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Research Article

Caveolin-1 is transcribed from a hypermethylated promoter to mediate colonocyte differentiation and apoptosis Nirmalya Dasgupta, Bhupesh Kumar Thakur, Atri Ta, Santasabuj Das

n

a

Department of Clinical Medicine, National Institute of Cholera and Enteric Diseases, P-33 C.I.T. Road, Scheme XM, Beliaghata, Kolkata 700010, India

article information

abstract

Article Chronology:

Caveolin-1(CAV1) is a tyrosine-phosphorylated scaffold protein of caveolae with multiple interact-

Received 16 January 2015

ing partners. It functions both as an oncogene and a tumour suppressor depending upon the cellular

Received in revised form

contexts. In the early stage of colorectal cancers (CRC), CAV1 suppresses tumour progression, while

23 March 2015

over-expression of CAV1 reduced the tumourigenicity of colon carcinoma cells. In contrast, elevated

Accepted 24 March 2015

level of CAV1 was reported in stage III CRC. To address this ambiguity, we studied the functional role

Available online 1 April 2015

and the regulation of CAV1 expression during colonocyte differentiation and apoptosis. Here, we

Keywords: Caveolin-1 Colonocyte differentiation Sodium butyrate DNA methylation Histone acetylation Colorectal cancer

reported for the first time that CAV1 expression was increased during colonocyte differentiation and mediated butyrate-induced differentiation and apoptosis of HT29 cells. CAV1 expression was silenced by promoter hypermethylation in HT-29 cells and reactivated by prolonged histone hyperacetylation of the promoter upon treatment of the cells with butyrate. However, the methylation status was unaltered by butyrate. We for the first time showed that HDAC inhibitormediated transactivation of CAV1 was regulated by methylation density of the promoter. Our study further explains the underlying mechanisms of the anti-cancer property of butyrate in CRC.

Introduction Caveolin-1(CAV1) is a 22-kDa tyrosine-phosphorylated scaffold protein of caveolae. CAV1 interacts with a variety of proteins, including Src-family tyrosine kinases, growth factor receptors, G protein and G-protein-coupled receptors, H-Ras, protein kinase C and MAP kinases, suggesting that CAV1 functions as a ‘molecular hub’ to integrate multiple signaling pathways [1,2].

& 2015 Elsevier Inc. All rights reserved.

In the past two decades, a large volume of data became available that point towards the tumour suppressor role of CAV1 by blocking cell cycle and activating intrinsic cell death pathway [1]. Expression of CAV1 is reduced in several human tumors, including breast, ovarian, colon and lung carcinomas, soft-tissue sarcomas and osteosarcomas [1,3]. CAV1 expression is down-regulated in breast cancer and the tumor size is inversely correlated with the expression [2]. Multiple transcription factors including FoxO3a [4], EGR1 [5],Sp1,

Abbreviations: Ac, Acetylated; APC, Adenomatous polyposis coli; CRC, Colorectal Cancer; CAV1, Caveolin-1; DNMT, DNA methyl transferase; DNMTi, DNA methyl transferase inhibitor; Dox, Doxycycline; H3/4, Histone3/4; HDAC, Histone deacetylase; HDACi, Histone deacetylase inhibitor; NaB, Sodium butyrate; POL2A, RNA polymerase II; TSS, Transcription start site n

Corresponding author. Fax: þ91 33 23632398. E-mail addresses: [email protected], [email protected] (S. Das).

http://dx.doi.org/10.1016/j.yexcr.2015.03.020 0014-4827/& 2015 Elsevier Inc. All rights reserved.

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Tp53 and PPAR-γ [1] and promoter shore CpG methylation [2] regulate the CAV1 expression in breast cancer.DNA methylation also downregulates CAV1 in the ovarian cancer [6]. The immunoreactivity of CAV1 in lung carcinoma is histotype-dependent and CAV1 expression is negative in 85% of lung carcinomas [7]. The majority of primary osteosarcoma and alveolar rhabdomyosarcoma tumors showed significantly lower levels of CAV1 than normal tissues, suggesting its role as a tumour suppressor [8]. In addition, CAV1 expression is associated with better overall survival for osteosarcoma patients [8]. Contrasting the tumour suppressor role, many reports suggested an oncogenic role for CAV1 in prostate and pancreatic cancers, meningiomas, squamous cell carcinoma of the esophagus, glioblastoma, renal cell carcinoma and non-small cell lung carcinoma (NSCLC) [1,3]. Recurrence-free survival was significantly lower in patients with higher expression of CAV1 in prostate [9] and NSCLC [10]. In both carcinomas, CAV1 expression is regulated by EGF/EGFR signaling (3). In addition, the PKCɛ/MEK/ c-Myc signaling also regulates CAV1 in prostate cancer cells (3). In pancreatic cancer cells, p53 along with the transcription factors E2F/DP-1 and Sp1 directly bind to the caveolin-1 promoter to induce expression [11]. Higher expression is also correlated with advanced pathologic stage and poor prognosis of prostate cancer [11], pulmonary squamous cell [12] and renal cell carcinomas [13]. Despite the function of CAV1 as both tumour suppressor and an oncogene, the molecular determinants that allow one role to prevail over the other remain essentially undefined [1]. According to WHO fact sheet (Fact sheet N1297; last updated on Nov, 2014), colorectal cancer (CRC) is the third most common cancer and the fourth commonest cause of cancer-related death worldwide. CAV1 plays an important role in CRC development and progression. About 45–70% of human colon cancers show reduced expression of CAV1 [14,15] and the reduction is more frequent in stage-I and II tumors [14]. This may be due to CAV1 regulation by Adenomatous polyposis coli (APC) [16,17], which is frequently mutated in CRC [18]. The transcription factor FOXO1a, which is increased by wild-type APC expression, induces CAV1 expression, whereas the c-Myc protein, which is reduced in the presence of wild-type APC, represses CAV1 expression in colorectal carcinoma cells [16]. Friedrich T et al. reported that deficiency of CAV1 in Apcmin/þ mice promotes colorectal tumourigenesis and CAV1 mRNA and protein levels were diminished in the tumors compared with the healthy colonic tissues of these mice [17]. In contrast, CAV1 over-expression in colon carcinoma cell lines reduced the tumourigenicity when the cells were injected into the nude mice [15]. Several published studies also suggested tumour-promoting role of CAV1. Elevated levels of CAV1 were reported in colonic adenocarcinoma [19–21], particularly in stage-III masses [14]. CAV1 was suggested to promote tumour growth and progression through the regulation of glucose metabolism [14,20]. However, no consistent relations between CAV1 expression and CRC prognosis have been reported [21]. Regulation and biological significance of CAV1 expression in colorectal carcinogenesis is poorly defined [14]. We addressed this question from a different angle by studying CAV1 regulation during CRC differentiation and apoptosis. We used the HT-29 cell differentiation model, which represents a well-characterized model of CRC differentiation [22]. HT-29 cells start to polarize and form apical microvilli when treated with short-chain fatty acids (SCFA), such as butyrate, an abundant bacterial metabolite in the colon [23]. Butyrate functions as HDAC (Histone deacetylase)

