400-021), PR free RPMI medium 1640 (Phenol
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red free medium, 11835-030), Fetal bovine serum (16140-071) and CSS (Charcoal stripped FBS, 12676-011) were also from Life technologies. 2.2 Cell culture
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Two prostate cancer cell lines PC3 and LNCaP were obtained from National Centre for Cell Science, Pune, India. Both the cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) containing penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in the presence of 5% CO2 in a humidified environment. PC3 and LNCaP stable clones were maintained in the same medium with 600 µg/ml of G418. RWPE-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained on keratinocyte serum-free medium supplemented with bovine pituitary extract (0.05 mg/ml),
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epidermal growth factor (5 ng/ml) and gentamycin (0.4 mg/ml) as recommended by ATCC.
For steroid stimulation experiments, LNCaP -1133UTR stable clones were maintained in steroid
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free medium (PR free RPMI with 10% CSS) till they were ready for plating for luciferase assay.
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For the assay, cells were plated in a 12 well plate in complete growth medium or steroid free medium with or without 5nM DHT. Cells were allowed to grow till they reached confluence with
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change of growth medium every third day.
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2.3 Generation of CRISP3 promoter constructs
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Six different fragments comprising of region upstream of transcription start site (TSS) of human CRISP3 gene and ending either at the TSS (-247 to -1bp, -806 to -1bp, -1133 to -1bp) or at the
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5’UTR (-247 to +88bp, -806 to +88bp, -1133 to +88 bp) were amplified by PCR using specific primers and then cloned into the pGL3-basic luciferase reporter vector via NheI and XhoI restriction sites. These specific restriction sites were incorporated in forward and reverse primers
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respectively. The sequences of the primers are outlined in supplementary table (Table S1). All plasmids were verified by sequencing to confirm generation of respective reporter constructs. 2.4 Transfection and Luciferase reporter assay
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PC3 (1x105 cells/well) and LNCaP (2x105 cells/well) cells were plated in 24 well plate. Next day cells were co-transfected with 350 ng of respective CRISP3 promoter reporter constructs or with empty vector (pGL3) along with 35 ng of pRL-TK using XtremeGene HP transfection reagent in 1:1 ratio. Cell lysates were made 48h post transfection and used for measuring the firefly and Renilla luciferase activities on 96-well plate reader (Biotek Synergy 2, Winooski, VT) using
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luciferase reporter assay system according to the manufacturer’s protocol. The readings were normalized for the internal Renilla control and expressed as relative fold difference.
For luciferase assays involving stable clones including the DHT stimulation and TSA treatment experiments, cells were grown as mentioned in section 2.2. Firefly luciferase activity was measured in the lysates as indicated above. For normalization, total protein concentration in the
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cell lysate was estimated using BCA protein estimation kit (Pierce). Luciferase readings were
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normalized with protein estimation and expressed as relative fold difference.
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2.5 Generation of stable clones
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PC3 and LNCaP cells were plated in 35 mm dish and following day they were co-transfected either with 2 ug of -1133UTR construct or with pGL3-basic empty vector along with 200 ng of
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pcDNA3.1+ vector for neomycin selection, using X-tremeGENE HP transfection reagent in 1:1
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ratio. Next day, cells were split 1:3 and subjected to G418 (600 ug/ml) selection for 2 weeks. The clones were screened for the presence of stable integration of the luciferase construct by PCR.
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2.6 RT PCR
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LNCaP cells were plated in four 35 mm dishes in complete growth medium. After 72 h, the medium of two dishes were replaced with steroid free medium and from remaining two dishes with steroid free medium containing 5 nM DHT. Cells were kept in the presence of DHT for two
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different time points (72 h and 96 h). Cells were harvested in Trizol reagent . PC3 and RWPE-1 cells were grown in 60 mm dishes and next day either 100 ng/ml Trichostatin A (TSA) was added or they were kept as it is. After 48 h, cells were harvested in Trizol reagent. Total RNA was isolated according to the manufacturer’s instructions. The first strand of cDNA was generated from 2 μg of total RNA using SuperScript III Reverse Transcriptase and the companion reagents according to the manufacturer's instructions. PCR reactions were performed 6
using template from the reverse transcription reaction (1 μl), 2.5 units Taq polymerase (Invitrogen), 1 μM gene-specific primers, and the companion reagents for 30 cycles. The primer sequences used were indicated in Table S1. GAPDH was chosen as the internal control.
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2.7 Western blotting LNCaP cells were plated (8x105 cells/well) in 6 well plate in complete growth medium. After 72 h of plating, the medium of two wells was replaced with steroid free medium and from other two wells with steroid free medium containing 5 nM DHT. After 72 h or 96 h incubation time in respective medium, cells were incubated in serum free medium. Conditioned media were harvested after 48 h and secreted proteins were precipitated using trichloroacetic acid (TCA) as described earlier [17]. Total secreted proteins were separated on 12.5% SDS-PAGE and
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transferred onto nitrocellulose membrane for detection of CRISP3 and PAP (Prostatic acid
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phosphtase). The blots were probed with anti-CRISP3 antibody (1:1000) and anti-PAP antibody
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(1:500) followed by their respective secondary antibodies (1:2000) and detection using chemiluminence. For PC3 cells, conditioned media from TSA (100ng/ml) treated or untreated
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cells were harvested after 48 h and used for TCA precipitation followed by Western blotting as described above. 1/5th of the TCA precipitated proteins were separated on a parallel gel which
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was silver stained to be used as loading control. 2.8 Chromatin immunoprecipitation (ChIP)
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For Chromatin immunoprecipitation assay, PC3 and LNCaP cells were grown in 10 cm dishes (3 dishes for each cell line) on complete growth medium and for DHT stimulation experiment
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LNCaP cells were grown either in steroid free medium or medium containing 5 nM DHT. Cells were crosslinked in 1% formaldehyde and incubated for 10 min at RT with gentle shaking
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followed by quenching with 0.125 M glycine. Cells were washed twice with cold 1X PBS and then harvested in 1X PBS supplemented with protease inhibitors. Cells were pelleted at 1,000 rpm for 5 min at 4°C. After the supernatant was aspirated, the cell pellets were lysed in ChIP lysis buffer (50 mM Tris-HCl/pH 8.1, 10 mM EDTA, 1% SDS, and a protease inhibitor cocktail). Lysates were sonicated for 10 mins, consisting of 30 second “on” pulses, followed by 30 seconds “resting time” to obtain fragments ranging from 200 to 1000 bp. The sonicated cell
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lysate was centrifuged, 1/10th fraction of the supernatant was removed for using as input and the remaining supernatant was divided in three tubes, diluted to 1ml with ChIP dilution buffer (16.7 mM Tris-HCl/pH 8.1, 167 mM NaCl, 0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, and a protease inhibitor cocktail) and taken for immunoprecipitation. After pre-cleaning of supernatant by Salmon sperm DNA Protein A agarose beads and centrifugation, the supernatant was
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incubated with 2 ug of respective antibodies overnight at 40C. The supernatant incubated with only agarose beads served as a negative control. Immunoprecipitation was performed at 4°C for 2 h to isolate antibody-bound chromatin by incubating the supernatant with Salmon sperm DNA Protein A agarose beads. The immunoprecipitated complexes were collected by centrifugation and washed once with low salt buffer (20 mM Tris-HCl/pH 8.1, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, and 2 mM EDTA), high salt buffer (20 mM Tris-HCl/pH 8.1, 500 mM NaCl, 0.1%
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SDS, 1% Triton X-100, and 2 mM EDTA), LiCl wash buffer (10 mM Tris-HCl/pH 8.1, 1% NP-
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40, 0.25 M LiCl, 1% sodium deoxycholate, and 1 mM EDTA) and twice with TE buffer (10 mM Tris-HCl/pH 8.1 and 1 mM EDTA). ChIP elution buffer (1% SDS and 100 mM NaHCO3) was
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then added to the pellet and kept on rotator for 15 min. The complex crosslinking was reversed
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by heating the supernatant with 20 µl of 5M NaCl at 65°C overnight. DNA was released by incubation with proteinase K (100 µg) and glycogen (25 µg) at 45oC for 2 h. DNA was recovered
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by phenol/chloroform extraction and ethanol precipitation. Purified DNA was subsequently used
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for conventional PCR using specific primers listed in Table S1.
