Cyr61/CCN1 signaling is critical for epithelial-mesenchymal transition and stemness and promotes pancreatic carcinogenesis

To determine the status of Cyr61 mRNA in PDAC, we evaluated high grade primary pancreatic adenocarcinoma tissue samples (N = 16) along with adjacent normal pancreas. We found ~81% (13 out of 16) pancreatic cancer specimens exhibited over-expression of Cyr61 mRNA as compared to adjacent normal samples where expression was either undetected or minimal. The distribution of Cyr61 mRNA was mainly in the infiltrating ducts and acini of the tumor area (Figure1A, upper panel). To validate the results of in situ hybridization, we then determined Cyr61 protein status in PDAC by immunohistochemistry using a tissue array slide and a Cyr61 specific antibody. Each slide contained 63 specimens and these included: ductal adenocarcinoma Grade I (N = 10), Grade II (N = 15) and Grade III (N = 23) in addition to normal adjacent pancreas (N = 3), chronic pancreatitis ( N = 3), mucus and digestive tumor cells (N = 2), islet cell carcinoma ( N = 6), fibrous tissue and fatty tissue (N = 1). Data on chronic pancreatitis, mucinous and islet cell carcinoma were excluded from this study. We found ~85% (53 out of 63) PDAC samples were Cyr61 positive and the level of Cyr61 protein was markedly higher in PDAC specimens as compared to adjacent normal tissues where its expression was minimal (Figures1Alower panel and 1B). Cyr61 is distributed in the cytoplasm of tumor cells of the infiltrating pancreatic ducts and acinar cells. The intensity of the staining increased markedly as the disease progressed from Grade-I to Grade III. However, the expression profile was not grade-dependent (Figures1Aand1B). Moreover, increased level of Cyr61 protein was also detected in histologically defined precursor lesions (PanINs; PanIN-1A-PanIN-3) (Figure1C).

Our next goal was to determine the status of Cyr61 mRNA and protein in different pancreatic cancer cell lines. These included BxPC-3, Capan-1, Aspc-1, and Panc-1. These cells were well-characterized from less aggressive (i.e. BXPC-3 and Capan-1) to highly aggressive cell lines (i.e. Aspc-1, and Panc-1) with varied degrees of EMT markers [20]. Quantitative real-time PCR, Northern blotting and Western blotting analysis revealed that Cyr61 mRNA and protein were detected in BxPC-1, Capan-1, AsPC-1 and Panc-1 with varying degrees of expression (Figures2Aand2B). The highest expression of RNA and protein was detected in Panc-1 cells followed by AsPC-1, Capan-1 and BxPC-3 (Figure2).