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inhibitor and plays a key role in intestinal homeostasis [24]. It arrests cell cycle progression and induces differentiation and apoptosis of CRC cells in vitro at concentrations similar to those found in the colon [24]. We investigated if butyrate exerted its role in CRC through modulation of CAV1 expression and if so, how this expression was regulated. We showed for the first time that CAV1 expression was enhanced during butyrate-induced differentiation of HT-29 cells and mediated the role of butyrate in cellular differentiation and apoptosis. We also reported that CAV1 was silenced by promoter methylation, and butyrate-induced prolonged histone hyperacetylation was able to reactivate the silenced promoter without altering the promoter methylation status. We had a novel observation that HDAC inhibitor-mediated transactivation of CAV1 was regulated by the methylation density of the promoter. This study offered, an APCindependent regulation of CAV1, since APC is mutated in HT-29 cells. Moreover, it suggests that butyrate exerts its anti-cancer role in CRC through the regulation of CAV1 expression.

Material and methods Cells and reagents HT29, Caco-2 and SW480 cell lines were purchased from the American Type Culture Collection (ATCC). Rest of the cell lines were the kind gift from Dr. P. Tsichlis, Tufts University. Sodium butyrate (NaB), Cycloheximide (CHX), Actinomycin D (Act-D), 5-Aza-20 dC, HDAC1 and Caveolin-1 antibodies were purchased from Sigma. Antibodies against acetylated Histone3 and 4 were purchased from Millipore. Antibody against α-Tubulin and β-Actin was purchased from Santracruz biotechnology. Oligonucleotides for the real-time PCR, ChIP and MSP were custom-synthesized from IDT, USA. psPAX2 (Plasmid#12260), pMD2.G (Plasmid#12259) and Tet-pLKO-puro (Plasmid#21915) were procured from Addgene.

Cell culture and stimulation Colo-205 cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% FBS, whereas Caco-2 was cultured in MEM (Invitrogen) supplemented with 20% FBS. Rest of the cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS. All the cell lines were maintained at 37 1C incubator with 5% CO2. Cells near confluence were cultured overnight in serum-free media followed by Sodium Butyrate treatment (4 mM). For 5-Aza-20 dC treatment, cells were split 12–18 h before the treatment with 5-Aza-20 dC (1–10 mM) or the vehicle (DMSO) for 24– 120 h replacing the medium every 24 h.

Total RNA extraction and cDNA synthesis Total RNA was extracted with TRI reagent (Sigma) following the manufacturer's instructions. cDNA was prepared from 1 μg of extracted RNA using RevertAidTM reverse transcriptase (Fermentas) following the manufacturer's protocol.

SYBR-Greens Real Time PCR Real time quantitative PCR (RT-real-time PCR) was performed by Power SYBR-Green (Applied Biosystems) using StepOnePlus

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Real-Time PCR Systems (Applied Biosystems). β-Actin and/or GAPDH levels were taken for normalization, and fold change was calculated using 2  ΔΔCt as described earlier [25]. Data was represented as fold changes relative to untreated. Primers used for real-time PCR amplification: CAV1 expression (FP, 50 -GATTCAGTGCATCAGCCGTGTC-30 ; RP, 50 -GCGGACATTGCTGAATATTTTCCC-30 ), GAPDH (FP,50 CA TGTTCGTCATGGGTGTGAACCA-30 ; RP,50 -AGTGATGGCATGGACTGTGGTCAT-30 ), β-Actin (FP, 50 -CTGGCCGGGACCTGACTGACT-30 ; RP, 50 GCCGTGGCCATCTCTTGCTCG-30 ).

Membrane fraction isolation Membrane fraction of the cells was isolated as described elsewhere with modifications [26]. Cells grown in 100 mm plate were washed with ice-cold 1X PBS and harvested by scraping into Tris–EGTA lysis buffer [20 mM Tris–HCl (pH 7.4), 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 2% protease cocktail Inhibitor (Sigma)]. Cells were disrupted by Dounce homogenization (50 strokes) and nuclei were pelleted down by centrifugation at 800Xg for 10 min at 4 1C. The supernatants were centrifuged again at 12,000Xg for 10 min at 4 1C to pellet down the mitochondria. Plasma membrane was pelleted down from the supernatants by centrifugation at 100,000Xg for 1 h at 4 1C and the pellet was dissolved in 2%SDS by heating at 65 1C for 10 min. After quantification of total protein by BCA reagent (Pierce), protein samples were equalized, boiled in 5X Laemmli buffer and resolved in SDS-PAGE.

Western Blot analysis Western Blot was performed as described earlier [25]. Cell lysates were prepared by harvesting in NP-40 lysis buffer. After clearing the lysate by centrifugation at 14,000 rpm for 10 min, supernatants were collected and total protein content was measured by the BCA reagent (Pierce). 30 μg of total protein per well was resolved in a 12% SDS-PAGE and electrotransferred to a 0.45 μM polyvinylidenedifluoride membrane at 100 mV for 45 min. The blot was probed with the specific primary antibody followed by HRP-conjugated secondary antibody (Pierce) and developed using SuperSignal WestPico chemiluminescence substrate (Pierce).