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3. Results
3.1 Analyzing the human CRISP3 promoter activity in prostate cancer cell lines
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A sequence of ~1700bp upstream of TSS of human CRISP3 gene was subjected to ConTra promoter analysis [18] which identified two Androgen Response Elements (AREs). In order to
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study the transcriptional regulation of CRISP3 in prostate cancer cells, three overlapping CRISP3 promoter fragments were cloned in pGL3 basic vector upstream of luciferase reporter gene. The fragments were numbered as per the position of the base pairs from the TSS. The three CRISP3 promoter constructs which ended at the TSS contained fragments of -247 bp, -806 bp and -1133 bp respectively from the TSS (Fig. 1). The constructs which ended at the 5’ UTR (included 88 bp more than the earlier constructs) were labeled as -247 UTR, -806 UTR and -1133 UTR. The 8
longest promoter fragment contained two androgen response elements (AREs) as indicated in Fig. 1. All the six constructs were transfected in LNCaP (endogenous CRISP3 positive) and PC3 (CRISP3 negative) cells and luciferase activity was evaluated 48 h post transfection. Transfection efficiency was normalized by co-transfection of the cells with a construct expressing Renilla luciferase. All the three constructs without UTR region showed no luciferase
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activity in both the cell lines (Fig. 2) whereas all the three constructs with UTR showed strong reporter activity in both the cell lines suggesting that inclusion of this region restores the inititator (Inr) element along with probable downstream promoter element and imparts the basal promoter activity. As in transient transfections the CRISP3 promoter constructs were found to be active in both- CRISP3 expressing and non-expressing cell lines (with activity difference of only ~4 fold between them), it appeared that the endogenous promoter is probably regulated
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differently in PC3 and LNCaP cells. As the promoter inherently has strong basal activity, the
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endogenous promoter might be silenced by histone deacetylation in PC3 cells. .
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3.2 Evaluating epigenetic regulation of CRISP3 promoter in prostate cancer cells
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To investigate if CRISP3 promoter is regulated by histone deacetylation mediated silencing in PC3 cells, they were treated with Trichostatin-A (TSA, histone deacetylase inhibitor). It was
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observed that TSA treatment resulted in expression of CRISP3 transcript in PC3 cells (Fig. 3A).
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RWPE-1 cells which are immortalized normal prostate epithelial cells (and do not express CRISP3) were also treated with TSA and like PC3 they also showed CRISP3 expression by RT-
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PCR (Fig. 3B). TSA treatment of PC3 cells showed expression of CRISP3 protein though in very low amount (Fig. 3C). This data suggested that CRISP3 promoter is under histone
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deacetylase (HDAC) mediated regulation in prostate cancer cells. Treatment of PC3 cells with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, did not result in increase in CRISP3
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transcription (data not shown). In order to study the epigenetic regulation of CRISP3 promoter, stable clones of PC3 and LNCaP cells carrying the longest CRISP3 promoter fragment (-1133 UTR) with the luciferase reporter were generated. Clones were screened by PCR for the presence of the construct (data not shown). Three stable clones each of LNCaP (clone 2, 3, 5) and PC3 (clone K, J, M) along with an empty vector clone (LNCaP clone a and PC3 clone 8) were evaluated for the promoter 9
activity by the luciferase assay (Fig. 3D). In accordance with their endogenous CRISP3 expression status, LNCaP stable clones showed ~87 fold higher promoter activity than PC3 stable clones (Fig. 3D) demonstrating that stably integrated -1133UTR fragment was under epigenetic regulation like the endogenous CRISP3 promoter. Further, treatment of PC3 stable clones with TSA resulted in upregulation of the promoter activity similar to the effect observed
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on endogenous CRISP3 mRNA expression upon TSA treatment (Fig. 3E). 3.3 Effect of DHT on CRISP3 expression in LNCaP
As LNCaP is an androgen-sensitive cell line, effect of AR agonist DHT on CRISP3 expression was studied by growing it either in the presence or absence of DHT and evaluating CRISP3 expression at RNA (Fig. 4A) and protein level (Fig. 4B). DHT treatment of LNCaP cells led to
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significant increase in the CRISP3 RNA and protein levels. LNCaP cells growing in the absence
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of steroids were also subjected to TSA to check whether the downregulation of CRISP3
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expression by androgen deprivation is via histone deacetylation. TSA could reverse the downregulation of CRISP3 expression suggesting that DHT mediated effect on CRISP3
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expression involves histone acetylation.
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3.4 Effect of DHT on CRISP3 promoter activity
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To evaluate if 1133 bp CRISP3 promoter responds to DHT, luciferase assay was used to compare the promoter activity of the clones grown in complete growth medium, in medium lacking
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steroids or in medium supplemented with DHT. CRISP3 promoter activity was found to be reduced in cells grown in steroid free medium, as compared to the cells which were grown in
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complete growth medium (Fig. 5). DHT supplementation of the steroid-free medium resulted in an increase in the CRISP3 promoter activity which became almost similar to the cells grown in
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the complete growth medium. This data clearly demonstrated that CRISP3 promoter is regulated by DHT in prostate cancer cells. 3.5 Acetylation status of histone H3 at CRISP3 promoter and its response to DHT CRISP3 upregulation upon TSA treatment of PC3 cells suggested that CRISP3 promoter is regulated by HDACs. In order to confirm the histone H3 acetylation status at the endogenous 10
CRISP3 promoter, PC3 and LNCaP cells were subjected to ChIP assay. The three different primer sets used for ChIP-PCR are schematically shown in Fig. 6A. ChIP-PCR data revealed that CRISP3 promoter indeed shows more histone H3 acetylation in LNCaP cells than in PC3 cells in accordance with the expression status of the CRISP3 gene in these two cell lines (Fig. 6B).
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Further to determine the occupancy of CRISP3 promoter by AR, LNCaP cells grown in the presence or absence of DHT were subjected to ChIP using anti-acetyl histone H3, anti-histone H3 and anti-AR antibody. Growth of cells in the presence of DHT increased the occupancy of AR at both the AREs in CRISP3 promoter and also resulted in concomitant increase in the histone H3 acetylation at the proximal region of the CRISP3 promoter (Fig. 6C). This data
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provided evidence for androgen dependent epigenetic regulation of CRISP3 promoter.
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4. Discussion
CRISP3 which belongs to CRISP family of proteins is predominantly expressed in the salivary
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glands [15], neutrophils [19] and the prostate and to a lower extent in epididymis and ovary [20].
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Its expression is highly upregulated in prostate cancer and is associated with disease recurrence. CRISP3 overexpression was found to be associated with ERG fusion positive cancers [3, 16] and
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CRISP3 was shown to be a direct target of ERG. But irrespective of ERG fusion status, CRISP3
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overexpression was reported to be associated with PSA recurrence in all prostate tumors [3] suggesting an alternate regulatory mechanisms for CRISP3 expression. Previous study from our laboratory has also shown that CRISP3 and PSA are co-regulated as knockdown of CRISP3
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resulted in reduction in PSA expression. In this study we attempted to characterize CRISP3 promoter and understand the role of AR in regulation of CRISP3 expression in prostate cancer
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cells.
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Six different promoter-reporter constructs were tested for promoter activity. The shortest promoter fragment which included 247 bp above TSS lacked ARE and the longest fragment which included 1133 bp above TSS contained both the AREs. By luciferase reporter assays, we observed that promoter constructs ending at the TSS had no promoter activity which might be due to disruption of the initiator (Inr) element. Inr element usually encompasses -2 to +4 bp with respect to TSS and is found in both TATA- containing and TATA-less promoters [21]. We also 11
observed that this region contained only one ETS binding site in contrast to three sites reported by Ribeiro et al. [16]. Careful evaluation of the sequence of the 3 ETS binding sites given by Ribeiro et al. revealed that the authors had looked at the sequence upstream of the first ATG of the mature CRISP3 protein which is encoded by the second exon (not considering the ATG in the first exon which codes for the signal peptide). Two of the three reported ETS binding sites
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actually map to the first intron of the CRISP3 gene and the third ETS site maps to the first exon encompassing the 5’UTR of the CRISP3 transcript and hence those are not part of the upstream promoter region.
Upon transient transfection, the three promoter constructs showed promoter activity in PC3 cells which do not endogenously express CRISP3 which was only ~4 fold lower than that observed in
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the LNCaP cells. The difference in the transient activity of the promoter in both these cell lines,
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though small, might be due to the presence of AR and ETV1 in LNCaP cells [22] but not in PC3. These results also indicated that the basal activity of CRISP3 promoter is strong and in non-
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expressing cells, it could be regulated by epigenetic mechanism involving histone
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acetylation/deaetylation. AR is reported to regulate gene expression by epigenetic mechanisms. Epigenetic regulation of CRISP3 gene via histone deacetylation was also indicated by its re-
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expression in TSA treated PC3 and RWPE1 cells. In order to study if the cloned CRISP3
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promoter fragment was also subjected to epigenetic regulation; it needed to be stably integrated in the genome. Hence, stable clones were generated with the longest CRISP3 promoter reporter construct in PC3 and LNCaP cells. Interestingly, the difference in the promoter activity of stable
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clones of LNCaP and PC3 was much higher (~87 fold) than that obtained after transient transfection of these two cell lines (~4 fold), further supporting the epigenetic regulation of the
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CRISP3 promoter in these cells.