To investigate the pathobiological role of Cyr61 in pancreatic cancer, first, we determined the morphology as well as the status of epithelial and mesenchymal/stem cell markers in BxPC-3, Capan-1, AsPC-1 and Panc-1. Consistent with previous work [20], we found that BxPC-3 and Capan-1 cells are morphologically epithelial in nature (Figure3A), but these cells differentially express both epithelial (i.e., Keratin-19, E-cadherin and β-catenin) and mesenchymal (i.e., Vimentin) markers (Figure3B). In contrast, AsPC-1 and Panc-1 cells are mixed populations of epithelial and spindle-shaped mesenchymal type cells along with stemness and express epithelial, mesenchymal and stem cell markers with some exclusion in Panc-1 cells. These cells express only Keratin-19 and high levels of Vimentin, Notch-1 and Oct-4 (Figure3B). E-cadherin and β-catenin expression was undetected or minimally detected in Panc-1 cells and AsPC-1 cells. Taken together, these and previous studies suggest that these pancreatic cancer cell lines are a phenotypically mixed population but not identical because they express epithelial and mesenchymal markers with varying degrees and their aggressive/metastatic behaviors are different. Therefore, these cells can be categorized from less aggressive (i.e., BxPC-3 and Capan-1 cell lines) to highly aggressive (i.e., AsPC-1 and Panc-1) with varied degrees of epithelial-mesenchymal transition (EMT) and stemness (Figure3C). Next, we attempted to determine the role of Cyr61 in the morphological and behavioral alterations of pancreatic cancer cells by focusing on in vitro migration. To do so, we blocked the Cyr61 expression in Panc-1 cells by stable transfection of a pSilencer 5.1-U6-retroviral vector-containing Cyr61-specific shRNA, and we evaluated the expression of Cyr61 in these cells. We found that more than 95% of Cyr61's expression was suppressed by stable transfection of Cyr61-shRNA while this effect was undetected in mismatched-shRNA transfected cells (Figure4A). After confirmation, the morphology of Cyr61 positive (Panc-1 ^Cyr61+ ) and Cyr61 knockout (Panc-1 ^KOCyr61 ) cells was evaluated. We found that the morphology of the Panc-1 cells was markedly altered with a transition from the mesenchymal/fibroblast-type to the epithelial-type (Figure4B). Finally, we determined whether Cyr61 has any role in in vitro migration of these cells. To do so, we seeded Panc-1^Cyr61+ and Panc-1^KOCyr61 cells in the upper chamber of the Boyden chamber to test in vitro migration toward the serum for 24 h. We stained the migrated cells with Crystal violet, and then performed a colorimetric (quantitative) analysis using an ELISA plate reader. This study reveals that the migration of Panc-1^KOCyr61 cells toward the serum was significantly less as compared to Panc-1^Cyr61+ cells (Figure4C). The results were consistent when the paracrine action of Cyr61 was blocked by adding a human polyclonal anti-rabbit Cyr61 blocking antibody in the media (Figure4D). We repeated the experiments in AsPC-1 cells and also found that the silencing of Cyr61 alters the morphology of AsPC-1 cells as well as significantly blocks the in vitro migration of these cells (data not shown). Finally, we evaluated the impact of Cyr61 on pancreatic cancer cell proliferation. We found that blocking Cyr61 in Panc-1 cells by shRNA or a Cyr61-specific antibody has no effect on the proliferation of Panc-1 cells (data not shown).

An EMT event is involved in the formation of motile cells from parent epithelial cells that are not themselves motile [21]. EMT is not only crucial for embryogenesis, but this event is a prerequisite for the progression of carcinogenesis as well. Because we found that Cyr61 is essential for the morphological alteration (i.e. EMT) and in vitro migration of PDAC cells, we sought to determine if Cyr61 modulates the expression of EMT and stem cell molecular markers. To do so, expression profiles of different epithelial and mesenchymal markers were evaluated in Panc-1^Cyr61+ and Panc-1^KOCyr61 cells. In the absence of Cyr61, the epithelial markers (i.e., E-cadherin, β-catenin) expressions significantly increased, while the expressions of mesenchymal/stem cell markers (i.e., Vimentin, Notch-1, Oct-4, ABCG2 and CD44) markedly decreased (Figures5Aand5B). We found consistent results when Cyr61 paracrine action was blocked by the addition of Cyr61 antibody (different concentrations) in the media of Panc-1 cell cultures (Figure5B). We found consistent results in AsPC-1 cell line (data not included). In order to confirm this phenomenon, Cyr61 negative or minimal expressing BxPC3 cells were exposed to Cyr61 recombinant protein (100 ng/ml)[22] for 48 h and morphology as well as molecular markers of EMT and stemness were characterized. We found that addition of Cyr61 in the culture media markedly enhanced the EMT and stemness in these cells (Figure6).