Cloning of Cav1-shRNA and luciferase reporter plasmid To generate Cav1-shRNA clone, we inserted the annealed CAV1shRNA sequence (FP: 50 -CCGGGACGTGGTCAAGATTGACTTTCTCGAGAAAGTCAATCTTGACCACGTCTTTTTG-30 ; RP:50 -AATTCAAAAAGACGT GGTCAAGATTGACTTTCTCGAGAAAGTCAATCTTGACCACGTC-30 Validated sequence, Sigma) into the Tet-pLKO-puro vector [27] at AgeIEcoR1 restriction sites. The positive clone was confirmed by two close bands of  200 bp after digestion of the plasmid with XhoI. To generate a luciferase reporter construct under the control of the CAV1 promoter, two genomic DNA fragments were amplified by PCR using the primers for the upstream regulatory sequences (FP1, 50 -GGTACCTCCCTCTTACTCCCAACCC-30 ; FP2, 50 -GGTACCTAAAGGT TCCTAGCCGTC-30 and RP, 50 -CTCGAGCAGTCGGGATATTTGGAGAGG-30 ). The fragments from nucleotides  1189 to þ218 and  439 to þ218 were amplified using the forward primers FP1 and FP2, respectively. PCR products were cloned into pTZ57R/T vector (Fermentas) followed by blue-white screening.

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Positive clones were confirmed by sequencing in an ABI automated sequencer. The reporter constructs of CAV1 promoter sequences were generated by sub-cloning into the pGL3-Basic vector (Promega) upstream of the luciferase gene via KpnI and XhoI digestion (Fermentas).

Production and infection of lentivirus For production and infection of lentivirus, we used Addgene pLKO.1 Protocol. Briefly, CAV1-shRNA plasmid was co-transfected with lentiviral packaging mix (pSPAX and pMD2.G) into 60–70% HEK293 T cells in a 60 mm dish by using Lipofectamine2000 (Invitrogen). The medium was replaced with a fresh medium after 24 h. After 48 h of transfection, the lentivirus-containing medium was collected and filtered with 0.45 μm of syringe filter and stored at 4 1C. For infection, HT-29 cell was seeded at 40–50% confluency. After overnight incubation, the media was replaced with Lentivirus containing media and DMEM complete media (1:1) with 5 μg/mL polybrene to infect the cells. Infection was repeated after 24 h of first infection. After 48 h of first infection, 100 nM Doxycycline(Dox) was added to express the CAV1-shRNA. After 72 h of first infection, media was replaced by serum-free media with or without Dox. We maintained the parental-type without adding Dox in infected cell. After overnight incubation cell was treated with butyrate for 24 or 48 h for further experiments. We checked the effectiveness of the CAV1-shRNA by real-time PCR and Western blot analysis. We tried to make stable cell line with CAV1-shRNA with puromycin treatment. But, surprisingly, we found the cells were not growing after 2–3 passages. Hence, we took the strategy to infect the cell and immediately use that.

Alkaline phosphatase (ALPase) assay by p-Nitrophenylphosphate (pNPP) method HT-29 cell was seeded in 96-well plate for CAV1-shRNA-lentiviral infection and butyrate treatment, as mentioned above. After 48 h butyrate treatment, cells were lysed in 20 μL 1X Passive Lysis Buffer (Promega). ALPase activity was measured by incubating 10–15 μL lysate with 200 ml of an substrate solution containing 0.025 mM glycine, 8.62 mM MgCl2 and 3.2 mM pNPP (pH 9.6) for 1–2 h at 37 1C in 96-well ELISA plate. Absorbance of the end product p-nitrophenol was determined at 405 nm using ELISA reader. Protein content was determined by BCA (Pierce) method to calculate specific activity.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay HT-29 cells were infected in 35-mm dish for CAV1-shRNAlentiviral infection and butyrate treatment, as mentioned before. After 24 h butyrate treatment, cells were fixed in 1% paraformaldehyde (w/v) containing PBS buffer and TUNEL assay was performed using flow cytometry with Apo-Direct Kit (BD Pharmingen) according to manufacturer's protocol.

MTS assay To check the cell viability, we performed MTS assay (Promega) as per manufacture's protocol with modifications. Briefly, HT-29 cells were infected with CAV1-shRNA-lentivirus and treated

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Fig. 1 – CAV1 is induced to promote HT-29 differentiation: (A) CAV1 mRNA expressions was measured by Real-time PCR after 24 h of NaB with the indicated doses. Wilcoxon Signed-rank (WS) test was used for statistical analysis. (B) CAV1 mRNA expression was quantified in serum starved HT-29 cell line by Real-time PCR after 0–24 h with 4 mM sodium butyrate (NaB) treatment. WS-test was used for statistical analysis. (C) Immunoblot analysis of CAV1 protein expression in cell membrane fractions after treatment with NaB (4 mM) for the indicated time points. Cell membrane bound β-Actin was used as loading control. (D) CAV1 Protein and (E) mRNA expression showed the efficiency of CAV1-shRNA. (F) Specific activity of Alkaline phosphatise (ALPase) was determined by pNPP method after 48 h butyrate treatment. Mann–Whitney (MW) test was performed for statistical analysis. (G) CDKN1A (p21, Cip1) expression was determined by Real-time PCR. Fold change calculation was done by considering  Dox/-NaB sample as 1.WStest was performed to assess the significant up-regulation. Difference in  Dox/þNaB vs. þDox/þNaB was tested by MW-test. Dox represents the parental cell, whereas þDox is the CAV1-knocked-down HT-29 cell. For (A), (B), (E–G), data are presented as the means7the SD. Real-time PCR data were normalized by GAPDH and/or β-Actin expression. n, po0.05; nn, po0.01.

with butyrate in a 96-well plate, as mentioned in the earlier sections. After 48 h butyrate treatment, the media was aspirated and cells were washed with 1X PBS. We used 200 μL MTS/ PTS solution per well. We prepared MTS/PTS solution by mixing 1 μL PTS and 20 μL MTS in phenol red free (to reduce background absorbance) 180 μL DMEM media (Sigma). After 0.5–1 h incubation, we aspirated the media in a fresh 96-well ELISA plate. The reaction was stopped by adding 10%SDS and absorbance at 490 nm was taken using ELISA reader.