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AR is reported to stimulate gene expression through epigenetic mechanisms by means of recruiting histone acetyl transferases (HATs) like SRC-1, 2 & 3 (steroid receptor coactivator), CBP, p300 and PCAF [23]. Apart from its role as transcriptional activator, AR is also shown to epigenetically repress gene expression. AR mediated epigenetic gene repression occurs by its interaction with proteins like Alien, Hey1 and SHP that mediate histone deacetylation or with proteins like EZH2 or LSD1 which mediate histone methylation and demethylation respectively 12
[23]. In order to confirm the involvement of AR in regulating the CRISP3 expression, effect of DHT on CRISP3 expression was first evaluated in LNCaP cells. CRISP3 expression was enhanced in response to DHT at RNA and protein level. Further luciferase activity of the LNCaP stable clones carrying CRISP3 promoter reporter construct was found to be significantly reduced in cells grown in steroid-free medium and was restored upon treatment of cells with DHT. This
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data indicated that AR positively regulated CRISP3 expression in ligand-dependent manner and the AREs predicted at -327 bp and -815 bp of the CRISP3 gene were functional. Histone H3 acetylation status of the endogenous CRISP3 promoter in LNCaP and PC3 cells was determined by ChIP. In accordance with the absence of CRISP3 protein in PC3 and their response to TSA, histone H3 acetylation was lower in PC3 as compared to LNCaP cells. At the same time histone H3 occupancy at the TSS was higher in PC3 than in LNCaP (Fig. 6B). This is in agreement with
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the fact that nucleosome positioning at transcription start sites regulates gene expression by
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controlling the access of specific transcription factors and RNA polymerase [24]. H3 occupancy is found to be lower at the TSS in a transcriptionally active gene than in an inactive gene. A zone
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of nucleosomal depletion or a “dip” in histone H3 occupancy has been demonstrated by Lin et al.
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at MLH1 1a promoter region in MLH1 expressing cells as compared to non-expressing cells
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whereas acetylated histone H3 occupancy was overall higher in the expressing cells [25].
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LNCaP cells express mutant AR (T877A mutation) which is responsive to DHT [26]. Occupancy of the CRISP3 promoter by AR was demonstrated in LNCaP cells by ChIP. AR was found to occupy the AREs in the CRISP3 promoter when LNCaP cells were grown in presence of DHT
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but not in cells grown in steroid-free medium. AR binding to promoter corresponded with the increased histone H3 acetylation at the promoter in the cells grown in presence of DHT. Based
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on this data we postulate that in AR positive prostate cancer cells, AR occupies CRISP3 promoter in ligand-dependent manner and with the help of HATs brings about acetylation of
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histones at the proximal CRISP3 promoter resulting in nucleosomal repositioning and CRISP3 transcription. Dalhman et al. [7] have compared the effect of short-term and long-term ADT on CRISP3 expression and found that CRISP3 upregulation persists during therapy. This was due to parallel high levels of AR which is known to confer resistance to antiandrogens and maintain expression of androgen-responsive genes like PSA [27]. In case of normal prostate cells where CRISP3 expression is absent or minimal, AR may still be occupying the promoter in ligand13
dependent manner but may have different interacting partners which impart repressive role to AR. This possibility needs to be evaluated in normal prostate tissue by ChIP assay. On the other hand, in case of AR-independent prostate cancer cells like PC3, CRISP3 gene remains silenced probably due to maintenance of histone deacetylation. Whether over-expression of AR in AR-
independent CRISP3 regulation in them cannot be ruled out.
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independent cells can upregulate CRISP3 remains to be investigated. Possibility of AR-
In conclusion, CRISP3 gene is under epigenetic regulation and its expression is responsive to DHT in AR positive prostate cancer cells. The two AREs present in the CRISP3 promoter are functional and occupied by AR in LNCaP cells in a ligand-dependent manner. The -1133 UTR fragment of human CRISP3 promoter is subjected to the similar epigenetic regulation like the
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endogenous CRISP3 promoter. This promoter-reporter construct appears to be specifically active
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in AR positive prostate cancer cells. Its specificity needs to be validated in other androgenresponsive cancer cell lines in order to ascertain its utility in bioassays aimed at screening of
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novel AR agonists/antagonists as well as in prostate cancer specific transgenic strategies.
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Acknowledgements
The research work related to this publication (RA/485/05-2017) was supported by grants from
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the Indian Council of Medical Research (ICMR) and DAE-BRNS, India (2011/37B/40/BRNS). Technical assistance provided by Ms. Summaiya Patel who was a trainee in the laboratory is
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acknowledged. We acknowledge Dr. Srabani Mukherjee and Ms. Nanda Joshi for their help with
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DNA sequencing.
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References: 1. Asmann YW, Kosari F, Wang K, Cheville JC, Vasmatzis G. Identification of differentially expressed genes in normal and malignant prostate by electronic profiling of expressed sequence tags. Cancer Res 2002; 62: 3308–14.
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2. Bjartell AS, Al-Ahmadie H, Serio AM, Eastham JA, Eggener SE, Fine SW, Udby L, Gerald WL, Vickers AJ, Lilja H, Reuter VE, Scardino PT. Association of cysteine-rich secretory protein 3 and beta-microseminoprotein with outcome after radical prostatectomy. Clin Cancer Res. 2007; 13: 4130-8.
3. Grupp K, Kohl S, Sirma H, Simon R, Steurer S, Becker A, Adam M, Izbicki J, Sauter G, Minner S, Schlomm T, Tsourlakis MC. Cysteine-rich secretory protein 3 overexpression
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4. Noh BJ, Sung JY, Kim YW, Chang SG, Park YK. Prognostic value of ERG, PTEN,
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CRISP3 and SPINK1 in predicting biochemical recurrence in prostate cancer. Oncol Lett.
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5. Pathak BR, Breed AA, Apte S, Acharya K, Mahale SD. Cysteine-rich secretory protein 3
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6. Bjartell A, Johansson R, Bjo¨rk T, Gadaleanu V, Lundwall A, Lilja H, Kjeldsen L, Udby L. Immunohistochemical detection of cysteine rich secretory protein 3 in tissue and in
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7. Dahlman A, Edsjo¨ A, Hallde´n C, Persson JL, Fine SW, Lilja H, Gerald W, Bjartell A. Effect of androgen deprivation therapy on the expression of prostate cancer biomarkers
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MSMB and MSMB binding protein CRISP3. Prostate Cancer Prostatic Dis 2010; 13: 369–75.
8. Perrotti M, Pantuck A, Rabbani F, Israeli RS, Weiss RE. Review of staging modalities in clinically localized prostate cancer. Urology 1999; 54: 208-14. 9. Perner S, Cronauer MV, Schrader AJ, Klocker H, Culig Z, Baniahmad A. Adaptive responses of androgen receptor signaling in castration-resistant prostate cancer. Oncotarget 2015; 6: 35542-55. 15
10. Armstrong CM, Gao AC. Drug resistance in castration resistant prostate cancer: resistance mechanisms and emerging treatment strategies. Am J ClinExp Urol. 2015; 3: 64-76. 11. Kobayashi T, Inoue T, Kamba T, Ogawa O. Experimental evidence of persistent androgen-receptor-dependency in castration-resistant prostate cancer. Int J Mol Sci.
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2013; 14: 15615-35.
12. Haendler B, Krätzschmar J, Theuring F, Schleuning WD. Transcripts for cysteine-rich secretory protein-1 (CRISP-1; DE/AEG) and the novel related CRISP-3 are expressed under androgen control in the mouse salivary gland. Endocrinology 1993; 133: 192-8.
13. Haendler B, Toda I, Sullivan DA, Schleuning WD. Expression of transcripts for cysteinerich secretory proteins (CRISPs) in the murine lacrimal gland. J Cell Physiol. 1999; 178:
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14. Schwidetzky U, Haendler B, Schleuning WD. Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-3 (CRISP-3 gene). Biochem J.
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15. Laine M, Porola P, Udby L, Kjeldsen L, Cowland JB, Borregaard N, Hietanen J, Ståhle M, Pihakari A, Konttinen YT. Low salivary dehydroepiandrosterone and androgen-
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regulated cysteine-rich secretory protein 3 levels in Sjögren's syndrome. Arthritis Rheum.