Recently, different laboratories have characterized side population (SP) of pancreatic cancer cells [23, 24]. The SP cells are either isolated from cell lines or xenograft tumor cells. The SP is enriched with cancer stem cells, forms tumors in xenograft models and exhibits chemoresistance. Our goal was to examine the status of Cyr61 in the SP of a pancreatic cancer cell line, Panc-1. To do so, first, SP cells were isolated from Panc-1^Cyr61+ and Panc-1^KOCyr61 cells using a dye-cycle-violet (DCV)-488 Alexa stained Panc-1 cells using a flow cytometry technique [24]. DCV negative cells are considered SP cells and stained cells are NSP cells (Figure7A). Consistent with previous works [23, 24], our studies demonstrate that there are about 10-15% SP cells in Panc-1 and this population can significantly be reduced by several folds in the presence of Verapamil (Figure7Aand7B), an inhibitor of ABC transporter and known to inhibit side population in various cancer cells [25]. Moreover, negligible percent (less than 3%) of SP cells were detected in Panc-1^KOCyr61 cells and these cells are Verapamil insensitive (Figure7Aand7B). Finally, after isolation, both SP and non-SP cells were grown for the determination of Cyr61, epithelial and mesenchymal/stem cell markers using Western blotting. SP cells are morphologically spindle-shaped (Figure8A), and over express Cyr61 along with CD44, Notch-1 and Oct-4. CD24, Keratin-19 and β-catenin are down regulated in SP cells (Figure8B). Blocking the expression of Cyr61 by shRNA or a Cyr61 neutralizing antibody reduces the expression of stemness markers (i.e., CD44, Notch-1 and Oct-4) (data not shown). Collectively, the data is consistent with previous studies [23, 24] indicating that SP cells are enriched with stem cell properties. Cyr61 may play critical role in the formation of SP in Panc-1 cell lines.

To observe the in vivo tumor growth potential of SP cells, both SP and NSP cells (5 × 10⁴) cells with Matrigel were injected into the rear flank of the each mouse (5-9 mice/exp). At the site where the SP cells were injected, nine of 9 mice demonstrated tumor formation 10-15 days post injection with evidence of angiogenesis (Figure9). The xenograft tumors overexpressed Cyr61 and other markers. SP-tumors were developed in all nine mice 20 days post injection. However, tumors were not detected in the animals injected with NSP cells (Figure9). Tumor formation was detected in two out of 5 mice 30 days post the injection of unsorted Panc-1 cells (data not shown). Our next goal was to find the role of Cyr61 in tumor formation of SP cells in the xenograft model. To do so, Cyr61 was silenced in SP cells by stable transfection of a Cyr61-shRNA containing retroviral. SP cells and Cyr61 silenced SP cells (SP^KOCyr61) were injected into nude mice and tumor growth was evaluated 15-20 days post injection. Like NSP, SP^KOCyr61 cells were unable to form tumor in the xenograft even after 20 days post injection (Figure9). Collectively, these studies suggest that Cyr61 may play a critical role in stemness and tumor initiating capability in a population of pancreatic cells.

To gain further insight into the factors critically regulated by Cyr61 in pancreatic cancer cells, we established quantitative miRNA expression profiles of candidate markers in Cyr61 knockout Panc-1 cells. Prior to microarray analysis, the quality of each RNA sample was verified by determining the quality of RNA (Figure10). The miRNA expression analysis was carried out in three independent culture samples and we performed pair wise comparisons of each culture. Data was converted into log2 ratios comparing levels of miRNA expression in Cyr61⁺ and Cyr61 knockout Panc-1 cells. We observed a dramatic alteration in the miRNA expression profiles in Cyr61 knockout Panc-1 cells and we identified miRNAs that are critically involved in EMT, migration and invasion, and stemness (Figures10Band10C). In particular, we noticed an increased in the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) in Panc-1^KOCyr61 cells (Figure10B). This family of miRNAs is known to regulate EMT and tumor aggressiveness [26]. Furthermore, the microRNAs, which are associated with the inhibition of stemness [26‐28] are upregulated, while those responsible for stem cell generation [29] are down regulated in Cyr61-shRNA transfected Panc-1 cells (Figure10C). While future systematic Northern blots and in situ hybridization screens in these cells and human tissue samples are required to validate all miRNAs, we corroborated the differential expression by qPCR in these cells and human pancreatic cancer cells (data not shown).