Transient transfection and reporter assay Transfection of HT29 cells was done by using Lipofectamine 2000 (Invitrogen) following the manufacture's protocol. Briefly, cells (1  105/well) were seeded in 24-well plates 18 h prior to transfection and cultured in Opti-MEM supplemented with 4% fetal bovine serum. Cells in each well were incubated with transfection mix containing 1 μg of total DNA for 6 h followed by addition of complete DMEM. Cells were treated with butyrate 48 h post-

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transfection and reporter assay was performed 24 h later using dual luciferase assay kit (Promega) following the manufacturer's protocol. Briefly, cells were washed two times with 1XPBS followed by lysis with 1X passive lysis buffer (Promega). The lysate was cleared by brief centrifugation, and firefly luciferase reporter activity in the clear supernatant was measured with a luminometer (Berthold). Renilla luciferase activity in the same lysate was used as transfection control.

sets, we used Mann–Whitney (MW) test, the nonparametric counterpart of independent t-test. For comparing more than two data sets, we used Kruskal–Wallis (KW) test which is the nonparametric counterpart of one-way ANOVA. The results were considered significant at po0.05. Data are expressed as Mean7SD; representan tive of at least three-independent experiments. , po0.05, nn, po0.01, nnn, po0.001.

Chromatin immunoprecipitation (ChIP)

Results

ChIP assays were performed using EZChIPTM chromatin immunoprecipitation kit (Millipore) following the manufacturer's instructions. Briefly, HT29 cells were cross-linked using formaldehyde after treatment and harvested by scraping. The cells were lysed and cross-linked DNA was sheared to  200–1000 bp fragments by giving 5 pulses of 30 s duration each with 1 min interval after every pulse. Sheared chromatin was immunoprecipitated using specific antibodies and immunoprecipitated genomic DNA of CAV1 promoter was amplified by real-time PCR using primers (FP,50 -CCACCCCTGCTGAGATGATGC-30 ;RP,50 -GCCCGCCAAAGGTTTG TTCTGC-30 ).The GAPDH promoter was also used as housekeeping gene. The ‘percent input’ value represents the enrichment of DNAProtein binding on specific region of CAV1 promoter.

CAV1 expression is induced by butyrate to promote differentiation of HT-29 cells

Bisulfite treatment and methylation analysis Methylation of CAV1 promoter was analyzed by bisulfite treatment of DNA followed by Nested Methylation- specific PCR (MSP) analysis. Genomic DNA was prepared from cells using QIAamp DNA mini-kit (Qiagen) following the manufacturer's protocol. Sodium bisulfite conversion of 5 μg of DNA was carried out using MethylEasy™ Xceed kit (Human Genetic Signatures). MSP for CAV1 was performed as described elsewhere [28]. New set of CAV1 MSP-specific primers were designed by Methprimer [29]. The location of the DNA fragments is shown in Fig. 5A. 1st round PCR (25 mL) was performed by Go-Taq PCR Kit (Promega) with 5 mL converted DNA using primers FP,50 -AGGATAGGGTAGGATTGTGG-30 and RP,50 -CATAAAACATTCCTAACTTCTC TTCACCTC-30 (Annealing temp 53 1C). 5 mL of diluted (1:50) 1st round PCR products were used as template for 25 mL of the 2nd round MSP reactions. Primers used for 2nd round PCR were M1 (FP,50 -TTCGTTTTTTTCGGGACGTTTTTCG-30 ; RP,50 -CGCCAAAAATTT ATTCTACTCGCGAT-30 ;), U1 (FP, 50 -TTGTTTTTTTTGGGATGTTTTTT M2(FP,50 GG-30 ;RP,50 -CCCACCAAAAATTTATTCTACTCACAAT-30 ), GGTATTTTTGTAGGCGCGTC-30 ;RP,50 -CTAACAACAAAAAACGAAAA ACG-30 )and U2(FP,50 -GTTTATATTGGGTATTTTTGTAGGTGTGT-30 ;RP, 50 -TCCCCAAAATTCTAACAACAAAAAACAAAAAAC-30 ).PCR products were separated using 1.5% agarose gel electrophoresis. Annealing temperature was set 50 1C for M2 and 55 1C for others.

Statistical analysis Statistical significance was analyzed by the nonparametric method using GraphPad Prism 5 Software. For real-time PCR data, we considered the value for untreated control as 1 as per convention and calculate the fold change of treated sample. We checked whether the fold change significantly differs from 1 by Wilcoxon Signed Rank test. Wilcoxon Signed Rank (WS) test is the nonparametric counterpart of one-sample t-test. For comparing two data

To investigate if butyrate could alter CAV1 expression in CRC cell line HT-29 during differentiation, we stimulated the cells for 24 h with different concentrations of butyrate at the physiological concentrations. The results showed maximum CAV1 expression with 4 mM of butyrate (Fig. 1A), which is readily available in the gut [24]. Next, we stimulated the cells with butyrate (4 mM) for various durations. Butyrate enhanced CAV1 mRNA expression in a time-dependent manner with the optimal effects observed after 12 h of treatment (Fig. 1B). Western blot analysis with the cell membrane fractions showed that up-regulation of CAV1 transcripts contributed to an increase in the expression levels of CAV1 protein (Fig. 1C). Since butyrate was demonstrated as a wellestablished differentiating agent for CRC cells, we investigated whether CAV1 induction played any role in mediating butyrateinduced differentiation. To this end, we knocked-down CAV1 in HT-29 cells using CAV1 shRNA under the control of a tetracyclineresponsive promoter. Addition of Doxycycline (100 nM) to the cultured cells resulted in around 75% reduction of CAV1 mRNA/ Protein expression, which was accompanied by a similar decrease in alkaline phosphatase (ALPase)-specific activity, a marker of intestinal epithelial cell (IEC) differentiation after butyrate treatment (Fig. 1D–F). This indicated that butyrate-induced differentiation of the above cell was suppressed by CAV1 deficiency. Previous studies suggested CDKN1A (p21, Cip1) as a major regulator of butyrate-induced colonocyte differentiation. We investigated if p21 induction by butyrate was CAV1 dependent. Butyrate significantly enhanced p21 mRNA expression, but the levels were unaltered by silencing of the CAV1 gene (Fig. 1E). Suppression of differentiation despite p21 induction was consistent with the earlier report that p21 is dispensable for intestinal cell differentiation [22]. Taken together, the above data suggested that butyrate induced the expression of CAV1 to promote colonocyte differentiation in a p21-independent manner.

