The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis

Accepted Manuscript The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis

Morgane Placet, Guillaume Arguin, Caroline M. Molle, JeanPhilippe Babeu, Christine Jones, Julie C. Carrier, Bernand Robaye, Sameh Geha, Francois Boudreau, Fernand-Pierre Gendron PII: DOI: Reference:

S0925-4439(18)30057-7 doi:10.1016/j.bbadis.2018.02.008 BBADIS 65056

To appear in: Received date: Revised date: Accepted date:

18 October 2017 24 January 2018 12 February 2018

Please cite this article as: Morgane Placet, Guillaume Arguin, Caroline M. Molle, JeanPhilippe Babeu, Christine Jones, Julie C. Carrier, Bernand Robaye, Sameh Geha, Francois Boudreau, Fernand-Pierre Gendron , The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbadis(2018), doi:10.1016/j.bbadis.2018.02.008

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ACCEPTED MANUSCRIPT The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis

Morgane Placet1, Guillaume Arguin1, Caroline M. Molle1, Jean-Philippe Babeu1, Christine

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Jones1, Julie C. Carrier2, Bernand Robaye3, Sameh Geha4, Francois Boudreau1, Fernand-Pierre

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Gendron1*

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1. Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada

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2. Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

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3. Institute of Interdisciplinary Research, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, Gosselies, Belgium

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4. Department of Pathology, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada

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Running title: The P2Y6 receptor promotes colorectal cancer cell survival

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* Corresponding author: Pr. Fernand-Pierre Gendron, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3201, rue JeanMignault, Sherbrooke, QC, J1E 4K8, Canada. Phone: 1-819-821-8000 (75272); Fax. 1-819-820-6831

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ACCEPTED MANUSCRIPT Abstract Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5’-diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-

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promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC)

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was chemically induced in P2ry6 knockout (P2ry6-/-) mice using azoxymethane and dextran

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sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also

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prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in

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resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6-/- mice developed a reduced number

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of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was

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significantly lower in P2ry6-/- animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNF-induced apoptosis. This protective effect was

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mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP)

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by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the

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cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.

Keywords: P2Y receptors, cancer, apoptosis, drug resistance, intestine, GPCR

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ACCEPTED MANUSCRIPT 1. Highlights Absence of P2Y6R expression reduced tumor load in a mouse model of colorectal cancer

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The presence of P2Y6R was associated with augmented dysplastic grade

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P2Y6 receptor protected colorectal cancerous cells from apoptosis via XIAP signaling

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Activation of P2Y6R protected proliferative cells from 5-FU-induced apoptosis

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ACCEPTED MANUSCRIPT 2. Introduction Colorectal cancer (CRC), the third most prevalent form of cancer worldwide [1], originates from the accumulation of genetic mutations and epigenetic deregulation of tumor suppressor genes and oncogenes, most prominently APC, K-RAS and TP53 genes [2]. As a result, transformed

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intestinal epithelial cells (IECs) acquire evolutionary growth advantages leading to the formation

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of tumors [2]. Tumor resistance to apoptosis is one of the hallmarks of cancer potentially

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contributing to tumor progression and resistance to treatment [3]. The cancerous epithelial cells acquire this capacity by increasing the expression of anti-apoptotic proteins and by altering their

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response to the TNF- family of death receptors [3]. This acquired resistance may be associated

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with the ability of cancer cells to survive in a pro-inflammatory environment that should normally be detrimental to their survival. Indeed, as in other solid tumors, various types of tumor-

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promoting molecules (e.g. TNF-α, IL-6, CCL2, CXCL8) are found in the vicinity of colorectal

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tumors (see Terzic et al. [4] and references therein) as well as a high concentration of extracellular nucleotides [5]. Accordingly, these molecules have recently been identified as

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important modulators of tumor biology favoring cancerous cell proliferation and dissemination

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[6, 7].

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The presence of uridine 5’-triphosphate (UDP) in the extracellular environment is of particular interest since it is the unique endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R). In the intestine, this receptor has immunomodulatory functions as previously reported by our group [8, 9]. In cancer, stimulation of CRC-derived Caco-2 and HCT9 cells [10], as well as in PANC-1 pancreatic cancer cells [11], with UDP increases cell proliferation. On the other hand, receptor stimulation had no effect in BT549 breast cancer cells [12] or in A375 human

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ACCEPTED MANUSCRIPT melanoma cells [13], while it has anti-proliferative properties in gastric cancer cells [14]. Furthermore, UDP has been reported to exert cytostatic and cytotoxic actions on human neuroblastoma SH-SY5Y cells overexpressing a recombinant P2Y6R [15]. In contrast, P2Y6R activity was associated with resistance to TNF-induced apoptosis in astrocytes [16] and skeletal

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muscle cells [17]. Accordingly, P2Y6R activity was also found to amplify the cellular responses

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to DNA damages caused by gamma irradiation of A549 human adenocarcinoma cells [18]. It

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would thus appear that the functions of P2Y6R in cancer may be linked to the affected organ and

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cell types, hence the need to clarify the action of this receptor in CRC.

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The X-linked inhibitor of apoptosis protein (XIAP) is a critical anti-apoptotic factor and regulator of TNF-induced cell death in inflammatory diseases and cancer [19]. XIAP directly binds to

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caspase-3, -7, and -9 to block their activities [20]. Irrespective of its anti-apoptotic effect, XIAP

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can also interact with Bone morphogenetic protein receptor-1A, TAK1 binding protein 1 [21] and TAK1 [22]. More recently, it has been suggested that XIAP lacking the RING domain may

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promote anchorage-independent cancer cell growth by interacting with the E2F1 transcription

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factor [23]. XIAP has been identified as the most potent anti-apoptotic protein of the IAP family members [19] in addition to being recognized as a potential target for abrogating drug resistance

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[19]. Indeed, its increased expression in rectal cancer is an indicator of neoadjuvant radiochemotherapy resistance [24].