Human polyclonal anti-rabbit Cyr61 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-mouse GAPDH antibody was purchased from Applied Biosystems (Foster City, CA). Mouse monoclonal Vimentin antibody was obtained from Lab Vision (Fremont, CA), mouse monoclonal anti-β-catenin and rabbit polyclonal Keratin-19 were purchased from BD Transduction Laboratory (San Jose, CA), rabbit polyclonal anti-human Oct-4 and CD44 were purchased from Cell Signaling (Boston, MA). Rabbit polyclonal anti-human Notch-1, mouse monoclonal anti-human CD24, polyclonal goat anti-rabbit IgG-HRP and monoclonal goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cyr61 recombinant protein was purchased from Thermo Fisher Scientific (Waltham, MA). pSilencer™ 5.1-U6 retroviral vector and siPORT™ XP-1 transfection agent were obtained from Applied Biosystems (Foster City, CA). Matrigel was purchased from BD Biosciences (Bedford, MA). All other chemicals were obtained either from Sigma (St. Louis, MO) or Fisher Scientific (Houston, TX).

Archived, 4% formalin-fixed, paraffin-embedded primary pancreatic adenocarcinoma samples and chronic pancreatitis tissue samples were obtained from the Department of Surgical Pathology of VA Medical Center. The pancreatic tissue array containing adjacent normal (NP), chronic pancreatitis (CP) and different grades of ductal adenocarcinoma (Gr-I, Gr-II and Gr-III) were evaluated in this study. The tissue arrays were purchased from Cybrdi Inc (Frederick, Maryland).

All Pancreatic cancer cell lines (i.e., BxPC-3, AsPC-1, Capan-1, and Panc-1) were purchased from American Type Culture Collection (ATCC, Manassas, VA). The cell lines were cultured in Dulbecco's modified Eagle's medium (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM glutamine, 100 units/ml penicillin and 100units/ml streptomycin (Sigma) at 37°C incubator in the presence of 5% CO₂. Ampho-pak 293 packaging cell line was purchased from Clontech (California, USA), and was maintained in high glucose DMEM containing 10% FBS. Cells were used for the experiment between four and six passages.

The Immunohistochemistry was performed on 4% formalin fixed-paraffin-embedded tissue sections according to our previous method [46, 47]. Briefly, tissue sections were deparaffinized in Xylene, rehydrated in different grades of alcohol, washed with PBS and blocked with tissue blocker (Zymed Laboratories, CA) for 10 minutes and immunostained by polyclonal human anti-rabbit Cyr61 antibody (1:300) overnight. The clinical stages obtained from database were reviewed and reconfirmed by a pathologist using adjacent hematoxylin and eosin stained slides. The sections were imaged with a Leica photomicroscope. All samples were used according to VA Medical Center and University guidelines after receiving Institutional Review Board approval.

Total RNA extraction was essentially the same as that previously described [46, 47]. cDNA synthesis and probe preparation were done according to the method described by Banerjee et al [46].

In situ hybridization for Cyr61 mRNA expression was performed on formalin-fixed, paraffin embedded tissue sections according to the method that is described earlier by us [48]. Briefly, the paraffin sections were dewaxed in xylene, rehydrated through different grades of alcohol and digested with proteinase K followed by post-fixation in 10% formaldehyde solution at room temperature. The sections were thoroughly washed with RNAse-free water and incubated with Digoxigenin (DIG)-labeled PCR generated Cyr61 specific non-radioactive probe (250 ng/ml) overnight at 37°C in a humidified hybridization chamber. The hybridized probe was detected using alkaline phosphatase-conjugated anti-DIG antibody (InnoGenex) and visualized with chromogen combination 5-bromo-4chloro-3-indolyl phosphate NBT.

For Northern blotting, an established method previously reported by us was used [49]. Briefly, Total RNA (10 μg) of each sample was separated by formaldehyde/agarose gel electrophoresis and transferred to a nylon membrane. The membranes were hybridized with nonradioactive digoxigeninlabeled, PCR-generated probes. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Relative expressions of mRNA were calculated by densitometric analyses using One-dimensional Image Analysis Software version 3.6 (Eastman Kodak Co., Rochester, NY).