CAV1 mediates butyrate-induced apoptosis of HT-29 cells Butyrate causes apoptosis of HT-29 cells, which underlies the anti-cancer property of butyrate. However, this is not considered a direct consequence of terminal differentiation of the colonocyte [30].We used TUNEL-flow cytometry assay to analyze apoptosis of the cells by quantification of DNA fragmentation. CAV1 gene silencing per se exerted no role in apoptosis. However, butyrate-induced apoptosis after 24 h was significantly reduced when CAV1 expression was silenced (42.6% in the parental vs. 26.8% in CAV1 knocked down cells) (Fig. 2A).

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Fig. 2 – CAV1 promotes NaB-mediated apoptosis in HT-29 cell: (A) TUNEL-Assay was performed by flow cytometry after 24 h NaB treatment. Unstained is represented by shaded curve. (B) Cell viability after 48 h butyrate treatment was determined by MTS assay. Data are presented as the means7the SD. Mann–Whitney (MW) test was performed for statistical analysis. (C) Survivin (BIRC5) and Cyclin D1 (CCND1) expression was determined by Real-time PCR. þ/  Dox represents the cell types, as mentioned in Fig. 1. Statistical test used for (C) is exactly same, as said in Fig. 1G. n, po0.05; nn, po0.01. Further analysis by MTS assay complemented the above findings by showing lower number of viable parental cell counts after 48 h (41% vs. 62%survival) (Fig. 2B). Pro-apoptotic role of CAV1 may be mediated through the suppression of Survivin (BIRC5), an inhibitor of apoptosis and cell cycle protein Cyclin D1 (CCND1) transcription [31]. The same factors also contributed to butyrate-induced HT-29 cell differentiation [32]. We checked whether BIRC5 and CCND1 downregulation by butyrate was CAV1-dependent. Silencing of CAV1 gene resulted in higher expression of BIRC5 ( 4 fold) and CCND1 (5 fold) compared with the butyrate-treated parental cells (Fig. 2C), suggesting that the repression by butyrate was mediated through CAV1 and might contribute to apoptosis of the cells.

CAV1 expression was transcriptionally regulated by prolonged histone hyperacetylation The above results indicated that butyrate enhanced CAV1 expression, which served an important biological role for colonocyte differentiation and apoptosis. Regulation of CAV1 expression during differentiation was never studied earlier. APC was suggested as a major regulator of CAV1 [16,17]. However, frequent mutation of APC in colon cancers suggested, an APC-independent regulation. HT-29 cell also posses mutated APC. We inhibited transcription by pre-treating HT-29 cells with Actinomycin-D to exclude enhanced mRNA stability as the underlying reason for the increase in CAV1 mRNA levels in butyrate-treated cells. The degradation rate of CAV1 mRNA as determined by Real-time

PCR was found to be similar with or without butyrate treatment, resulting in t1/2 of 5.5 h. GAPDH mRNA was used as a control for its long half-life (more than 24 h) [33](Fig. 3A). To further investigate if the above induction was purely transcriptional, we checked RNA Polemerase II (POL2A) occupancy of the CAV1 promoter near the transcription start site (TSS) by ChIP assays at different time intervals after butyrate treatment. Cells were crosslinked with formaldehyde and chromatin was immunoprecipitated using POL2A or IgG (negative control) antibody. Amplification of the immunoprecipitated genomic DNA of the CAV1 promoter by Realtime PCR showed time-dependent enrichment of POL2A occupancy (Fig. 3B). These results suggested that CAV1 induction by butyrate was transcriptionally regulated. Number of recent studies have shown that microRNAs, such as miR-802 [34] miR-199a-5p [35] and miR-203 [36] directly suppress the expression of CAV1 by binding to its 30 -UTR region. Butyrate may also alter miRNA expression in the cells [24]. However, mRNA stability and POL2A occupancy experiments ruled out any involvement of miRNA in CAV1 regulation by butyrate. We reported earlier that butyrate may regulate gene expression by altering the expression of other proteins [25]. To address if de novo protein synthesis was required to mediate CAV1 regulation, cells were pre-treated with protein synthesis inhibitor, cycloheximide (CHX). Butyrate-induced CAV1 expression was not inhibited by CHX, indicating that new protein synthesis was not required (Fig. 3C). Interestingly, treatment of HT29 cells with CHX alone up-regulated CAV1 expression. This indicated that basal transcription machinery of CAV1 might be under the control of repressor(s)

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Fig. 3 – Prolonged histone hyperacetylation is required for CAV1 expression: (A) HT29 cells were treated with Act-D (10 lg/mL) for the indicated time points either alone or after 24 h of NaB treatment. After each time point, CAV1 mRNA expression was measured by Real-time PCR taking GAPDH as normalization control. RNA without treatment of Act-D in NaB-treated and untreated cells was considered as initial RNA content. (B) Quantitative ChIP for POL2A occupancy of the CAV1 promoter near TSS was performed at the indicated time intervals after NaB treatment. Kruskal–Wallis (KW) test was performed for statistical analysis. n, po0.05; nn, po0.01 vs. 0 h. (C) Cells were treated with or without 50 lg/mL of cycloheximide (CHX) concomitantly followed by butyrate (4 mM) for 24 h. The cells were harvested for RNA extraction followed by Real-time PCR. WS and MW-test was applied as mentioned in Fig. 1G. n , po0.05; nn, po0.01 vs. Untreated. (D) Luciferase reporter construct of CAV1 putative promoter was transiently over-expressed in the HT29 cells followed by stimulation of the cells with 4 mM NaB for 24 h. Reporter activities are presented as the Relative Light Unit/sec (RLU/s) (Firefly luciferase), normalized against the Renilla luciferase reporter activities, of the treated and untreated cells. MW-test was performed between NaB-treated vs.Untreated. (E) 800 nM TSA was added to the cells after 16 h of serum starvation. Repeated doses of TSA were added at 6 h and 8 h intervals for two and three times, respectively. Cells were harvested in TRI reagent at indicated time intervals. WS-test was performed to check the statistical significance of fold change for (E) as mentioned earlier figures. n, po0.05 vs. Untreated.