16. Ribeiro FR, Paulo P, Costa VL, Barros-Silva JD, Ramalho-Carvalho J, JerónimoC, Henrique R, Lind GE, Skotheim RI, Lothe RA, Teixeira MR. Cysteine-rich secretory
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protein-3 (CRISP3) is strongly up-regulated in prostate carcinomas with the TMPRSS2ERG fusion gene. PLoS One. 2011; 6: e22317.
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17. Pathak BR, Breed AA, Nakhawa VH, Jagtap DD, Mahale SD. Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific. Asian J Androl 2010;
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12: 677–89.
18. Broos S, Hulpiau P, Galle J, Hooghe B, Van Roy F, De Bleser P. ConTra v2: a tool to identify transcription factor binding sites across species, update 2011. Nucleic Acids Res. 2011; 39 (Web Server issue):W74-8. 19. Kjeldsen L, Cowland JB, Johnsen AH, Borregaard N. SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis16
specific gene product and a rodent sperm-coating glycoprotein. FEBS Lett 1996; 380: 246–50. 20. Kra¨tzschmar J, Haendler B, Eberspaecher U, Roosterman D, Donner P, Schleuning WD. The human cysteine-rich secretary protein (CRISP) family. Primary structure and tissue distribution of CRISP-1, CRISP-2 and CRISP-3. Eur J Biochem 1996; 236: 827–36.
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21. Butler JE, Kadonaga JT. The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 2002; 16: 2583-92.
22. Tomlins SA, Laxman B, Dhanasekaran SM, Helgeson BE, Cao X, Morris DS, Menon A, Jing X, Cao Q, Han B, Yu J, Wang L, Montie JE, Rubin MA, Pienta KJ, Roulston D, Shah RB, Varambally S, Mehra R, Chinnaiyan AM. Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer. Nature 2007; 448:
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595-9.
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23. Cai C, Yuan X, Balk SP. Androgen receptor epigenetics. Transl Androl Urol. 2013; 2: 148-57.
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24. Andreu-Vieyra C, Lai J, Berman BP, Frenkel B, Jia L, Jones PA, Coetzee GA. Dynamic
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nucleosome-depleted regions at androgen receptor enhancers in the absence of ligand in prostate cancer cells. Mol Cell Biol. 2011; 31: 4648-62.
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25. Lin JC, Jeong S, Liang G, Takai D, Fatemi M, Tsai YC, Egger G, Gal-Yam EN, Jones
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PA. Role of nucleosomal occupancy in the epigenetic silencing of the MLH1 CpG island. Cancer Cell 2007; 12: 432-44. 26. Koochekpour S. Androgen receptor signaling and mutations in prostate cancer. Asian J
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Androl. 2010; 12: 639-57.
27. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers
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CL. Molecular determinants of resistance to antiandrogen therapy. Nat. Med. 2004;
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10:33-9
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Legends Fig.1 Human CRISP3 promoter sequence Sequence of 1133 bp upstream and 88 bp downstream of TSS of human CRISP3 gene is shown. In the given sequence, 5’ UTR region (88 bp) is underlined and position of two androgen elements
(AREs)
as
identified
through
ConTra
Promoter
analysis
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response
(http://bioit.dmbr.ugent.be/contrav2/index.php?v2) are shown in a box. Location of -247 bp, -806 bp and -1133 bp is indicated by an arrow. Fig.2 Analysis of human CRISP3 promoter activity
PC3 and LNCaP cell lines were transiently transfected using constructs with 5’ UTR (-247 UTR,
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-806 UTR, -1133 UTR) and without 5’ UTR (-247, -806, -1133) along with Renilla luciferase
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expressing vector pRL-TK. The firefly luciferase activity was measured in cell lysates 48 h post transfection and normalized with Renilla luciferase activity. Normalized values obtained for cells
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transfected with empty vector (pGL3) were considered as one and relative fold difference was
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plotted. Each experiment was performed thrice and data plotted as mean ± SEM of three experiments. Statistical analysis was performed using One way ANOVA test. ***p<0.001
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compared to pGL3.
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Fig.3 Regulation of CRISP3 expression by epigenetic mechanism A&B) RT-PCR for CRISP3 (149 bp) and GAPDH (330 bp) in PC3 (A) and RWPE-1 (B) cells
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treated with 100 ng/ml of TSA for 24 h (Lanes 2 and 5) or left untreated as control (Lanes 1 and 4). RNA extracted from these cells was converted to cDNA and used for PCR. CRISP3
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expression was evaluated by singlet and biplex PCR where GAPDH ampliconwas a loading control. Lanes 3 and 6 are water control. C) Detection of CRISP3 protein in the TCA precipitated
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proteins from the conditioned media of untreated and TSA treated PC3 cells by Western blotting (top panel). Silver stained gel (bottom panel) is shown for loading control D) Luciferase activity of LNCaP -1133 UTR stable clones (-1133 UTR clone 2, 3, 5 and empty vector clone a) and PC3 -1133 UTR stable clones (-1133 UTR clone K, J, M and empty vector clone 8) was measured by luciferase assay. Parental LNCaP and PC3 cells (untransfected) were taken as a negative control. Firefly luciferase activity was normalized with protein estimation of respective clones. 18
Normalized values obtained for untransfected LNCaP and PC3 were considered as one and relative fold difference was plotted. E) PC3 -1133 UTR stable clones (J and M) were treated with TSA at indicated doses for 48 h. Firefly luciferase activity was normalized with protein estimation. Normalized values obtained for untreated PC3 stable clones were considered one and relative fold difference was plotted. Each luciferase assay (D and E) was performed at least
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thrice and data plotted as mean ± SEM of three experiments. Statistical analysis was performed using One way ANOVA test. **p<0.01, ***p<0.001 compared to untransfected parental cell line (D) and untreated PC3 stable clone (E). Fig.4 CRISP3 expression is androgen dependent
A) LNCaP cells were either grown in the presence of 5 nM DHT or in medium lacking steroids
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for two different time points (72 h and 96 h). CRISP3 expression was evaluated by RT PCR
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where GAPDH amplicon was a loading control. Lane 1 is 100 bp ladder. Lanes 2 and 6 represent RT reactions of cells grown in steroid free medium for 72 h and 96 h respectively whereas lanes
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3 and 7 are “no RT” reactions of the same samples. Lanes 4 and 8 represent RT reactions of cells
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grown in DHT supplemented medium for 72 h and 96 h respectively whereas lanes 5 and 9 are “no RT” reactions of the same samples. Lane 10 is water control. B) Western blot analysis of
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CRISP3 protein expression in TCA precipitated conditioned media of the cells grown as
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mentioned above. The blot was probed with anti-PAP antibody (top panel) and anti-CRISP3 antibody (bottom panel). PAP (Prostatic acid phosphatase) whose expression is not affected by DHT was used as a loading control. Arrows on the left indicate position of the protein molecular
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weight marker. C) Western blot analysis of CRISP3 protein expression in TCA precipitated conditioned media of the cells grown in steroid free medium alone or in presence of two different
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concentration of TSA. The blot was probed as indicated above.
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Fig.5 1133 UTR-luciferase construct shows androgen responsiveness LNCaP stable clones (Cl.3 & Cl.5) carrying -1133 UTR luciferase construct were grown either in complete growth medium or in medium lacking steroids or in steroid-free medium supplemented with DHT. Luciferase activity was measured and normalized with protein estimation. Normalized values obtained for cells grown on complete growth medium were considered as one and relative fold difference was plotted. Each experiment was performed thrice and data plotted 19
as mean ± SEM of all three experiments. Statistical analysis was performed using One way ANOVA test. ‘*’ indicates p <0.001 compared to cells grown in complete growth medium and ‘#’ indicates p <0.001 compared to cells grown in DHT containing medium. Fig.6 Transcriptionally active CRISP3 promoter shows histone H3 acetylation which is
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responsive to DHT A) Schematic outline of CRISP3 promoter region and the location of the primers used for ChIPPCR. Set 1, Set 2 and Set 3 are three primer pairs which cover the TSS with 5’ UTR and the two AREs respectively. B) PC3 and LNCaP cells were subjected to ChIP using anti-histone H3 (H3) and anti-acetylated H3 (AcH3) antibody. Immunoprecipitated DNA was analyzed by PCR with primer Set 1. Immunoprecipitation with “No antibody” (only beads) served as a negative control.