Briefly, total RNA was extracted from different pancreatic cancer cell lines using TRIZOL (Invitrogen, Carlsbad, CA). cDNA was prepared from total RNA by using Taqman Reverse Transcription kit. Real-time PCR was performed from cDNA products using Taqman universal PCR and Taqman assay kit by Applied Biosystem Step One real-time PCR system. C_T values for Cyr61 were normalized to human GAPDH by subtracting the average C_T value for each sample. Relative quantification (RQ) values for Cyr61 in each sample were determined using the 2^-ΔΔCT method [47]. Each PCR reaction was performed in triplicate.

Cell lysates from different pancreatic cell lines were prepared for Immuno-Western blotting according to our previous method [46]. Briefly, cells were washed with phosphate-buffer saline (PBS) and lysed in RIPA buffer (50 mM tris-Cl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate) containing the protease inhibitors, 0.5 mM phenylmethylsulfonyl fluoride, 1μM leupeptin, 1μM aprotinin. The lysates were centrifuged at 18000 rpm for 60 min at 4°C. Equal amounts of protein (50 μg), as determined by Coomassie blue reagent assay (Bio-Rad, Richmond, CA), were subjected to 10% SDS-PAGE, and the gel-fractionated proteins were transferred to nitrocellulose membranes (Bio-Rad) and reacted with appropriate antibodies. Signals were detected with Super Signal ULTRA chemiluminescent substrate (Pierce, Rockford, IL) by using one dimensional Image analysis, version 3.6 (Eastman Kodak Co., Rochester, NY).

Human Cyr61-shRNA primers were designed using vector NTI software from Invitrogen. The shRNAs sequences of human Cyr61 are: shRNA-1, forward: 5'- GAT CCG GGA AAG TTT CCA GCC CAA TTC AAG AGA AGT TGG GCT GGA AAC TTT CCC TTT TTT GGA AA -3'; reverse: 5'- AGC TTT TCC AAA AAA GGG AAA GTT TCC AGC CCA ACT TCT CTT GAA TTG GGC TGG AAA CTT TCC C -3'; shRNA-2, forward: 5'- GAT CCG CCC AAC TGT AAA CAT CAG TTC AAG AGA CAC TGA TGT TTA CAG TTG GGC TTT TTT GGA AA -3'; reverse: 5'- AGC TTT TCC AAA AAA GCC CAA CTG TAA ACA TCA GTG TCT CTT GAA CTG ATG TTT ACA GTT GGG C -3'. Recombinant clones of Cyr61shRNA are produced using pSilencer™5.1-U6 Retro-viral Vector from Ambion (Austin, TX) as per the protocol described in the instruction manual. Recombinant clones were confirmed by sequencing using sequencing primers. Scrambled control provided in the kit was used as a control. Briefly, Cyr61shRNA is transfected in Amphopak ™293 packaging cell line using siPORT™ XP-1 transfection agent. Supernatant containing viral particles was collected after 72 h. ~60% Panc-1 cells were infected with Cyr61-shRNA containing viral supernatant or scrambled control with different dilutions (from 1:10¹ to 1:10⁵) and incubated for 72 hrs. Stable cell lines were prepared after prolonged puromycin treatment. Stable cells were then cultured in regular DMEM media with 10% FBS and harvested for western or northern blot analysis to check the transfection efficiency.