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Fig. 4 – CAV1 is induced by Histone-3/4 acetylation of a CpG methylated promoter: (A) Schematic representation of CpG Island (CGI) on 50 -UTR-region of CAV1 promoter near TSS (gray box). Black vertical bars under gray box are the indicator of CpG bases. U1, U2, M1, M2 indicate the regions on the promoter that were examined by methylation-specific PCR using unmethylated (U) and methylated (M) primers sets. PCR product region for ChIP is also indicated in the diagram. (B) HT29 cells were seeded in 24-well plate. After 16–18 h, growing cells were exposed to the indicated concentrations of 5-Aza-20 dC or the vehicle (DMSO) for 3 days. (C) Experiments as in (B) were performed with 5 lM of 5-Aza-20 dC for 0–5 days. Total RNAs were extracted and CAV1 mRNA levels were measured by Real-time PCR. WS-test was performed for (B,C). n,po0.05; nn, po0.01 vs. DMSO. (D) CpG methylation status on CAV1-CGI of HT29 cells was measured by nested-MSP. After 5 lM 5-Aza-dC treatment for 0–3 days, genomic DNA was isolated from HT29 cells. Methylation status was checked by nested-MSP with the Bisulphite converted genomic DNA. M1 and U1, 2 denotes the 2nd round PCR product with their corresponding primer sets. U2M2 represents the PCR product using U2 forward and M2 reverse primer. (E) After NaB treatment for different durations, sheared Chromatin was pulled-down by Rabbit-IgG, Acetyl-Histone3 and Acetyl-Histone-4 antibody to check Histone acetylation on the CAV1 promoter near TSS. Real time PCR was performed with primer set flanking the CGI region of CAV1promoter. Kruskal–Wallis (KW) test was performed for statistical analysis. n, po0.05; nn, po0.01 vs. 0 h. (F) HT29 cells were seeded in a 35 mm plate. After serum starvation, cells near confluence were treated with butyrate for various durations. Cell lysates were prepared and subjected to immunoblot analysis using Ac-H3, Ac-H4-specific antibodies. αTubulin was used as loading control.

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Fig. 5 – NaB induces CAV1 expression without altering the CpG methylation status: (A) After 3 days of 5 lM 5-Aza-20 dC treatment and 24 h of 4 mM Butyrate treatment to HT-29 cells, HDAC1 enrichment on CGI of the CAV1 promoter was studied by quantitative ChIP assay. nn, po0.01 vs. Untreated. For 5-Aza-20 dC, Untreated denotes DMSO Control. (B) POL2A occupancy and H3 acetylation status on CAV1 promoter in the above experiment of 5-Aza-20 dC was measured by ChIP-qPCR. nn, po0.01 vs. DMSO. (C) Nested-MSP with the Bisulphite converted genomic DNA after HT-29 cells were treated with 4 mM of NaB for 24 h. (D) Schematic representation of the mechanism of CAV1 induction by butyrate and 5-Aza-20 dC. The methylation (closed circle) status was unchanged by NaB and hence HDAC1 was not removed from the repressor complex. Despite that, NaB chemically inhibited HDAC1 directly and acetylated Histone to open-up chromatin structure for the binding of Transcription factors (TFs) and RNA POLII to start the transcription. In contrast, 5-Aza-20 dC treatment caused demethylation of CpG (open circle). The repressor complex was unable to bind demethylated DNA. Hence, repressor complex and HDAC1 were removed from the promoter. This resulted in histone acetylation followed by binding of TFs and RNA POLII for transcription initiation.

and suppression of protein synthesis led to increased CAV1 transcription by depleting the repressors. Together the above findings suggest that butyrate upregulated CAV1 through transcriptional de-repression or promotion of transactivation by other proteins. CAV1 is known to be regulated by several transcription factors including FOXO1a, c-Myc, Sterolresponsive element (SRE)-binding proteins (SREBP), Tp53,KLF11 and Sp1 [1]. To address this issue, reporter constructs of serial deletions of the 50 UTR of CAV1 promoter were transfected into the HT-29 cells. No significant changes in the reporter activities were noted after butyrate treatment (Fig. 3D). This excluded the possibility of activation by transcription factors or transcriptional derepression. On the other hand, butyrate might function through epigenetic mechanisms by inducing chromatin remodeling. This would not alter reporter activities, since transiently-transfected plasmids are not efficiently packaged into the chromatin [37]. Butyrate is a potent HDAC inhibitor, but also functions through multiple other mechanisms. To further address epigenetic regulation of CAV1 expression, we used specific HDAC inhibitor, Trichostatin A (TSA). While butyrate induced persistent histone H3 and H4

acetylation beyond 24–72 h, acetylation by TSA was more transient, reaching a maximum at 4–8 h and returning to the baseline by 24 h due to its rapid metabolism by the cells [38,39]. However, repeat doses of TSA may induce a state of prolonged histone hyperacetylation and differentiation [40]. We treated HT-29 cells with two and three repeat doses of TSA (800 nM) at 6 h and 8 h intervals, respectively. Histone acetylation by TSA induced CAV1 after 6 h, which returned to the baseline after 12–24 h. However, repeat doses of TSA resulted in sustained CAV1 induction in HT29 cells (Fig. 3E). These observations indicated that butyrate-mediated CAV1 induction was a consequence of prolonged histone acetylation of the promoter near the TSS.

CAV1 was induced by histone 3 and 4 acetylation of a CpG hypermethylated promoter near the TSS CpG methylation critically regulates CAV1 expression in CRC [14]. CpG island prediction by MethPrimer suggested that the 50 UTR of CAV1 is a TATA-less promoter (Fig. 4A) [41]. We found that treatment with CpG methylation inhibitor, 5-Aza-20 dC dose

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dependently restored CAV1 mRNA expression in cultured HT29 cells with the optimal effects observed at a dose of 5 mM (Fig. 4B). The maximal re-expression was noted after 2–3 days of the above treatment (Fig. 4C). A temporal reduction of methylation density was seen with concomitant increase in de-methylation of CGI after 5-Aza-20 dC treatment (Fig. 4D). These data suggested that CGI hypermethylation was responsible for the reduced CAV1 transcription in HT29 cells. However, PCR using a different primer set (U2 & M2) revealed that the distant 50 end of CGI, which is frequently methylated in other cell lines was not methylated in HT-29 [28,42–44]. Promoter hypermethylation leads to hypoacetylation of the histones, which silences gene expression. We asked whether butyrate transactivated CAV1 expression by inducing acetylation of the core histones (Hitone3 and 4) at the CpG methylated region of the promoter. We performed ChIP to analyze the acetylation status of H3/H4 in the promoter. Chromatin was precipitated from butyrate-treated HT29 cells at various time points using anti-AcH3/H4 antibodies. Real-time PCR assays showed that CAV1 promoter regions near the TSS in butyrate-treated HT-29 cells were significantly enriched in acetylated H3 at 12–24 h and H4 at 24 h (Fig. 4E). However, H3 acetylation was predominant over H4, comparing percent input value and acetylation time. Enhanced promoter acetylation pattern was consistent with POL2A occupancy (Fig. 3B), total histone acetylation of the cells (Fig. 4F) and CAV1 expression within them (Fig. 1A and C).