In the present study, we show that deletion of the P2ry6 gene leads to a reduction in the number and volume of tumors in mice and that this effect is putatively linked to P2Y6R-dependent stimulation of the anti-apoptotic protein XIAP through a PI3K/AKT-dependent mechanism in

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ACCEPTED MANUSCRIPT cancerous IECs. In addition, using tumoroids derived from mouse colorectal tumors, we show that stimulating P2Y6 receptor activity using the P2Y6R agonist MRS2693 partially blocks 5-FU cytotoxicity.

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3. Material and Methods

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3.1 Cell Culture. Human colon carcinoma HT-29 cells (ATCC, HTB-38) were grown in

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Dulbecco’s modified Eagle Medium (DMEM, Wisent Inc., St. Bruno, QC) supplemented with 10% FBS, 1% penicillin-streptomycin, 1% HEPES (Wisent Inc.) and 1% GlutaMAX (Thermo

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Fisher Scientific, Burlington, ON) at 37oC in a 5% CO2 humidified atmosphere, as previously

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described [9]. To study the effect of P2Y6 receptor activation on apoptosis at confluence, P2Y6R was activated with either 10 M UDP (Sigma-Aldrich, Oakville, ON) or 1.5 M MRS2693

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(Cederlane, Burlington, ON), a selective P2Y6R pharmacological agonist, for 15 min. Apoptosis

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was induced using 20 ng/μL TNFα for 4h (BioShop Canada Inc., Burlington, ON) in the presence

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of 5 M cycloheximide (CHX, Sigma-Aldrich) [25]. After the 4h treatment with TNF, cell medium was replaced for 16h by fresh medium containing UDP or MRS2693, CHX and 1% FBS

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along with the supplements described above.

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3.2 Generation of shP2Y6R cells. The 21mer shRNA constructs directed against human P2RY6 (NM_176797.1) were purchased from Sigma-Aldrich MISSION shRNA (cat. # SHCLNGNM_176797; St. Louis, MO). Lentiviruses were produced in HEK293T cells and used for HT-29 cell infection as previously described [8, 26]. To validate shRNA efficiency, HT-29 cells were harvested and P2RY6 gene expression was analyzed by quantitative real-time PCR (qPCR). Briefly, qPCR was realized as previously reporter [26] using the human sequence-specific

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ACCEPTED MANUSCRIPT primers

for

P2RY6:

5'-CCAGAGCAAGGTTTAGGGTGTA-3'

and

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GTGTCTACCGCGAGAACTTCA-3'. Gene expression was normalized to glyceraldehyde 3phosphate dehydrogenase (GAPDH) gene expression as reported [9].

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3.3 Human colorectal tissues. Samples of 12 CRC and paired normal tissues (at least 10 cm the

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tumor) were obtained from patients undergoing surgical resection without neoadjuvant therapy.

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Tissues were collected after obtaining the patient’s written informed consent according to the protocol approved by the Institutional Human Subject Review Board of the Centre Hospitalier

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Universitaire de Sherbrooke. Paired tissues were collected and processed as previously described

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[27, 28]. Total RNA was isolated with the Totally RNA kit (Thermo Fisher Scientific) according to manufacturer instructions, complementary DNA prepared as previously presented [26]. The

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qPCR analyses were performed as describe above.

3.4 Animals. The P2ry6 knockout mice (P2ry6-/-) were kindly provided by Pr. Bernard Robaye

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(Université Libre de Bruxelles, IRIBHM, Gosselies) [29]. The mice were backcrossed in a

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C57BL6 background at the Université de Sherbrooke Pavilion of Applied Research on Cancer transgenic mice unit and maintained in this facility thereafter. Twelve-week-old P2ry6-/- mice (F8

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to F12) and control littermates (P2ry6+/+) were used for the ensuing experiments.

3.5 Azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colorectal tumor model. Mice were injected intraperitoneally with 6 mg/kg body weight of AOM (NCI, Frederick, MD) on day one. After one week, mice were given a solution of 1.5% (w/v) DSS (MW 36–50 kDa, MP Biomedicals, Solon, OH) as drinking water for 7 days. A recovery phase of 14 days was allowed followed by a 2nd and 3rd cycle of 1.5% DSS treatments and recovery. Following the last 7

ACCEPTED MANUSCRIPT 14-day recovery period, mice were euthanized by cervical dislocation under Isoflurane (Baxter Corp., Mississauga, ON) anesthesia and the colons harvested, washed with phosphate-buffered saline and opened longitudinally. The number of tumors was counted and their volume estimated as V = 1/6d3. The disease state, expressed as disease activity index (DAI), was monitored

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weekly as previously described [8]. All procedures were approved by the Université de

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Sherbrooke Animal Care Committee and performed according to the Canadian Guidelines for

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Care and Use of Experimental Animals.