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Data shown is from a representative experiment and the individual experiment was performed
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twice. C) LNCaP cells grown either in steroid free medium or in medium containing DHT were
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subjected to ChIP using anti-AR antibody, anti-histone H3 and anti-AcH3 antibody. Immunoprecipitated DNA was analyzed by PCR with all three primer sets. Data shown is from a
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S0960-0760(18)30108-0 https://doi.org/10.1016/j.jsbmb.2018.02.012 SBMB 5123
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
9-6-2017 9-2-2018 20-2-2018
Please cite this article as: Pathak BR, Breed AA, Deshmukh P, Mahale SD, Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells, Journal of Steroid Biochemistry and Molecular Biology (2010), https://doi.org/10.1016/j.jsbmb.2018.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells Bhakti R Pathak1#, Ananya A Breed1, Priyanka Deshmukh and Smita D Mahale
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Division of Structural Biology, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Mumbai, India. 1
#
Equal contribution by both authors Correspondence to:
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Dr. Bhakti R Pathak
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Division of Structural Biology,
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National Institute for Research in Reproductive Health (ICMR)
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Jehangir Merwanji Street, Parel, Mumbai-400012, India.
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Phone: 91-22-24192014, Fax: 91-22-24139412
Highlights
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Email: [email protected], [email protected]
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Three overlapping fragments of human CRISP-3 promoter were cloned and characterized for promoter activity. Stable clones of LNCaP and PC3 cell line carrying 1133 bp CRISP-3 promoter –reporter construct were generated and high reporter activity was observed in LNCaP cells which endogenously express CRISP-3 Endogenous CRISP-3 expression was responsive to DHT in LNCaP CRISP-3 promoter showed histone H3 acetylation in LNCaP but not in PC3 cells AR occupied CRISP-3 promoter in LNCaP cells and was associated with acetylated histone H3
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Abstract Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer
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(CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1
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cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency
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of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide
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molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP
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PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is
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epigenetically regulated by AR.
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Keywords: - CRISP3; Androgen receptor; DHT; Prostate cancer
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1. Introduction Prostate cancer is the most commonly diagnosed cancer among men in Western countries. CRISP3 (Cysteine Rich Secretory Protein-3) is upregulated in prostate cancer [1, 2] and appears to be a promising prognostic marker for prostate cancer. Overexpression of CRISP3
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along with loss of PTEN has been shown to define a subgroup of prostate cancer which shows recurrence and worst prognosis [3, 4]. Association of overexpression of CRISP3 with disease recurrence is probably due to its role in prostate cancer invasion [5]. CRISP3 upregulation occurs right from the early stages of prostate tumorigenesis that is high-grade intraepithelial neoplasia up to the most advanced stage of castration resistant prostate cancer (CRPC) [6, 7]. As proliferation of prostate cancer cells depends on androgen signaling, androgen deprivation
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therapy (ADT) is the first line of therapy for prostate cancer [8]. Eventually 10-20% of the patients who undergo ADT, show castration-resistant prostate cancer (CRPC) with proliferation
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of tumor cells at the castrate levels of circulating testosterone and increase in PSA levels [9].
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Androgen receptor (AR) still remains a key player in progression and maintenance of CRPC
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where it shows hyperactivation either due to mutation/s, overexpression or alterations in interacting proteins [10, 11]. Whether AR directly regulates expression of CRISP3 during
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prostate cancer development and progression still remains unexplored.
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CRISP3 was originally described as an androgen-dependent protein in mouse salivary gland [12] and lacrimal glands [13]. In mice, males expressed high CRISP3 mRNA in the
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salivary glands than females and the levels significantly reduced post castration. Both mouse and human CRISP3 genes have shown presence of androgen response elements (ARE) in the
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promoter suggesting possible regulation by AR [14, 15]. Stimulation of human submandibular gland cells with dehydroepiandrosterone (DHEA) has been shown to increase CRISP3 mRNA [15]. Though there is no direct evidence for CRISP3 expression to be regulated by AR in human
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prostate cancer cells, a study by Bjartell et al. [6] which looked at levels of PSA and CRISP3 in the sera of prostate cancer patients undergoing orchiectomy suggested that CRISP3 expression by the prostate cancer cells may not be as strongly regulated by androgens as PSA. On the other hand, CRISP3 overexpression was shown to be frequently associated with TMPRSS-ERG fusion positive prostate tumors and CRISP3 was shown as a direct target of the ERG transcription
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factor [16]. Using prostate cancer cell line as a model, we have earlier shown that overexpression of CRISP3 in prostate tumor may maintain higher PSA expression [5]. In order to study the regulation of CRISP3 expression in prostate cancer cells, different overlapping fragments of CRISP3 promoter were cloned in a luciferase reporter vector and their
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activity was assessed in LNCaP and PC3 cells. Luciferase activity was evaluated upon transient as well as stable transfections and in the presence or absence of DHT. Effect of DHT on expression of endogenous CRISP3 was also investigated in androgen-dependent LNCaP cells. CRISP3 promoter was found to be epigenetically regulated which was demonstrated by ChIP in LNCaP and PC3 cells.
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2. Materials and Methods
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2.1 Reagents and chemicals
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pGL3 basic luciferase reporter vector (E1751), pRL-TK (Renilla luciferase reporter vector,
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E2241), firefly luciferase assay system and Renilla luciferase assay system (E2810) were purchased from Promega Corp (Madison, WI). X-tremeGENE HP DNA transfection reagent
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(06366236001) was purchased from Roche diagnostics (Mannheim, Germany). The antibodies used for this study included anti-CRISP3 antibody (AF2397, R&D Systems, Minneapolis, MN,
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USA), anti-PAP antibody (P905470A, US Biological, Swampscott, MA), HRP-conjugated rabbit anti-goat (Santacruz Biotech, CA, USA) and rabbit anti-mouse secondary antibodies (Dako,
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Agilent technologies, Santa Clara, CA, USA), ChIP grade anti-Histone H3 (06-755, Merck Millipore, Bangalore, India) and anti-acetyl-Histone H3 (06-599, Millipore). ECL Plus Western
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Blotting substrate (32132) was obtained from Pierce (Rockford, IL, USA). The reagents used for ChIP assay were as follows: Formaldehyde (12765, Qualigens, Thermo Fisher scientific, India),
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Glycine (24755, Thermo Fisher scientific), Salmon sperm DNA Protein A agarose beads (16157, Millipore), Proteinase K (03115836001, Roche diagnostics), Glycogen (G0885, Sigma, St. Louis, MO, USA) and Phenol/chloroform (77618, Sigma). Trizol, DNase I, oligo dT primer, and Superscript III reverse transcriptase were purchased from Life technologies (Invitrogen, Carlsbad, CA, USA). RPMI medium 1640 (23400-021), PR free RPMI medium 1640 (Phenol
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red free medium, 11835-030), Fetal bovine serum (16140-071) and CSS (Charcoal stripped FBS, 12676-011) were also from Life technologies. 2.2 Cell culture
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Two prostate cancer cell lines PC3 and LNCaP were obtained from National Centre for Cell Science, Pune, India. Both the cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) containing penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in the presence of 5% CO2 in a humidified environment. PC3 and LNCaP stable clones were maintained in the same medium with 600 µg/ml of G418. RWPE-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained on keratinocyte serum-free medium supplemented with bovine pituitary extract (0.05 mg/ml),
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epidermal growth factor (5 ng/ml) and gentamycin (0.4 mg/ml) as recommended by ATCC.
For steroid stimulation experiments, LNCaP -1133UTR stable clones were maintained in steroid
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free medium (PR free RPMI with 10% CSS) till they were ready for plating for luciferase assay.
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For the assay, cells were plated in a 12 well plate in complete growth medium or steroid free medium with or without 5nM DHT. Cells were allowed to grow till they reached confluence with
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change of growth medium every third day.
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2.3 Generation of CRISP3 promoter constructs
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Six different fragments comprising of region upstream of transcription start site (TSS) of human CRISP3 gene and ending either at the TSS (-247 to -1bp, -806 to -1bp, -1133 to -1bp) or at the
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5’UTR (-247 to +88bp, -806 to +88bp, -1133 to +88 bp) were amplified by PCR using specific primers and then cloned into the pGL3-basic luciferase reporter vector via NheI and XhoI restriction sites. These specific restriction sites were incorporated in forward and reverse primers
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respectively. The sequences of the primers are outlined in supplementary table (Table S1). All plasmids were verified by sequencing to confirm generation of respective reporter constructs. 2.4 Transfection and Luciferase reporter assay
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PC3 (1x105 cells/well) and LNCaP (2x105 cells/well) cells were plated in 24 well plate. Next day cells were co-transfected with 350 ng of respective CRISP3 promoter reporter constructs or with empty vector (pGL3) along with 35 ng of pRL-TK using XtremeGene HP transfection reagent in 1:1 ratio. Cell lysates were made 48h post transfection and used for measuring the firefly and Renilla luciferase activities on 96-well plate reader (Biotek Synergy 2, Winooski, VT) using
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luciferase reporter assay system according to the manufacturer’s protocol. The readings were normalized for the internal Renilla control and expressed as relative fold difference.