LoVo, SW480 and SW620 cells and lower levels in Colo-205 cells in comparison to HT-29 cells (Fig. 6A and B). Similar results were observed when the levels of CAV1 protein in the cell membrane fractions was compared by western blot analysis (Fig. 6C). In agreement with the published reports, MSP study showed that the differential expression pattern of CAV1 directly correlated with CpG methylation status of the CAV1 promoter (Fig. 6D) [14]. When these cells were treated with butyrate or 5-Aza-20 dC, CAV1 expression was unaltered in LoVo, SW480 and SW620 cells (Fig. 6E and F). This indicates that CAV1 promoter was already hypomethylated and carried no HDAC1/2-corepressor complexes. As a result, treatment with methylation and HDAC inhibitors exerted no effects on CAV1 expression. This is in contrast to HT-29 cells, where both agents were effective (Fig. 6E and F). On the other hand, only 5-Aza-20 dC induced CAV1 in Colo-205 cells (Fig. 6E and F). CAV1 promoter in Colo-205 cells is more heavily methylated than HT-29 cells (Fig. 6D). This high methylation density may deter butyrate to act on the corepressor complex. To further support this hypothesis, we took another APC  /  cell line, Caco-2, which has denser hypermethylation compared with Colo205 cells [14]. 5-Aza-20 dC treatment induced CAV1 in CaCo-2 cells, but butyrate failed to do so (Fig. 6E and F). Together the above data suggest that not the CpG methylation status alone, but methylation density is also critical to transactivate CAV1 by butyrate.

CAV1 promoter is reactivated by butyrate without alteration of the CpG methylation status

Discussion

DNA methylation recruits HDAC1/2 co-repressor complexes to render the chromatin transcriptionally incompetent [45]. We investigated whether HDAC1/2 co-repressor complexes were directly associated with the CAV1 promoter. ChIP assays showed that 5-Aza-20 dC treatment led to reduced HDAC1-binding to CGI (Fig. 5A). This confirmed the presence of HDAC1/2-corepressor complexes on the promoter, which were perhaps removed by demethylation of CGI. This resulted in H3 acetylation to open the nucleosome for POL2A binding (Fig. 5B). In contrast, HDAC1 occupancy of the CGI remained the same after 24 h of butyrate treatment (Fig. 5A). We hypothesized that butyrate treatment failed to decrease CAV1 promoter methylation. PCR showed comparable methylation before and 24 h after butyrate treatment of the cells (Fig. 5C), indicating that DNA-methyltransferase enzymes (DNMTs) were not deterred from the hyper-acetylated DNA [46]. Thus, butyrate induces CAV1 transcription from a hyper-methylated promoter. Butyrate was earlier reported to preferentially inhibit HDAC1 and HDAC2 [47]. This suggested that butyrate remodeled the chromatin structure by chemically inhibiting HDACs rather than removing the co-repressor complexes from the promoter and promoted histone acetylation to initiate transcription. In contrast, 5-Aza-20 dC treatment removed corepressor complexes from the promoter by reducing CpG methylation, followed by histone acetylation and transcription (Fig. 5D).

CAV1 induction by butyrate in APC  /  cells is dependent on CpG methylation density of the promoter We checked whether butyrate induced CAV1 induction in other APC  /  CRC cell lines. We found higher CAV1 mRNA levels in

Intestinal epithelium cell differentiation and apoptosis are highly regulated processes controlled by APC [48]. Loss of wild type APC perturbs differentiation of cells, which no longer express villus cell markers like alkaline phosphatase [48]. However, dietary fibre-derived metabolite butyrate induces differentiation and apoptosis of colonic epithelial cells in the absence of APC. This makes butyrate a protective agent for lowering colon cancer risks, considering that APC is mutated in most CRC [49]. We observed that CAV1 is induced during differentiation of APC-mutated HT-29 cells by butyrate and mediates butyrate effects on the differentiation and apoptosis of the cells. This is in agreement with the CAV1 expression patterns that roughly correlate with the degree of differentiation of the five CRC cell lines we tested. LoVo is the most well-differentiated cell line [50] with the highest expression levels and Colo-205 is the least differentiated cells [51] with the lowest CAV1 expression. Moderately differentiated cell lines, such as SW480 and SW620 [50] and the less differentiated HT-29 cells [51,52] also maintain the gradient of CAV1 expression. However, controversy existed for over a decade regarding the role of CAV1 in CRC. Some investigators reported a suppressive function in CRC development and progression, while others found it to promote tumorigenesis. Our study reinforces the role of CAV1 as a tumour suppressor in CRC, at least in vitro. This role is explained by its association with Wnt/β-catenin/Lef-1 pathways. CAV1 participates in this pathway by forming a multi-protein complex, which includes Ecadherin/β-catenin and helps to sequester β-catenin to the membrane, thereby precluding β-catenin/Tcf-Lef-dependent transcription of antiapoptotic and cell cycle regulatory genes, such as BIRC5 and CCND1 [31]. Transcription of these genes is inhibited by wild-type APC [53,54]. We found that BIRC5 and CCND1 expression in APCdeficient HT-29 cells is suppressed by butyrate and this is mediated