3.6 Tumoroid preparation, maintenance and treatments. Tumoroids were prepared from

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colorectal tumors isolated from AOM-DSS-treated P2ry6+/+ mice following the protocol described by Sato et al. [30]. Briefly, tumors were resected from the colon and incubated on a

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rocker for 1h at RT in a PBS/2 mM EDTA solution. Cleansed resected tumors were digested in

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digestion solution consisting in Advanced DMEM/F12 (Gibco by Thermo Fisher Scientific),

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2.5% FBS (Wisent, Canada), Dispase type II (Roche Life Science, Laval, QC) [125 g/mL], and Collagenase IX (Roche Life Science) [10 mg/mL]) for 1 h à 37°C. Detached cells were

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centrifuged at 200 x g for 4 min, resuspended in matrigel (Gibco by Thermo Fisher Scientific)

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and placed in culture in the tumoroid culture medium (Advance DMEM F12, 50 g/ml EGF (Thermo Fisher Scientific), 1x B27 supplement (Gibco by Thermo Fisher Scientific), 1x N2 supplement (Gibco by Thermo Fisher Scientific) and 500 mM n-acetylcysteine (Sigma-Aldrich). Tumoroids were maintained in culture for 6 days prior to replating. The tumoroid culture medium was renewed every 2 days. For the experiments, at 72 h post replating, 1.5 M MRS2693 or vehicle was added to the tumoroid medium 30 min prior to the addition of 10 M or 100 M 5FU for 24h. Following treatments, phase contrast micrographs were acquired using a Zeiss

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ACCEPTED MANUSCRIPT Axiovert 200M microscope, after which tumoroid morphology was assessed and the number of living or dying tumoroids counted.

3.7 Tissue processing, histopathology, immunohistochemistry and indirect

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immunofluorescence. Following the macroscopic assessment, colons were fixed using

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methacarn solution (60% methanol, 30% chloroform and 10% glacial acetic acid) and sent to the

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Electron Microscopy and Histology Research Core for paraffin embedding, sectioning (5m

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sections) and hematoxylin & eosin (H&E) staining. Histological analyses were performed by a clinical pathologist (SG). The dysplastic state was score following this scoring system: 1 for

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region with regeneration, 2 for low grade dysplasia, 3 for low grade dysplasia with important inflammation and a score of 4 for region of high grade dysplasia. The results were presented as

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the mean score ± SEM. Immunohistochemistry (IHC) staining was performed with the Dako

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EnVision+ System-HRP (DAB) according to the manufacturer's protocol (Dako Canada ULC,

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Mississauga, ON). Tissue was immunostained overnight at 4oC with mouse anti--catenin (BD Transduction Laboratories, Canada, 1:500) or rabbit anti-c-MYC (Santa Cruz Biotech, Santa

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Cruz, CA, 1:500) as primary antibodies and for 60 min at RT using an anti-mouse or anti-rabbit labeled polymer HRP Dako Envision + System-HRP (DAB). Image acquisition was performed

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on a Hamamatsu Nanozoomer Digital Slides Scanner under bright field illumination. Tissue vascularization was assessed by indirect immunofluorescence. Tissues were stained overnight at 4oC with 1:500 of a polyclonal rabbit anti-CD31 antibody (Abcam Inc., Toronto, ON). Slides were washed and incubated 2h at RT with 1:500 Alexa Fluor 488 donkey anti-rabbit IgG (Abcam Inc.) as secondary antibody. Images were captured on a Leica DM2500 microscope mounted with a Hamamatsu ORCA-R2 digital camera. The dysplastic regions were attributed a score of 1 for

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ACCEPTED MANUSCRIPT regions with regeneration, 2 for low grade dysplasia, 3 for low grade dysplasia with important imflammation and a score of 4 for region of high grade dysplasia. The results were presented as the mean score ± SEM.

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3.8 High-Resolution confocal microscopy for visualization of EdU incorporation in

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tumoroids.

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Tumoroids were labeled for proliferative cells (S phase cells) using the Click-It EdU imaging C10338 kit according to the manufacturer’s protocol (Thermo Fisher Scientific). Tumoroids were

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plated on Ibidi -slide angiogenesis (Ibidi USA, Inc., Fitchburg, WI) and grown for 72 h as

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described above and thereafter treated with 100 M 5-FU for 24h in the presence of 1.5 M MRS2693 or vehicle. The P2Y6R agonist MRS2693 was added 30 min prior to 5-FU treatment.

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EdU incorporation and detection was carried out as described by the manufacturer’s protocol.

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Nuclei were counterstained with Hoechst 33342. Immunofluorescence was captured on a Leica

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TCS SP8 STED 3X/WLL\FLIM/FCS microscope.

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3.9 Western blot analysis. After treatment and stimulation, cells were harvested and used for Western blot analyses as described previously [9]. Alternatively, colons were dissected and

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quickly snap frozen in liquid nitrogen. Frozen colons were lysed at 4oC with Triton buffer (40 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium orthovanadate, 40 mM glycerophosphate, 0.1 mM phenylmethanesulfonyl fluoride and protease inhibitor mixture from Sigma-Aldrich) using a Qiagen TissueLyser LT (Qiagen, Toronto, ON). Cell or tissue lysates (20 g protein per lane) were used for SDS-PAGE and Western blot analyses. Immunodetection was performed using anti-cleaved PARP (Asp214, 1:1,000, Cell

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ACCEPTED MANUSCRIPT Signaling Technology (CST)) rabbit monoclonal antibody, anti-GAPDH (1:100,000, CST), antiXIAP (1:1,000, CST), anti-PUMA (1:500, CST), anti-Bax (1:500, CST), anti-Bid (1:500, CST), anti--actin (1:10,000, Millipore), anti-AKT (1:2,000, CST), anti-pAKT (Thr308, 1:500, CST), anti-pXIAP (Ser87, 1:500, Abcam) and anti--catenin (BD Transduction Laboratories, Canada,

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1:5,000) antibodies. After extensive washings with PBS/Tween-20 solution, the blots were

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incubated for 60 min at room temperature with HRP-conjugated secondary antibodies and bands

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revealed with the Millipore chemiluminescence system and HyBlot ES film (Denville Scientific

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Inc.) according to the manufacturer’s instructions. Western blot signals quantification by

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densitometry was realized as previously described [9].

3.10 Annexin V assay. HT-29 cells were seeded on sterile glass coverslips and apoptosis was

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induced at confluence as described above in the presence or absence of P2Y6R agonists.