For luciferase assays involving stable clones including the DHT stimulation and TSA treatment experiments, cells were grown as mentioned in section 2.2. Firefly luciferase activity was measured in the lysates as indicated above. For normalization, total protein concentration in the
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cell lysate was estimated using BCA protein estimation kit (Pierce). Luciferase readings were
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normalized with protein estimation and expressed as relative fold difference.
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2.5 Generation of stable clones
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PC3 and LNCaP cells were plated in 35 mm dish and following day they were co-transfected either with 2 ug of -1133UTR construct or with pGL3-basic empty vector along with 200 ng of
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pcDNA3.1+ vector for neomycin selection, using X-tremeGENE HP transfection reagent in 1:1
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ratio. Next day, cells were split 1:3 and subjected to G418 (600 ug/ml) selection for 2 weeks. The clones were screened for the presence of stable integration of the luciferase construct by PCR.
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2.6 RT PCR
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LNCaP cells were plated in four 35 mm dishes in complete growth medium. After 72 h, the medium of two dishes were replaced with steroid free medium and from remaining two dishes with steroid free medium containing 5 nM DHT. Cells were kept in the presence of DHT for two
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different time points (72 h and 96 h). Cells were harvested in Trizol reagent . PC3 and RWPE-1 cells were grown in 60 mm dishes and next day either 100 ng/ml Trichostatin A (TSA) was added or they were kept as it is. After 48 h, cells were harvested in Trizol reagent. Total RNA was isolated according to the manufacturer’s instructions. The first strand of cDNA was generated from 2 μg of total RNA using SuperScript III Reverse Transcriptase and the companion reagents according to the manufacturer's instructions. PCR reactions were performed 6
using template from the reverse transcription reaction (1 μl), 2.5 units Taq polymerase (Invitrogen), 1 μM gene-specific primers, and the companion reagents for 30 cycles. The primer sequences used were indicated in Table S1. GAPDH was chosen as the internal control.
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2.7 Western blotting LNCaP cells were plated (8x105 cells/well) in 6 well plate in complete growth medium. After 72 h of plating, the medium of two wells was replaced with steroid free medium and from other two wells with steroid free medium containing 5 nM DHT. After 72 h or 96 h incubation time in respective medium, cells were incubated in serum free medium. Conditioned media were harvested after 48 h and secreted proteins were precipitated using trichloroacetic acid (TCA) as described earlier [17]. Total secreted proteins were separated on 12.5% SDS-PAGE and
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transferred onto nitrocellulose membrane for detection of CRISP3 and PAP (Prostatic acid
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phosphtase). The blots were probed with anti-CRISP3 antibody (1:1000) and anti-PAP antibody
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(1:500) followed by their respective secondary antibodies (1:2000) and detection using chemiluminence. For PC3 cells, conditioned media from TSA (100ng/ml) treated or untreated
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cells were harvested after 48 h and used for TCA precipitation followed by Western blotting as described above. 1/5th of the TCA precipitated proteins were separated on a parallel gel which
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was silver stained to be used as loading control. 2.8 Chromatin immunoprecipitation (ChIP)
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For Chromatin immunoprecipitation assay, PC3 and LNCaP cells were grown in 10 cm dishes (3 dishes for each cell line) on complete growth medium and for DHT stimulation experiment
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LNCaP cells were grown either in steroid free medium or medium containing 5 nM DHT. Cells were crosslinked in 1% formaldehyde and incubated for 10 min at RT with gentle shaking
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followed by quenching with 0.125 M glycine. Cells were washed twice with cold 1X PBS and then harvested in 1X PBS supplemented with protease inhibitors. Cells were pelleted at 1,000 rpm for 5 min at 4°C. After the supernatant was aspirated, the cell pellets were lysed in ChIP lysis buffer (50 mM Tris-HCl/pH 8.1, 10 mM EDTA, 1% SDS, and a protease inhibitor cocktail). Lysates were sonicated for 10 mins, consisting of 30 second “on” pulses, followed by 30 seconds “resting time” to obtain fragments ranging from 200 to 1000 bp. The sonicated cell
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lysate was centrifuged, 1/10th fraction of the supernatant was removed for using as input and the remaining supernatant was divided in three tubes, diluted to 1ml with ChIP dilution buffer (16.7 mM Tris-HCl/pH 8.1, 167 mM NaCl, 0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, and a protease inhibitor cocktail) and taken for immunoprecipitation. After pre-cleaning of supernatant by Salmon sperm DNA Protein A agarose beads and centrifugation, the supernatant was
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incubated with 2 ug of respective antibodies overnight at 40C. The supernatant incubated with only agarose beads served as a negative control. Immunoprecipitation was performed at 4°C for 2 h to isolate antibody-bound chromatin by incubating the supernatant with Salmon sperm DNA Protein A agarose beads. The immunoprecipitated complexes were collected by centrifugation and washed once with low salt buffer (20 mM Tris-HCl/pH 8.1, 150 mM NaCl, 0.1% SDS, 1% TritonX-100, and 2 mM EDTA), high salt buffer (20 mM Tris-HCl/pH 8.1, 500 mM NaCl, 0.1%
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SDS, 1% Triton X-100, and 2 mM EDTA), LiCl wash buffer (10 mM Tris-HCl/pH 8.1, 1% NP-
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40, 0.25 M LiCl, 1% sodium deoxycholate, and 1 mM EDTA) and twice with TE buffer (10 mM Tris-HCl/pH 8.1 and 1 mM EDTA). ChIP elution buffer (1% SDS and 100 mM NaHCO3) was
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then added to the pellet and kept on rotator for 15 min. The complex crosslinking was reversed
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by heating the supernatant with 20 µl of 5M NaCl at 65°C overnight. DNA was released by incubation with proteinase K (100 µg) and glycogen (25 µg) at 45oC for 2 h. DNA was recovered
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by phenol/chloroform extraction and ethanol precipitation. Purified DNA was subsequently used
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for conventional PCR using specific primers listed in Table S1.
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3. Results
3.1 Analyzing the human CRISP3 promoter activity in prostate cancer cell lines
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A sequence of ~1700bp upstream of TSS of human CRISP3 gene was subjected to ConTra promoter analysis [18] which identified two Androgen Response Elements (AREs). In order to
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study the transcriptional regulation of CRISP3 in prostate cancer cells, three overlapping CRISP3 promoter fragments were cloned in pGL3 basic vector upstream of luciferase reporter gene. The fragments were numbered as per the position of the base pairs from the TSS. The three CRISP3 promoter constructs which ended at the TSS contained fragments of -247 bp, -806 bp and -1133 bp respectively from the TSS (Fig. 1). The constructs which ended at the 5’ UTR (included 88 bp more than the earlier constructs) were labeled as -247 UTR, -806 UTR and -1133 UTR. The 8
longest promoter fragment contained two androgen response elements (AREs) as indicated in Fig. 1. All the six constructs were transfected in LNCaP (endogenous CRISP3 positive) and PC3 (CRISP3 negative) cells and luciferase activity was evaluated 48 h post transfection. Transfection efficiency was normalized by co-transfection of the cells with a construct expressing Renilla luciferase. All the three constructs without UTR region showed no luciferase
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activity in both the cell lines (Fig. 2) whereas all the three constructs with UTR showed strong reporter activity in both the cell lines suggesting that inclusion of this region restores the inititator (Inr) element along with probable downstream promoter element and imparts the basal promoter activity. As in transient transfections the CRISP3 promoter constructs were found to be active in both- CRISP3 expressing and non-expressing cell lines (with activity difference of only ~4 fold between them), it appeared that the endogenous promoter is probably regulated
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differently in PC3 and LNCaP cells. As the promoter inherently has strong basal activity, the
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endogenous promoter might be silenced by histone deacetylation in PC3 cells. .
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3.2 Evaluating epigenetic regulation of CRISP3 promoter in prostate cancer cells
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To investigate if CRISP3 promoter is regulated by histone deacetylation mediated silencing in PC3 cells, they were treated with Trichostatin-A (TSA, histone deacetylase inhibitor). It was
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observed that TSA treatment resulted in expression of CRISP3 transcript in PC3 cells (Fig. 3A).
