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Table of Contents
Abstract
Introduction
Materials And Methods
Results
Discussion
Authors’ Contributions
Acknowledgements
Grant Support
Figure Legends
Abstract
1 Cancer Research Unit, VA Medical Center, Kansas City, MO, 64128, USA 2 Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA 3 Department of Ob/Gyn, University of Kansas Medical Center, Kansas City, Kansas, USA 4 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA 5 The Translational Genomics Research Institute (TGen), Phoenix, AZ 85004 6 Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105 7 The Sarah Cannon Cancer Center at HCA Midwest Health, Kansas City, Missouri 64131.
2 Pancreatic ductal adenocarcinoma (PDAC) develops extrinsic- and intrinsic-resistant phenotypes to prevent chemotherapies from entering into the cells by promoting desmoplastic reactions (DR) and metabolic malfunctions of the drugs. It is well established that these responses are also associated with pancreatic cancer cells’ gemcitabine (GEM) resistance. However, the mechanism by which these resistant pathways function in the pancreatic cancer cells remains poorly understood. In these studies, we show that CYR61/CCN1-signaling plays a vital role in making pancreatic cancer cells resistant to GEM in vitro and also in a tumor xenograft model. We proved that the catastrophic effect of GEM could significantly be increased in GEM-resistant PDAC cells when CYR61/CCN1 is depleted, while this effect can be suppressed in GEMsensitive neoplastic cells by treating them with CYR61/CCN1 recombinant protein. Ironically, non-transformed pancreatic cells, which are sensitive to GEM, cannot be resistant to GEM by CYR61/CCN1 protein treatment, showing a unique feature of CYR61/CCN-signaling that only influences PDAC cells to become resistant. Further, we demonstrated that CYR61/CCN1 suppresses the expression of the GEM-activating enzyme dCK while it induces the expression of a DR-promoting factor CTGF (connective tissue growth factor) in pancreatic cancer cells in vitro and in vivo. Thus, the previously described mechanisms (dCK and CTGF pathways) for GEM resistance may be two novel targets for CYR61/CCN1 to protect pancreatic cancer cells from GEM. Collectively, these studies reveal a novel paradigm in which CYR61/CCN1regulates both extrinsic and intrinsic GEM-resistance in PDAC cells by employing unique signaling pathways. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 3
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a common form of pancreatic cancer and now the third leading cause of cancer death in the United States, and an anticipated second leading cause of cancer-related death by 2030 (1-3). The prognosis of PDAC remains depressing and a difficult malignancy to treat (4). Although the location of the pancreas leads to late diagnosis and one of the vital weaknesses in treatment and unresectability, a major contributor to the poor clinical outcome is chemoresistance. The treatment of choice for PDAC in the early stage is surgery followed by adjuvant therapy. Regrettably, the surgery is not always a choice as most patients present with locally advanced unresectable disease or their disease has already metastasized to the distant organs, leaving them with a poor prognosis (5,6). Thus, GEM, a nucleoside analog, has been widely used as a firstline chemotherapeutic drug for PDAC with advanced stages. It can be used alone or in combination with other agents (7). It is reported that PDAC cells are comparatively more sensitive to GEM than other anticancer drugs (5). However, the majority of the PDAC patients eventually develop resistance to GEM with poorly known mechanisms (5). Thus, understanding the mechanism of resistance of GEM by PDAC cells is urgently needed. GEM resistance in pancreatic cancer cells is a complex and multifactorial process (8,9). GEMactivation in cancer cells needs a rate-limiting transition from the inactive prodrug to an active compound through a series of phosphorylations by a rate-limiting enzyme deoxycytidine kinase (dCK) (10,11). The epigenetic silencing or inactivating mutations of dCK plays a crucial role in GEM-resistance (12,13). However, how dCK is epigenetically inactivated in PDAC cells is not understood well. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 4 In addition, desmoplasia, a dynamic process regulated by the multiple signaling cross-talk of tumor cells and surrounding stroma, plays a critical role in chemoresistance (5,14-19). Desmoplasia leads to a significant increase in the production of dense and fibrous connective tissues, extracellular matrix proteins, stroma with high interstitial pressure around the tumors and proliferation of myofibroblast cells/active pancreatic stellates cells (PSCs) to maintain the tumor growth, metastasis, and a barrier to chemotherapy penetration (16). Multiple studies have shown that CTGF, a cysteine-rich, matricellular protein, is involved in GEM resistance in many cancer cells including PDAC (20). The PDAC cells’ secreted-CTGF activates stellate cells to induce the desmoplastic reaction. However, no molecule has yet been found that could regulate CTGF levels in PDAC cells. We and others have previously shown that CYR61/CCN1, which belongs to the CCN family of growth factors, acts as an oncoprotein in PDAC (21-25). CYR61/CCN1 promotes growth and invasive phenotypes of PDAC cells via integrin-dependent non-canonical pathways. Moreover, our studies also showed that CYR61/CCN1 reprograms the epithelial-mesenchymal transition (EMT) and maintains cancer stemness/tumor-initiating properties that are associated with metastasis and drug resistance (18,21,22,26-30). This provokes the hypothesis that PDAC cellsecreted CYR61/CCN1-signaling contribute to GEM resistance. In this study, we validated the premise and demonstrated that CYR61/CCN1 depleted PDAC cells are sensitive to GEM. Further, we show that CYR61/CCN1 can regulate dCK and CTGF in PDAC cells and stellate cells, and concurrently activate stellate cells. Based on these previously unrecognized mechanisms we suggest that CYR61/CCN1 promotes GEM resistance through the regulation of dCK and CTGF, and thus, CYR61/CCN1 signaling may represent a new target for sensitization of GEM in PDAC. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 5