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Following treatments, cells were washed with cold PBS and incubated in cold Cell Staining

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Buffer (BioLegend, San Diego, CA) for 15 min at room temperature. Cells were then stained for Annexin V (BioLegend) and the nuclei counterstained with Hoechst 33342 (ThermoFisher

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Scientific, Mississauga, ON) for 15 min at room temperature. The number of dead cells was determined by propidium iodide (PI) staining for 1 min. Slides were processed as previously

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described [31], and images were captured on a Leica DM2500 microscope using a Hamamatsu ORCA-R2 digital camera.

3.11 Statistical analysis. Results were expressed as the mean ± SEM. Statistical significance was determined using an unpaired t test or ANOVA with multiple comparisons post-test as described

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ACCEPTED MANUSCRIPT in figure legends. The number of replicates for each experiment is indicated in figure legends. A value of p < 0.05 was considered statistically significant.

4. Results

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The AOM/DSS model is a highly suitable animal model for recapitulating the human colorectal

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adenoma-carcinoma sequence by displaying neoplastic lesions, aberrant crypt foci and

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adenocarcinomas [32]. Using this model and large field histological observation of H&E-stained sections of the distal mouse colon, we can appreciate the extend of dysplastic regions and tumor

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load in P2ry6+/+ mouse (Fig. 1A) vs. P2ry6-/- animals (Fig. 1B). In fact, the absence of P2ry6

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expression in mice significantly reduced the number (Fig. 1C) and volume of colorectal tumors (Fig. 1D) as compared to P2ry6+/+ littermates. Assessment of the tumor dysplastic score was also

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significantly lower in P2ry6-/- mice (Fig. 1E). Histological analyses revealed that P2ry6+/+ mice

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featured dysplastic lesions with a high dysplastic grade (Fig. 1F), low-grade dysplastic lesions with signs of regeneration (Fig. 1G) or highly infiltrated dysplastic lesions (Fig. 1H). Conversely,

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the P2ry6-/- mouse lesions were characterized by regenerative crypts and cryptal proliferation

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(Fig. 1I), regenerative crypts with significant inflammation (Fig. 1J) or simple dysplasia (Fig. 1K). These differences in the histologic assessment do not seem associated to the inflammatory

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response of animals as both P2ry6+/+ and P2ry6-/- mice displayed similar DAI values (additional file 1). Overall, the results suggest that the absence of P2Y6R expression and activity are somewhat protective of tumorigenesis. In fact, human P2RY6 gene transcript was significantly increased in tumors isolated from CRC patients vs. matching resection margins (additional file 2), thus suggesting that P2Y6R expression was required for tumor development. However, these results must be interpreted with caution given the small sample size. Further supporting a role for P2Y6R in CRC, tumors in P2ry6+/+ mice were vascularized as shown by the increase in CD31 12

ACCEPTED MANUSCRIPT staining (Fig. 2A-C) when compared to the weak CD31 signaling observed in the lesions of P2ry6-/- animals (supplement Fig. 2D-E).

To further investigate the consequence of P2ry6 knock-down on tumorigenesis, the expression

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pattern of -catenin in colonic lesions was determined by IHC analyzes. These analyzes have

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revealed that -catenin expression was found to be delocalized from the plasma membrane

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toward the cytosol and nucleus of IECs forming the tumor in P2ry6+/+ mice colons (Fig. 3;

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additional file 3). Indeed, contrary to the adjacent margins where -catenin was located at the plasma membrane (Fig. 3A), cytosolic, perinuclear and nuclear staining was observed in the

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dysplastic regions (Fig. 3B), in accordance with the literature [33]. In P2ry6-/- mice, -catenin

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expression was observed at the plasma membrane of colonocytes in both margin (Fig. 3C) and regenerative areas (Fig. 3D) with little or no delocalization toward the cytoplasm and nucleus.

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The presence of -catenin in the nucleus was often associated with the aberrant expression of

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transcriptional targets such as the proto-oncogene c-MYC [34]. Indeed, high grade dysplastic lesions and intra-mucosal carcinoma, as found in the AOM/DSS model, are reported to have

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increased c-MYC expression [32]. Accordingly, immunohistochemistry analyses for c-MYC

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expression patterns revealed a nuclear expression of c-MYC in the dysplastic tissues of P2ry6+/+ mice, whereas nuclear c-MYC staining was virtually absent in P2ry6-/- lesions (Fig. 4). These findings are in agreement with observations reported in human colorectal tumors, which showed that the decreased expression of -catenin at the plasma membrane and in the cytosol was associated with poor outcomes in primary CRC patients [33].

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ACCEPTED MANUSCRIPT One of the hallmarks of colorectal cancer cells is the intrinsic or acquired resistance to apoptosis, such as the resistance toward TNF- [35]. This ability to evade apoptosis contributes to carcinogenesis, tumor progression and resistance to treatments [36]. Treatment of HT-29 cells with the P2Y6R agonist MRS2693 (1.5 M) prevented the apoptotic effect of TNF as shown by

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the increased number of Hoechst33342-positive cells (Fig. 5A) as well as reduced number of

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propidium iodide-positive stained cells (Fig. 5C). The overall number of positive annexin V cells

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appeared similar between both groups even in the presence of MRS2693 (Fig. 5B). This apparent lack of effect may be the result of the higher number of cells found in the P2Y6R activated group.

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Similar results were also obtained in response to 10 M UDP (additional file 4). The reduction in

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cleaved PARP in response to P2Y6R stimulation by UDP (additional file 4D-E) was correlated with a decrease in stimulated P2Y6R-dependent caspase 3 activity (additional file 4F). These

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results suggest that P2Y6R activity may prevent adenocarcinoma apoptosis in response to a TNF-

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 challenge.