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RWPE-1 cells which are immortalized normal prostate epithelial cells (and do not express CRISP3) were also treated with TSA and like PC3 they also showed CRISP3 expression by RT-
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PCR (Fig. 3B). TSA treatment of PC3 cells showed expression of CRISP3 protein though in very low amount (Fig. 3C). This data suggested that CRISP3 promoter is under histone
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deacetylase (HDAC) mediated regulation in prostate cancer cells. Treatment of PC3 cells with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, did not result in increase in CRISP3
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transcription (data not shown). In order to study the epigenetic regulation of CRISP3 promoter, stable clones of PC3 and LNCaP cells carrying the longest CRISP3 promoter fragment (-1133 UTR) with the luciferase reporter were generated. Clones were screened by PCR for the presence of the construct (data not shown). Three stable clones each of LNCaP (clone 2, 3, 5) and PC3 (clone K, J, M) along with an empty vector clone (LNCaP clone a and PC3 clone 8) were evaluated for the promoter 9
activity by the luciferase assay (Fig. 3D). In accordance with their endogenous CRISP3 expression status, LNCaP stable clones showed ~87 fold higher promoter activity than PC3 stable clones (Fig. 3D) demonstrating that stably integrated -1133UTR fragment was under epigenetic regulation like the endogenous CRISP3 promoter. Further, treatment of PC3 stable clones with TSA resulted in upregulation of the promoter activity similar to the effect observed
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on endogenous CRISP3 mRNA expression upon TSA treatment (Fig. 3E). 3.3 Effect of DHT on CRISP3 expression in LNCaP
As LNCaP is an androgen-sensitive cell line, effect of AR agonist DHT on CRISP3 expression was studied by growing it either in the presence or absence of DHT and evaluating CRISP3 expression at RNA (Fig. 4A) and protein level (Fig. 4B). DHT treatment of LNCaP cells led to
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significant increase in the CRISP3 RNA and protein levels. LNCaP cells growing in the absence
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of steroids were also subjected to TSA to check whether the downregulation of CRISP3
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expression by androgen deprivation is via histone deacetylation. TSA could reverse the downregulation of CRISP3 expression suggesting that DHT mediated effect on CRISP3
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expression involves histone acetylation.
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3.4 Effect of DHT on CRISP3 promoter activity
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To evaluate if 1133 bp CRISP3 promoter responds to DHT, luciferase assay was used to compare the promoter activity of the clones grown in complete growth medium, in medium lacking
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steroids or in medium supplemented with DHT. CRISP3 promoter activity was found to be reduced in cells grown in steroid free medium, as compared to the cells which were grown in
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complete growth medium (Fig. 5). DHT supplementation of the steroid-free medium resulted in an increase in the CRISP3 promoter activity which became almost similar to the cells grown in
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the complete growth medium. This data clearly demonstrated that CRISP3 promoter is regulated by DHT in prostate cancer cells. 3.5 Acetylation status of histone H3 at CRISP3 promoter and its response to DHT CRISP3 upregulation upon TSA treatment of PC3 cells suggested that CRISP3 promoter is regulated by HDACs. In order to confirm the histone H3 acetylation status at the endogenous 10
CRISP3 promoter, PC3 and LNCaP cells were subjected to ChIP assay. The three different primer sets used for ChIP-PCR are schematically shown in Fig. 6A. ChIP-PCR data revealed that CRISP3 promoter indeed shows more histone H3 acetylation in LNCaP cells than in PC3 cells in accordance with the expression status of the CRISP3 gene in these two cell lines (Fig. 6B).
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Further to determine the occupancy of CRISP3 promoter by AR, LNCaP cells grown in the presence or absence of DHT were subjected to ChIP using anti-acetyl histone H3, anti-histone H3 and anti-AR antibody. Growth of cells in the presence of DHT increased the occupancy of AR at both the AREs in CRISP3 promoter and also resulted in concomitant increase in the histone H3 acetylation at the proximal region of the CRISP3 promoter (Fig. 6C). This data
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provided evidence for androgen dependent epigenetic regulation of CRISP3 promoter.
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4. Discussion
CRISP3 which belongs to CRISP family of proteins is predominantly expressed in the salivary
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glands [15], neutrophils [19] and the prostate and to a lower extent in epididymis and ovary [20].
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Its expression is highly upregulated in prostate cancer and is associated with disease recurrence. CRISP3 overexpression was found to be associated with ERG fusion positive cancers [3, 16] and
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CRISP3 was shown to be a direct target of ERG. But irrespective of ERG fusion status, CRISP3
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overexpression was reported to be associated with PSA recurrence in all prostate tumors [3] suggesting an alternate regulatory mechanisms for CRISP3 expression. Previous study from our laboratory has also shown that CRISP3 and PSA are co-regulated as knockdown of CRISP3
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resulted in reduction in PSA expression. In this study we attempted to characterize CRISP3 promoter and understand the role of AR in regulation of CRISP3 expression in prostate cancer
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cells.
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Six different promoter-reporter constructs were tested for promoter activity. The shortest promoter fragment which included 247 bp above TSS lacked ARE and the longest fragment which included 1133 bp above TSS contained both the AREs. By luciferase reporter assays, we observed that promoter constructs ending at the TSS had no promoter activity which might be due to disruption of the initiator (Inr) element. Inr element usually encompasses -2 to +4 bp with respect to TSS and is found in both TATA- containing and TATA-less promoters [21]. We also 11
observed that this region contained only one ETS binding site in contrast to three sites reported by Ribeiro et al. [16]. Careful evaluation of the sequence of the 3 ETS binding sites given by Ribeiro et al. revealed that the authors had looked at the sequence upstream of the first ATG of the mature CRISP3 protein which is encoded by the second exon (not considering the ATG in the first exon which codes for the signal peptide). Two of the three reported ETS binding sites
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actually map to the first intron of the CRISP3 gene and the third ETS site maps to the first exon encompassing the 5’UTR of the CRISP3 transcript and hence those are not part of the upstream promoter region.
Upon transient transfection, the three promoter constructs showed promoter activity in PC3 cells which do not endogenously express CRISP3 which was only ~4 fold lower than that observed in
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the LNCaP cells. The difference in the transient activity of the promoter in both these cell lines,
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though small, might be due to the presence of AR and ETV1 in LNCaP cells [22] but not in PC3. These results also indicated that the basal activity of CRISP3 promoter is strong and in non-
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expressing cells, it could be regulated by epigenetic mechanism involving histone
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acetylation/deaetylation. AR is reported to regulate gene expression by epigenetic mechanisms. Epigenetic regulation of CRISP3 gene via histone deacetylation was also indicated by its re-
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expression in TSA treated PC3 and RWPE1 cells. In order to study if the cloned CRISP3
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promoter fragment was also subjected to epigenetic regulation; it needed to be stably integrated in the genome. Hence, stable clones were generated with the longest CRISP3 promoter reporter construct in PC3 and LNCaP cells. Interestingly, the difference in the promoter activity of stable
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clones of LNCaP and PC3 was much higher (~87 fold) than that obtained after transient transfection of these two cell lines (~4 fold), further supporting the epigenetic regulation of the
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CRISP3 promoter in these cells.
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AR is reported to stimulate gene expression through epigenetic mechanisms by means of recruiting histone acetyl transferases (HATs) like SRC-1, 2 & 3 (steroid receptor coactivator), CBP, p300 and PCAF [23]. Apart from its role as transcriptional activator, AR is also shown to epigenetically repress gene expression. AR mediated epigenetic gene repression occurs by its interaction with proteins like Alien, Hey1 and SHP that mediate histone deacetylation or with proteins like EZH2 or LSD1 which mediate histone methylation and demethylation respectively 12
[23]. In order to confirm the involvement of AR in regulating the CRISP3 expression, effect of DHT on CRISP3 expression was first evaluated in LNCaP cells. CRISP3 expression was enhanced in response to DHT at RNA and protein level. Further luciferase activity of the LNCaP stable clones carrying CRISP3 promoter reporter construct was found to be significantly reduced in cells grown in steroid-free medium and was restored upon treatment of cells with DHT. This
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data indicated that AR positively regulated CRISP3 expression in ligand-dependent manner and the AREs predicted at -327 bp and -815 bp of the CRISP3 gene were functional. Histone H3 acetylation status of the endogenous CRISP3 promoter in LNCaP and PC3 cells was determined by ChIP. In accordance with the absence of CRISP3 protein in PC3 and their response to TSA, histone H3 acetylation was lower in PC3 as compared to LNCaP cells. At the same time histone H3 occupancy at the TSS was higher in PC3 than in LNCaP (Fig. 6B). This is in agreement with
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the fact that nucleosome positioning at transcription start sites regulates gene expression by
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controlling the access of specific transcription factors and RNA polymerase [24]. H3 occupancy is found to be lower at the TSS in a transcriptionally active gene than in an inactive gene. A zone
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of nucleosomal depletion or a “dip” in histone H3 occupancy has been demonstrated by Lin et al.
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at MLH1 1a promoter region in MLH1 expressing cells as compared to non-expressing cells
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whereas acetylated histone H3 occupancy was overall higher in the expressing cells [25].