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Fig. 6 – CAV1 expression is dependent on CpG methylation density of the promoter: (A) Real-time PCR showing CAV1 expression in APC  /  colon carcinaoma cell lines. Ct values of β-Actin were subtracted from the Ct values of CAV1 for normalization. The values are plotted as ΔCt, where larger negative value represents the lower expression level. Horizontal bar represent the median of all  ΔCt values for each cell line. (B) Semi-quantitative RT-PCR of APC  /  Cell lines. Co, HT, Lo, S4 and S6 denote Colo-205, HT29, LoVo, SW480 and SW620, respectively. 27, 30, 33 cycles was used to determine the logarithmic phase of PCR-amplification. GAPDH was used as housekeeping gene. (C) Upper panel: Immunoblot analysis of basal levels of CAV1 protein expressions in the membrane fractions of the above cell lines. Cell membrane was isolated by differential centrifugation. Plasma membrane bound β-Actin was used as loading control. Lowe panel: β-Actin normalized relative band intensity is shown. The expression of HT-29 cell was considered as 1. (D) Nested MSP-PCR for APC  /  Cell lines. M1, 2 and U1, 2 denotes the 2nd round PCR product with their corresponding primer sets. (E) CAV1 expression was determined by Real-time PCR in the APC  /  cell lines after 3 days 5 μM 5-Aza20 -dC treatment. (F) CAV1 expression was determined by Real-time PCR in the APC  /  cell lines after 24 h 4 mM butyrate treatment. WS-test was performed for statistical analysis for (E, F). n, po0.05.

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through CAV1 induction. If we collate these facts, it may be hypothesized that CAV1 functions as a critical regulator of Wnt signaling pathways in the absence of wild-type APC and CAV1dependent Wnt signaling may be involved in butyrate-induced colonocyte differentiation and apoptosis. Since both BIRC5 and CCND1 are associated with the poor prognosis of CRC [55–57], our data provides a novel mechanism underlying the protective role of butyrate against CRC. CAV1 also participates in Hedgehog (Hh) signaling pathways [58], which is an antagonist of Wnt signaling in colonocyte differentiation [23]. In addition, it also takes part in several other signaling pathways involved in this process, such as PTK, MAPK/ ERK and GPCRs [2]. Full explanation of the role of CAV1 in differentiation of the colonic epithelial cells is an issue that requires further exploration. Researchers had shown that APC mutation in colon carcinoma causes CAV1 silencing, suggesting APC as a major regulator of CAV1. It is interesting and clinically important to study CAV1 expression in the absence of functional APC [18]. We showed here that chromatin remodeling by epigenetic mechanisms regulates CAV1 gene expression. DNA methylation of promoter CGIs results in transcriptional silencing, and deregulation of methylation is involved in oncogenesis and tumour progression. CpG Methylation of CAV1 is more frequent in the tumour tissues than the normal colonic mucosa [14,43]. We have shown that CAV1 is silenced by promoter CpG methylation in APC  / HT29 cells and may be induced by HDAC inhibitor butyrate without changes in the methylation status. In several other APC  / CRC lines, such as LoVo, SW480 and SW620, CGI is not methylated and significant levels of CAV1 is expressed (Fig. 6D) [14]. HDAC inhibitor had no effects on CAV1 promoter reactivation in these cells, since HDAC corepressor complexes were not recruited to the promoter. Together the above results suggest that CGI methylation, but not APC mutation may be the predominant factor behind CAV1 regulation in CRC cells. Unmethylated state of CpG sites within an island correlates with increased presence of acetylated histone H3 [59]. For CAV1 expression, Methyl-CpG binding proteins (MBD)/ HDAC binding is perturbed in de-methylated CpG after 5-aza-20 dC treatment, which is confirmed by the disappearance of HDAC1 from the promoter (Fig. 5D). This led to the acetylation of H3 followed by relaxation of chromatin structure for RNA polymerase binding to start transcription. Following butyrate treatment, HDAC1 is not removed from the promoter, but becomes inactive, leading to acetylated H3/H4 to initiate transcription [60]. It was believed for more than a decade that decreased methylation is a prerequisite for effective transcription after inhibition of HDACs, and hypermethylated genes cannot be transcriptionally reactivated with HDACi alone in tumour cells [61]. A recent study reported that vast majority of HDACi can induce genes, which are silenced by methylation without altering the methylation status [62]. We suggest that this ambiguity may be due to methylation density rather that the methylation status. We found that HDACi, such as Butyrate and TSA may induce hypermethylated CAV1 in HT29, suggesting that CpG demethylation and HDAC inhibition act independently to induce CAV1 expression in HT29 cells. However, this does not hold true for Colo-205 and CaCo-2 cells where CAV1 is also hypermethylated and may be induced by 5-Aza-20 dC, but not by butyrate (Fig. 6F). Methylation density was found to be very high in the latter cells and significantly denser than HT29 cells (Fig. 6D). We propose that the density of methylation is more critical than the methylation status per se

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for HDACi to reactivate hypermethylated promoters. However, if this phenomenon applies to other genes as well needs to be investigated. In conclusion, we are the first to address the regulation of CAV1 expression during colonocyte differentiation in vitro. Our study establishes the key molecular mechanisms of APC-independent epigenetic regulation of CAV1 expression in colon carcinoma cells. We report that CAV1 is up-regulated during butyrate-induced colonocyte differentiation and participates in cellular differentiation and apoptosis. We have shown that butyrate can reactivate CAV1 by prolonged histone hyper acetylation through direct inhibition of histone deacetylase activity without removing the deacetylase enzyme from the hypermethylated promoter. Simultaneously, it is not only methylation, but more importantly the density of methylation that determine HDAC inhibition and transcriptional activation of hypermethylated genes. It provides further insight into the molecular interactions between promoter methylation and HDAC inhibition in chromatin remodeling and the regulation of gene expression. Our findings show therapeutic implications of butyrate and other HDACi to reactivate tumor suppressor genes, which are silenced by CpG hypermethylation in cancer. Finally, CAV1 induction gives a novel explanation for the protective role against CRC by butyrate as well as dietary fibers.

Acknowledgment Authors acknowledge Dr. Didier Trono for his kind gift of Lentiviral packaging plasmids, psPAX2 (Addgene plasmid # 12260) and pMD2. G (Addgene plasmid # 12259). Authors acknowledge Indian Council of Medical Research (ICMR), Government of India for intramural grant. Author Nirmalya Dasgupta acknowledges ICMR for Fellowship (Grant no. ICMR:3/1/2/17/2013-Nut.).

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