The resistance to induced apoptosis is ensured by the down-regulation of pro-apoptotic proteins

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such as PUMA, Bax and Bid, or the up-regulation and activation of a number of anti-apoptotic

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proteins [36]. Activation of P2Y6R by 1.5 M MRS2693 increased the expression of the antiapoptotic protein XIAP, whereas PUMA, BAX, BID, BCL-2 and BCL-XL expression remained unchanged (Fig. 6; additional file 5). XIAP expression was significantly increased 15 min following P2Y6R activation by MRS2693 with its expression remaining significantly high up to 60 min when compared to non-stimulated HT-29 cells. This rapid increase in XIAP expression was not attributable to the synthesis of new protein, but was rather the result of XIAP protein stabilization as shown by its phosphorylation on Ser87 after 15 min of stimulation with 1.5 M

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ACCEPTED MANUSCRIPT MRS2693 (Fig. 7A). The addition of the PI3K inhibitor LY294002 30 min prior to the stimulation of P2Y6R with MRS2693 reduced the phosphorylation of AKT on Thr308 as well as the phosphorylation of XIAP on Ser 87 after 60 min (Fig. 7B). The increase in XIAP expression and phosphorylation was correlated with AKT phosphorylation on Thr308. We used shRNA

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targeting the P2RY6 gene to further confirm that the increase expression of XIAP was due to

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P2Y6R stimulation (Fig. 8). We knockdown P2RY6 gene expression in HT-29 cells using two

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different shRNA (Fig. 8A). The lost of P2RY6 expression blocked the stimulating effect of UDP, the endogenous P2Y6R agonist, as compared to control cells expressing a scrambled noncoding

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shRNA (Fig. 8B).

We then validated if the observed relocalization of -catenin toward the nucleus of IECs in the

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dysplastic tissues of P2ry6+/+ mice was accompanied by a modulation of its overall expression

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(Fig. 9). Distal colons were thus harvested from non-treated and AOM/DSS-treated P2ry6+/+ and P2ry6-/- mice and protein expression determined by Western blots. In fact, there was no

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significant difference in the overall expression of -catenin between animal groups and between

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non-treated and AOM/DSS-treated animals in a given group. Finally, we have shown that AKT phosphorylation on Thr308 was markedly reduced in the colon of P2ry6-/- mice vs. P2ry6+/+

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animals (Fig. 9). The overall level of AKT phosphorylation and AKT expression were not affected by the AOM/DSS treatment for a given group of animals.

Resistance of cancer cells to apoptosis contribute to anti-cancer chemotherapeutic drug resistance [36]. The chemotherapeutic agent 5-fluorouracil (5-FU) is widely used in the treatment of CRC, and despite its administration in combination with irinotecan or oxaliplatin and leucovorin to

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ACCEPTED MANUSCRIPT increase its efficiency, inherent and acquired resistance to 5-FU remains a challenge [37, 38]. In the present study, P2Y6R activity was found to participate in the resistance to 5-FU (Fig. 10), with the latter inducing the death of mouse-derived CRC cystic (Fig. 10A and 10C) and budding (Fig. 10B and 10C) tumoroids. This cytotoxic effect of 5-FU was partially prevented upon

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treatment of tumoroids with the P2Y6R agonist MRS2693 30 min prior to the addition of 5-FU

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for 24h (Fig. 10A-C). The number of degenerating tumoroids was reduced by 40%, whereas the

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number of living tumoroids was increased by more than 2-fold in the P2Y6R agonist-treated group (Fig. 10C). Hence, the stimulation of P2Y6R with MRS2693 prior to 5-FU treatment

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provided protection for proliferative cells as shown by the maintenance of EdU-stained cells in

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tumoroids (Fig. 11).

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5. Discussion

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The P2Y6 receptor is a modulator of the immune and inflammatory responses in inflammatory bowel diseases [8, 9] and in GI infection by Clostridium difficile [39]. Although the exact

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mechanism of UDP release has yet to be clearly established, this diphosphonucleotide acts as a

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danger signaling molecule and is putatively released by IEC in response to an inflammatory challenge [9]. More recently, exposure of epithelial breast cancer cells to the chemotherapeutic

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agent doxorubicin has been reported to also induce the release of UDP [40], thus suggesting that the presence of the P2Y6R agonist may contribute to the formation of a tumor-promoting microenvironment [40]. On the other hand, it was reported that P2Y6R activation suppressed gastric cancer cells growth via an anti-proliferative mechanism involving SOCE/Ca2+/-catenin signaling [14]. Given the particular biology of the intestinal epithelium that is characterized by its continuous renewal, the presence of a myriad of immune cells in both normal and pathological

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ACCEPTED MANUSCRIPT mucosa and exposure to a rich microbiota, our data are not surprising and further suggest that the role of the receptor is dependent of the environment and the cell type expressing P2Y6R as recently proposed [14]. Furthermore, the role of P2Y6R in CRC is in accordance with previous studies reporting a pro-tumorigenic effect and decreased survival probability in patients with a

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high P2RY6 mRNA expression in renal cancer as reported in Protein Atlas [41].