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LNCaP cells express mutant AR (T877A mutation) which is responsive to DHT [26]. Occupancy of the CRISP3 promoter by AR was demonstrated in LNCaP cells by ChIP. AR was found to occupy the AREs in the CRISP3 promoter when LNCaP cells were grown in presence of DHT
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but not in cells grown in steroid-free medium. AR binding to promoter corresponded with the increased histone H3 acetylation at the promoter in the cells grown in presence of DHT. Based
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on this data we postulate that in AR positive prostate cancer cells, AR occupies CRISP3 promoter in ligand-dependent manner and with the help of HATs brings about acetylation of
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histones at the proximal CRISP3 promoter resulting in nucleosomal repositioning and CRISP3 transcription. Dalhman et al. [7] have compared the effect of short-term and long-term ADT on CRISP3 expression and found that CRISP3 upregulation persists during therapy. This was due to parallel high levels of AR which is known to confer resistance to antiandrogens and maintain expression of androgen-responsive genes like PSA [27]. In case of normal prostate cells where CRISP3 expression is absent or minimal, AR may still be occupying the promoter in ligand13
dependent manner but may have different interacting partners which impart repressive role to AR. This possibility needs to be evaluated in normal prostate tissue by ChIP assay. On the other hand, in case of AR-independent prostate cancer cells like PC3, CRISP3 gene remains silenced probably due to maintenance of histone deacetylation. Whether over-expression of AR in AR-
independent CRISP3 regulation in them cannot be ruled out.
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independent cells can upregulate CRISP3 remains to be investigated. Possibility of AR-
In conclusion, CRISP3 gene is under epigenetic regulation and its expression is responsive to DHT in AR positive prostate cancer cells. The two AREs present in the CRISP3 promoter are functional and occupied by AR in LNCaP cells in a ligand-dependent manner. The -1133 UTR fragment of human CRISP3 promoter is subjected to the similar epigenetic regulation like the
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endogenous CRISP3 promoter. This promoter-reporter construct appears to be specifically active
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in AR positive prostate cancer cells. Its specificity needs to be validated in other androgenresponsive cancer cell lines in order to ascertain its utility in bioassays aimed at screening of
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novel AR agonists/antagonists as well as in prostate cancer specific transgenic strategies.
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Acknowledgements
The research work related to this publication (RA/485/05-2017) was supported by grants from
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the Indian Council of Medical Research (ICMR) and DAE-BRNS, India (2011/37B/40/BRNS). Technical assistance provided by Ms. Summaiya Patel who was a trainee in the laboratory is
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acknowledged. We acknowledge Dr. Srabani Mukherjee and Ms. Nanda Joshi for their help with
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DNA sequencing.
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10. Armstrong CM, Gao AC. Drug resistance in castration resistant prostate cancer: resistance mechanisms and emerging treatment strategies. Am J ClinExp Urol. 2015; 3: 64-76. 11. Kobayashi T, Inoue T, Kamba T, Ogawa O. Experimental evidence of persistent androgen-receptor-dependency in castration-resistant prostate cancer. Int J Mol Sci.
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Legends Fig.1 Human CRISP3 promoter sequence Sequence of 1133 bp upstream and 88 bp downstream of TSS of human CRISP3 gene is shown. In the given sequence, 5’ UTR region (88 bp) is underlined and position of two androgen elements
(AREs)
as
identified
through
ConTra
Promoter
analysis
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response
(http://bioit.dmbr.ugent.be/contrav2/index.php?v2) are shown in a box. Location of -247 bp, -806 bp and -1133 bp is indicated by an arrow. Fig.2 Analysis of human CRISP3 promoter activity
PC3 and LNCaP cell lines were transiently transfected using constructs with 5’ UTR (-247 UTR,
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-806 UTR, -1133 UTR) and without 5’ UTR (-247, -806, -1133) along with Renilla luciferase
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expressing vector pRL-TK. The firefly luciferase activity was measured in cell lysates 48 h post transfection and normalized with Renilla luciferase activity. Normalized values obtained for cells
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transfected with empty vector (pGL3) were considered as one and relative fold difference was
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plotted. Each experiment was performed thrice and data plotted as mean ± SEM of three experiments. Statistical analysis was performed using One way ANOVA test. ***p<0.001
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compared to pGL3.
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Fig.3 Regulation of CRISP3 expression by epigenetic mechanism A&B) RT-PCR for CRISP3 (149 bp) and GAPDH (330 bp) in PC3 (A) and RWPE-1 (B) cells
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treated with 100 ng/ml of TSA for 24 h (Lanes 2 and 5) or left untreated as control (Lanes 1 and 4). RNA extracted from these cells was converted to cDNA and used for PCR. CRISP3
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expression was evaluated by singlet and biplex PCR where GAPDH ampliconwas a loading control. Lanes 3 and 6 are water control. C) Detection of CRISP3 protein in the TCA precipitated
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proteins from the conditioned media of untreated and TSA treated PC3 cells by Western blotting (top panel). Silver stained gel (bottom panel) is shown for loading control D) Luciferase activity of LNCaP -1133 UTR stable clones (-1133 UTR clone 2, 3, 5 and empty vector clone a) and PC3 -1133 UTR stable clones (-1133 UTR clone K, J, M and empty vector clone 8) was measured by luciferase assay. Parental LNCaP and PC3 cells (untransfected) were taken as a negative control. Firefly luciferase activity was normalized with protein estimation of respective clones. 18
Normalized values obtained for untransfected LNCaP and PC3 were considered as one and relative fold difference was plotted. E) PC3 -1133 UTR stable clones (J and M) were treated with TSA at indicated doses for 48 h. Firefly luciferase activity was normalized with protein estimation. Normalized values obtained for untreated PC3 stable clones were considered one and relative fold difference was plotted. Each luciferase assay (D and E) was performed at least
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thrice and data plotted as mean ± SEM of three experiments. Statistical analysis was performed using One way ANOVA test. **p<0.01, ***p<0.001 compared to untransfected parental cell line (D) and untreated PC3 stable clone (E). Fig.4 CRISP3 expression is androgen dependent
A) LNCaP cells were either grown in the presence of 5 nM DHT or in medium lacking steroids
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for two different time points (72 h and 96 h). CRISP3 expression was evaluated by RT PCR
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where GAPDH amplicon was a loading control. Lane 1 is 100 bp ladder. Lanes 2 and 6 represent RT reactions of cells grown in steroid free medium for 72 h and 96 h respectively whereas lanes
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3 and 7 are “no RT” reactions of the same samples. Lanes 4 and 8 represent RT reactions of cells
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grown in DHT supplemented medium for 72 h and 96 h respectively whereas lanes 5 and 9 are “no RT” reactions of the same samples. Lane 10 is water control. B) Western blot analysis of
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CRISP3 protein expression in TCA precipitated conditioned media of the cells grown as
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mentioned above. The blot was probed with anti-PAP antibody (top panel) and anti-CRISP3 antibody (bottom panel). PAP (Prostatic acid phosphatase) whose expression is not affected by DHT was used as a loading control. Arrows on the left indicate position of the protein molecular
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weight marker. C) Western blot analysis of CRISP3 protein expression in TCA precipitated conditioned media of the cells grown in steroid free medium alone or in presence of two different
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concentration of TSA. The blot was probed as indicated above.
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Fig.5 1133 UTR-luciferase construct shows androgen responsiveness LNCaP stable clones (Cl.3 & Cl.5) carrying -1133 UTR luciferase construct were grown either in complete growth medium or in medium lacking steroids or in steroid-free medium supplemented with DHT. Luciferase activity was measured and normalized with protein estimation. Normalized values obtained for cells grown on complete growth medium were considered as one and relative fold difference was plotted. Each experiment was performed thrice and data plotted 19
as mean ± SEM of all three experiments. Statistical analysis was performed using One way ANOVA test. ‘*’ indicates p <0.001 compared to cells grown in complete growth medium and ‘#’ indicates p <0.001 compared to cells grown in DHT containing medium. Fig.6 Transcriptionally active CRISP3 promoter shows histone H3 acetylation which is
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responsive to DHT A) Schematic outline of CRISP3 promoter region and the location of the primers used for ChIPPCR. Set 1, Set 2 and Set 3 are three primer pairs which cover the TSS with 5’ UTR and the two AREs respectively. B) PC3 and LNCaP cells were subjected to ChIP using anti-histone H3 (H3) and anti-acetylated H3 (AcH3) antibody. Immunoprecipitated DNA was analyzed by PCR with primer Set 1. Immunoprecipitation with “No antibody” (only beads) served as a negative control.
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Data shown is from a representative experiment and the individual experiment was performed
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twice. C) LNCaP cells grown either in steroid free medium or in medium containing DHT were
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subjected to ChIP using anti-AR antibody, anti-histone H3 and anti-AcH3 antibody. Immunoprecipitated DNA was analyzed by PCR with all three primer sets. Data shown is from a
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