For miRNA array experiments, total RNA was isolated from mismatched-shRNA and Cyr61-shRNA transfected Panc-1 cell lines by Trizol method as described in the prior section. The integrity of total RNA was assessed by running RNA sample on a denaturing agarose gel stained with ethidium bromide. The 2:1 ratio of 28S and 18S are considered as a good indication of intact RNA. cDNA was synthesized using Megaplex RT PCR kit (Applied Biosystems, Foster City, CA) for Array A, which contains 384 stem-looped reverse transcription primers specifically binds to miRNAs. Briefly, 500 ng of total RNA and 4.5μl of RT reaction mix in a total volume of 7.5μl were processed for cDNA preparation at the following cycle conditions: 16°C for 2 min, 42°C for 1 min and 50°C for 1 min for total of 40 cycles followed by 85°C for 5 min and bringing the contents to 4°C. The human miRNA array system (Array A) (Applied Biosystems, Foster City, CA) was used for the detection and quantification of miRNA in mismatched-shRNA and Cyr61-shRNA transfected Panc1 cells. This miRNA-array kit consists of four plates of plate A and four plates of plate B which contain around 384 miRNAs including four internal controls. For this we used 6μl of cDNA synthesized by using Megaplex RT and 450 μl of TaqMan universal PCR master mix in a total of 900 μl of reaction volume and 100 μl of the reaction mixture was loaded into each port provided in the card (which has 8 ports for each card). The cards were run in Applied Biosystems Real-time PCR system (7900 HT) by selecting relative quantification (ΔΔCt) at following conditions: 95°C for 10 min, 95°C for 1 min and 60°C for 1 min for total of 40 cycles. All the samples were run in duplicates. Finally, all the raw data from each card was retrieved from the 7900HT machine and was run on Data Assist Software ver.1.2 (Applied Biosystems, Foster City, CA). The mean values for RQ (which is fold values of Cy61-shRNA transfected compared to mismatched-shRNA transfected panc1 cell lines) were used to plot the bar diagrams and heat map clusters.

The chemotaxis assay was conducted using a modified Boyden chamber technique as described previously [46]. Briefly, Panc-1 cells (20,000 cells/well), which were either infected with viral particles containing shRNA (Cyr61/scrambled) or treated with Cyr61 neutralizing antibody for 48 h, were added to the upper chambers of the Boyden chamber (8 micron) containing DMEM with 1% FBS. Lower chamber was loaded with DMEM with 10% FBS. Cells were allowed to migrate for 24 hrs. The migratory cells that were attached on the undersurface of Boyden chamber were stained with crystal violet solution for 10 min. Inserts were washed with tap water and then air dried for 30 minutes. Crystal violet stained cells were solubilized with 10% acetic acid and optical density is quantitated in Microplate reader at 600 nm. Three wells were examined for each condition and the experiments were repeated three times.

The side population/stem cells from Panc-1 cell line were isolated according to the previous methods with brief modifications [24]. Briefly, ~80 percent confluent cells were incubated with dissociation solution (Sigma Chemical Co. St. Louis) for 15 min at 37°C, and dissociated cells were counted and transferred to a 5 ml tube. Washed twice with pre-warmed DMEM containing 10% FBS. Finally, cells were resuspended in same media at concentration of 1 × 10⁶cells/100μl. Vybrant-Violet solution (10μM) and Verapamil (50 μM) solution were added into the sample and incubated at 37°C for 90 min. After incubation, cells were centrifuged, and resuspended in ice-cold 1 × PBS, pH 7.4. 2μg/ml propidium iodide was added immediately before flow cytometry analysis to exclude dead cells. SP cells were identified, sorted, and analyzed on a BD FACS Aria SORP flow cytometer (BD Biosciences) using ~405 nm excitation and 440 nm emission. Sorted cells (i.e., SP and Non-SP) were washed in serum-free medium and then cultured in DMEM with 10% FCS for several days in 5% CO₂ at 37°C. The cell lysate was extracted from semi-confluent cells for the analysis of different epithelial, mesenchymal and stem cell markers by Western blot analysis using specific antibodies.

Male athymic nude mice (nu/nu genotype), 6 to 8 weeks old, were obtained from Charles Rivers (Wilmington, MA) and acclimated in our facility for 1 week. The animal studies were conducted according to the approved Guidelines of the Animals Care and Use Committee of Kansas City VA Medical Center. Sorted cells (SP and NSP) were grown in DMEM with 10% FCS using the same procedure as described herein. Cells (5 × 10⁴) were injected s.c. into the right rear flank of each mouse (5-9 mice per group) and tumor growth was monitored after 2^nd days of injection and continued up to 21 days.

¹Cancer Research Unit, VA Medical Center, 4801 Linwood Blvd, Kansas City, Missouri 6 4128, USA. ²Division of Hematology and Oncology, 2330 Shawnee Mission Pkwy, Westwood, Kansas 66205, USA, ³Department of Anatomy and Cell Biology, 3901 Rainbow Blvd, Kansas City, KS 66205, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, Kansas 66160, USA.