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P2Y6R has furthermore been described for its anti-apoptotic properties in the 1321N1 astrocytoma cell line overexpressing a recombinant form of this receptor [16] as well as in

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C2C12 skeletal muscle cells [17]. Resistance to apoptosis is one of the hallmarks characterizing

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cancer cells [42]. This acquired phenomenon not only favors the chaotic growth of cancer cells, but also leads to chemoresistance and allows the survival of cancer cells in a usually detrimental

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pro-inflammatory microenvironment [43]. The adaptation of cancer cells to the presence of high

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levels of TNF in preneoplastic lesions of the inflamed colonic mucosa and in the microenvironment of CRC tumors [44] is a perfect example of acquired resistance to apoptosis

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by cancer cells. Typically, TNF induces apoptosis through the activation of caspase-8 and the

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downstream effectors caspases-1, -3, -6, and -7 [45]. Active caspase-3 is a key executioner of apoptosis since it is responsible for the proteolytic cleavage of several key proteins, including

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nuclear enzyme poly (ADP-ribose) polymerase (PARP) [45]. In the present study, we propose that P2Y6R contributes to TNF resistance by inducing the stabilization of the anti-apoptotic protein XIAP, which is known to block the effector function of caspase 3 [19], as shown herein by the loss of cleaved PARP in P2Y6-stimulated cells. In 1321N1 astrocytoma cells, the protective effect of the P2Y6 receptor toward TNF-induced apoptosis is mainly dependent on PKC activation, as well as partially dependent on ERK signaling, while seemingly independent of

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ACCEPTED MANUSCRIPT the PI3K/AKT pathway [16]. It is known from previous studies that P2Y6R can activate ERK1/2 and PKC signaling in IEC [8, 9, 39], thus, we cannot rule out that these pathways could also be involved in the survival of CRC-derived IEC. However, the present results establish that P2Y6R stimulation leads to XIAP phosphorylation on Ser87 by a pathway involving AKT. In fact, the

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expression of active AKT, which is characterized by its phosphorylation on Thr308, was

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markedly reduced in the colon of P2ry6-/- mice as compared to P2ry6+/+ animals. These results

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strongly suggest that AKT is a direct downstream effector of P2Y6R signaling. Furthermore, the phosphorylation of this particular XIAP serine residue was associated with protein stabilization

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and evasion from proteasomal degradation [46]. Indeed, the phosphorylation of XIAP by AKT

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has been associated with human ovarian cancer (OVCAR-3) cell survival and resistance to chemotherapy [47]. Similarly, the phosphorylation of XIAP on Ser87 increases SH-SY5Y

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neuroblastoma cell survival in response to the chemotherapeutic agent etoposide [46].

In the present study, we took advantage of P2ry6-/- mice and P2ry6+/+ littermates in which

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colorectal cancer was chemically induced with AOM and DSS treatments to elucidate the

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function of this receptor. Using this in vivo approach, we were able to show that the loss of P2Y6R expression in mice offered clear protection against chemically-induced colorectal

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carcinogenesis. In particular, macroscopic and histological characterization of colon sections isolated from P2ry6+/+ and P2ry6-/- mice with CRC showed an overall reduction in tumor load and dysplastic grade in KO animals. These findings are in accordance with the anti-apoptotic role of P2Y6R since knockout animals showed a limited number of tumors. The reduction in tumor load measured in P2ry6-/- animals is also in keeping with the previously reported in vitro positive growth effect of UDP in Caco-2 and HCT8 cell lines [10], as well as pancreatic PANC-1 cells [11]. We were not able to detect a significant proliferative effect in response to P2Y6 receptor 18

ACCEPTED MANUSCRIPT stimulation in vitro in HT-29 cells. However, histological analysis of colon sections showed the delocalization of the -catenin protein from the plasma membrane toward the cytosol and nucleus in the dysplastic tissues of P2ry6+/+ mice. In contrast, -catenin was mainly located at the plasma membrane of colonocytes in P2ry6-/- mice. The presence of -catenin in the cytosol and nucleus

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was not surprising given that this localization has been associated with proliferative and invasive

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phenotypes, as well as to poor outcomes in CRC patients [33]. Hence, the presence of -catenin

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in the nucleus of IECs of P2ry6+/+ dysplastic lesions was correlated with the increased expression

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of c-MYC, a direct transcriptional target of nuclear -catenin [48], thereby suggesting the presence of an active nuclear -catenin. The absence of -catenin delocalization in simple

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dysplasia isolated from P2ry6-/- mice suggests that this receptor may be involved in a mechanism

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modulating -catenin signaling. Indeed, the suppression of the -catenin pathway by P2Y6R has been reported in gastric cancer cells and associated with a reduction in gastric cancer cell growth

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[14]. While the relationship between P2Y6R and -catenin signaling requires further investigation

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in the context of CRC, its localization in the nucleus of cancer cells as observed herein in P2ry6+/+ mice colons nonetheless suggests that P2Y6R may also have a proliferative effect or be

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involved in tissue invasion. Furthermore, the co-expression of nuclear -catenin and c-MYC has

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been purported to be correlated with tumor size rather than with proliferative activity in colorectal adenomas [49], an effect also observed in our mouse model.

The increased resistance to apoptosis and the stabilization of XIAP via its phosphorylation is also indicative of a possible role for P2Y6R in chemoresistance. In CRC, the development of resistance to 5-FU treatments is often observed in cancer. To determine whether P2Y6R could be involved in 5-FU chemoresistance in CRC, we resected entire colorectal tumors from P2ry6+/+

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ACCEPTED MANUSCRIPT mice and placed them in culture in the form of tumoroids. The latter were treated with 5-FU for 24h in the presence or absence of the P2Y6R agonist MRS2693. As expected, 5-FU treatment killed most of the tumoroids whereas the addition of MRS2693 prior to 5-FU treatment allowed the survival of 50% of the tumoroids. These results are indicative of the protective effect of

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P2Y6R in the context of CRC treatment and of its involvement in chemoresistance to 5-FU.

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Furthermore, the activation of P2Y6R prior to the addition of 5-FU protected proliferative cells

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from 5-FU-induced apoptosis. Although we could not formally identify these proliferative cells, it is quite plausible that the latter represent stem-like cells. Indeed, intestinal stem cells have been

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reported to strongly retain proliferative staining for active S-phase markers such as BrdU and

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EdU [50]. It could be hypothesized that P2Y6R offers a protective mechanism to cancerous stem cells in order to evade chemically-induced apoptosis, which could favor the resurgence of

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cancerous cells following chemotherapy and further the progression of cancer. Based on our in

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vitro studies, we believe that the stabilization of XIAP expression in response to P2Y 6R stimulation was responsible for the resistance to the 5-FU regimen. In effect, XIAP expression

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has been associated with an increase in resistance to 5-FU in pancreatic carcinoma cells [51].

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Furthermore, the down-regulation of PPP2R1B by microRNA-587 has been associated with an increase in AKT phosphorylation and XIAP expression in colorectal cancer cells and resistance

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to 5-FU treatment [52], thus supporting our findings that P2Y6R-dependent stimulation of pAKT and stabilization of XIAP participate in the resistance to apoptosis and in increasing the resistance to 5-FU. This down-regulation of XIAP activity has furthermore been suggested as a means to sensitize cancerous colorectal epithelial cells [53] to apoptosis. Lastly, extracellular nucleotides, including UDP, have recently been described as emerging signaling molecules in tumor biology. The reported effects range from stimulation of cell proliferation to modulation of cancer cell dissemination and invasion (see Ferrari [6], and references therein). Hence, we 20

ACCEPTED MANUSCRIPT previously reported that P2Y6R stimulated the secretion of CXCL8 by IEC, which lead to the recruitment of neutrophils [8, 9]. This powerful chemokine also acts a paracrine factor to stimulate the carcinogenic process by directly stimulating cancerous cell proliferation and indirectly by promoting angiogenesis [54]. It is thus quite plausible that this mechanism could

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have also influenced tumorigenesis in our model. However, additional experiments will be

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required to validate this hypothesis.

6. Conclusion

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We have identified XIAP as a novel P2Y6R-dependent signaling effector in cancerous intestinal

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epithelial cells. In effect, we show that the apoptotic protective effect of P2Y6R in cancerous IEC was mediated by the stabilization of XIAP expression following its phosphorylation of Ser87 in

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response to a TNF challenge. We furthermore show in vivo that the absence of P2Y6R

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expression reduced both tumor load and the dysplastic grade of mice with chemically-induced CRC. Finally, using tumoroids derived from mouse CRC tumors, we demonstrate that the

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activation of P2Y6R provided resistance to the chemotherapeutic drug 5-FU, thereby suggesting

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that P2Y6R potentially contributes to the acquired resistance often observed in patients with CRD. With the recent development of new P2Y6R antagonists [55, 56], we believe that this

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receptor could be targeted to block XIAP activity and potentiate the effect of chemotherapeutic drugs such as 5-FU.

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ACCEPTED MANUSCRIPT 7. Acknowledgements The authors thank Mr. Pierre Pothier for the critical reading of the manuscript. This work was supported by a Canadian Institutes of Health Research operating grant [MOP-286567] to FPG. FPG is members of the FRQ-S-funded Centre de recherché du CHUS. The colorectal cancer

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tissues bank was supported by a team grant on digestive epithelium from the Canadian

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Institutes of Health Research. The authors thank Mr. Gérald Bernatchez (Université de

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Sherbrooke) for his technical assistance. The authors recognize the electron microscopy

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and histology platforms and the photon microscopy platform from the Faculty of

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medicine and health sciences at the Université de Sherbrooke for their services.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Invalidation of the P2ry6 gene in mice decreases colorectal carcinogenesis and reduces dysplastic grade. Colorectal cancer was induced in P2ry6 knockout mice (P2ry6-/-) and P2ry6+/+ littermates using azoxymethane (6 mg/kg) along with dextran sulfate sodium (1.5%)

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challenges. Typical large field examination of H&E-stained histological sections of distal colon

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for A) one non-treated (control) and two AOM-DSS treated P2ry6+/+ mice and B) one control

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and two AOM-DSS treated P2ry6-/- animals. Some of the dysplastic regions are indicated by arrows for the AOM-DSS treated P2ry6+/+ mice. Scale bars = 2.5 mm. C) Number of tumors, D)

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tumor volumes (measured as described in the Methods section) and E) dysplastic states were all

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significantly lower in P2ry6-/- mice compared to P2ry6+/+ animals (n = 5 mice per group). Dysplasia was assessed as described in the Methods section. Results are presented as the mean ±

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SEM and statistical significance determined by an unpaired t test, where *: p<0.05. F-K)

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Histological analysis on fixed and H&E-stained histological sections revealed high-grade dysplasia without immune cell infiltration and low-grade dysplasia in P2ry6+/+ mice under AOM-

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DSS treatment (F-H), while low-grade dysplasia and regenerative zones were observed in

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knockout mice (I-K). Scale bars = 250 m.

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Figure 2. Dysplastic lesions observed in P2ry6+/+ mice colon are vascularized comparatively to lesions found in P2ry6-/- animals. Indirect immunofluorescence for CD31 was used to identify vascular endothelial cells (stained in green). Nuclei were stained in blue using Hoechst33342. Three independent and representative micrographs obtained from P2ry6+/+ (A-C) and P2ry6-/- (D-F) mice are shown. Scale bars are provided on panels.

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ACCEPTED MANUSCRIPT Figure 3. Invalidation of the P2ry6 gene alters the localization of β-catenin in colonic dysplastic tissues. AOM-DSS was used to induce CRC in P2ry6+/+ and P2ry6-/- mice. The expression and localization of -catenin was analyzed by immunohistochemistry in both margin (A) and dysplastic area (B) of P2ry6+/+ animals as well as in the margin (C) and regenerative

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area (D) of P2ry6-/- mice. Typical micrographs are shown with scale bars (250 m) provided in

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each panel.

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Figure 4. Invalidation of P2ry6 gene expression affects the expression of C-MYC in colonic dysplastic tissues. AOM-DSS was used to induce CRC in P2ry6+/+ and P2ry6-/- mice. The

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expression and localization of C-MYC was analyzed by immunohistochemistry in both the margin and dysplastic area of P2ry6+/+ animals as well as in the margin and regenerative area of

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P2ry6-/- mice. Typical micrographs are shown for two P2ry6+/+ and two P2ry6-/- mice. Scale bars

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= 50 m.

Figure 5. P2Y6R activation protects HT-29 colorectal carcinoma cells from TNFα-induced

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apoptosis. Apoptosis was induced by a combined treatment of 20 ng/μL TNFα and 5 M CHX

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for 16 hours. P2Y6R was activated by 1.5 M MRS2693 15 min prior to the addition of TNF and CHX. A) Nuclear staining with Hoechst 33342 showed a marked reduction in cell number following TNF and CHX treatment, whereas P2Y6R activation partially prevented this reduction. B) Annexin V staining and C) propidium iodide incorporation strongly stained TNFand CHX-treated cells. The presence of the P2Y6R agonist decreased the number of dying cells. Scale bars = 345 m. D) PARP cleavage was used as an indicator of active caspase 3. TNF and CHX treatment induced PARP cleavage, which was prevented by the presence of the P2Y6

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ACCEPTED MANUSCRIPT receptor agonist MRS2693. GAPDH expression was used to ensure equal protein loading and lysate integrity. E) Densitometric quantification of 3 independent Western blot experiments for cleaved PARP. Results are presented as the mean ± SEM and statistical significance determined

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by an unpaired t test, where *: p < 0.05.

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Figure 6. Expression of anti- and pro-apoptotic proteins following the activation of P2Y6R.

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Expression levels of the anti-apoptotic proteins XIAP, Bcl-XL and Bcl-2, and of the pro-apoptotic proteins PUMA, Bax and Bid were determined as a function of time by Western blot analysis

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following the stimulation of P2Y6R with 1.5 M MRS2693. GAPDH and -actin expression was

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used to ensure equal protein loading and integrity. The presented blot is typical of three separate

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sets of experiments.

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Figure 7. Activation of the AKT signaling pathway by P2Y6R leads to the stabilization of

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XIAP expression. A) Western blot analyses showed that the stimulation of P2Y6R with 1.5 M MRS2693 induced the phosphorylation of XIAP on the Ser87 residue, which was correlated with

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an increase and maintenance of XIAP over time. The observed increased phosphorylation of XIAP was concomitant with the phosphorylation of the AKT Thr308 residue. GAPDH

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expression was used to ensure equal protein loading and integrity. B) Inhibition of PI3K/AKT signaling in HT29 cells by 20 M LY294002, added 30 min prior to P2Y6R activation with 1.5 M for 30 min, reduced the phosphorylation of AKT and XIAP. GAPDH expression was used to ensure equal protein loading and integrity. The blots are typical of three independent sets of experiments.

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ACCEPTED MANUSCRIPT Figure 8. The expression of XIAP in response to UDP was blocked in HT-29 cells stably expressing shRNA against the human P2Y6R. HT-29 cells were infected with lentivirus containing specific shRNA targeting the human P2Y6R (shP2Y6#76, shP2Y6#77) and scrambled nontarget shRNA (shNT). A) The expression of P2RY6 transcript was reduced by targeting

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shRNA as compared to shNT. The expression of the transcript was determined by qPCR. Results

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were normalized to the expression of TATA-binding protein mRNA. B) Typical Western blot

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results showed that increase in XIAP expression following 15 min of stimulation with 10 M

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UDP was blocked in HT-29 cells expressing shP2Y6#76 and shP2Y6#77. GAPDH expression

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was used to ensure equal protein loading and integrity.

Figure 9. The invalidation of the P2ry6 gene modulates AKT signaling, but not the

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expression of -catenin. Distal colons were isolated from control untreated animals or mice

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treated with AOM-DSS as described. The expression of -catenin, AKT and AKT

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phosphorylation on Thr308 was determined by Western blots. GAPDH expression was used to

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ensure equal protein loading and lysate integrity. Results are shown for three mice per groups.

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Figure 10. Activation of P2Y6R-induced resistance to the cytotoxic effect of 5-fluorouracil in mouse CRC-derived tumoroids. A-B) Bright field microscopy showing (A) cyst-like and (B) budding tumoroids. The addition of 10 M 5-FU for 24h led to the appearance of blebs typical of dying tumoroids, which was partially prevented by the activation of P2Y6R for 30 min with 1.5 M MRS2693 prior to the addition of 5-FU. Scale bars = 5000 m. C) The number of living and dying (presence of blebs) tumoroids was counted in blinded manner and reported as the mean ± SEM. Statistical significance determined by an unpaired t test, where *: p < 0.05. Data are

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ACCEPTED MANUSCRIPT representative of 4 independent experiments with statistical significance determined by a 2-way ANOVA test with a multiple comparison Tukey post-test. P values are presented in the table. NT, vehicle-treated tumoroids; MRS, MRS2693; n.s., non-significant.

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Figure 11. P2Y6R stimulation offers protection to proliferative cells in the presence of 5-FU.

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A) High-resolution confocal microscopy showing the incorporation of EdU in proliferative cells

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(S phase, stained in red) in non-treated tumoroids. B) EdU incorporation in tumoroids treated

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with 100 M 5-FU for 24h. Note the low number of EdU-stained cells. C) Immunofluorescence micrograph showing the incorporation of EdU in tumoroids in which P2Y6R was activated with

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1.5 M MRS2693 for 30 min prior to the addition of 100 M 5-FU for 24h. The presented micrographs are 3D reconstitutions of high-resolution confocal acquisitions and are

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representative of 10 different tumoroids. In all micrographs, EdU staining is shown in red and

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nuclei, stained with Hoechst 33342, are presented in blue color. Scale bars = 50 M.

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