Materials and Methods
The Care and Maintenance of Animals: Animal protocols were approved by the KCVAMC Animal Care and Use Committee, in accordance with the AAALAC animal care guidelines, and NIH Guide for the Care and Use of Laboratory Animals. Male and female athymic nude mice (nu/nu genotype), 6 to 8 weeks old, were purchased from Jackson Laboratories (Sacramento, CA) and acclimated in our facility for 1 week. Chemicals and Antibodies: GEM (Eli Lilly, and Company, Indiana, USA) was purchased through the VA pharmacy. Crystal Violet stain was purchased from Sigma (Millipore-Sigma, St. Louis, MO, USA). Human polyclonal anti-rabbit CYR61, CTGF and dCK antibodies and polyclonal goat anti-rabbit IgG-HRP and monoclonal goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Thermo Fisher, respectively. CYR61 human recombinant protein (hrCYR61) was purchased from PeproTech (Rocky Hill, NJ, USA). All other chemicals were purchased from either Sigma or Fisher Scientific. Authentication of the antibodies, specifically, CTGF and CYR61, which are closely related and belong to the same family, were confirmed, and found no cross-reactivity of the antibodies (Fig S1). Human Cell lines and Maintenance: Pancreatic cancer cell lines BxPC-3, Capan-1, AsPC-1, and Panc-1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were maintained as described previously (22). Human Pancreatic stellate cells (HPaSteC) was purchased from ScienCell (ScienCell Research Laboratory, Carlsbad, CA, USA) and maintained in Stellate cell media and FBS. FEF3, a fetal human esophageal fibroblasts cells, were a gift and maintained in high glucose DMEM with FBS. Authentication of the cell lines were performed by determining short tandem repeat (STR) profiles using the Promega on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 6 PowerPlex 16 system. This was performed once and compared with external STR profiles of the cell lines (when available). Cell lines were also figured out to be mycoplasma-free prior to use. Gene connectivity maps-The Connectivity Map represents the network of functional connection of the drug-resistant genes with CYR61/CCN1 in pancreatic cancer patient samples. The network was designed in IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis), using its knowledge base of known, experimentally validated, molecular interactions. The network reflects the potential pathways by which CYR61 interacts with drug resistance genes. Generation of CYR61-knockout stable pancreatic cancer Cell line using Short hairpin RNA (shRNA) mediated gene silencing technique- The CYR61-knockout stable Panc-1cell line was generated using our previous method (21,22). Generation of CYR61 gene knock-out using CRISPR/Cas9 gene editing: Human CYR61 gene knockout AsPC-1 cells were generated using CYR61/CCN1 Human Gene Knockout Kit (CRISPR) (OriGene, Rockville, MD). Three separate transfections were carried out in AsPC-1 cells. Donor DNA was co-transfected with either one of two guide RNA (gRNA) vectors or with scrambled control using EndoFectin TM Max Transfection Reagent (GeneCopoeia, Maryland, MD). Equilibrate gRNA vectors (1 and 2 each 5 μg) and donor DNA (5 μg) and endofectin were diluted in 250 l of Opti-Mem I (Invitrogen, Carlsbad, CA) and incubated for 20 mins at room temperature to form DNA-Transfection reagent complexes,and added to the cells. After selection by Puromycin (0.5 μg/ml), the status of CYR61 was determined using Western blotting, and one of the clones (clone 1), which exhibits complete ablation of CYR61 (Fig. S2), used for further studies on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 7 Isolation of Side Population (SP) by Flow Cytometry: The side population/stem cells from Panc-1 cell line were isolated according to the earlier methods (21). Briefly, cells were suspended in the culture media at concentration of 1 × 10 6 cells/100μl. Vybrant-Violet solution (10μM) and Verapamil (50 μM) solution were added into the sample and incubated at 37°C for 90 min. The cells were incubated with Propidium iodide (2μg/ml) immediately to exclude dead cells before flow cytometry analysis. SP cells were identified, sorted, and analyzed on a BD FACS Aria SORP flow cytometer (BD Biosciences) using ~405 nm excitation and 440 nm emissions. 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. Cell Viability Assay: Cell viability assay was conducted as previously described (22). Briefly, the cells were exposed to different concentrations of GEM (0, 0.10, 0.25, 0.5, 1.0, and 5 M) for 72 hours. The cells were then stained with crystal violet solution. The absorbance was measured at 600 nm using Microplate Reader (Spectra Max 340) available from Molecular Devices, CA. Generation of Gemcitabine Resistant Panc-1 CYR61 (+) and Panc-1 CYR61 (-) cells: To create stable GEM resistant Panc-1 CYR61 (+) and Panc-1 CYR61 (-) cell line, cells were regularly exposed to increasing doses of Gemcitabine. Cells were first treated with 50 nM of Gemcitabine for 6 days continuously and triggered cell death more than 60%. After 8 weeks of continuous exposure of increasing concentrations of Gemcitabine (100 nM - 250 nM), the cells were considered as stable to Gemcitabine. Then cells were cultured after withdrawal of drug for 7-8 passages and resistant stability was checked after exposing the cells to GEM (250nM) for another 7 days. GEM treatment dose was applied at the time of medium change at 2-3 days interval. Transfection of dCK gene in Panc-1 cells: Vectors containing dCK gene (plasmid pDONR223DCK) or vector alone (Addgene, Cambridge, MA, USA) was transiently transfected in Panc-1 on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 8 cells using EndoFectinTM Max Transfection Reagent (GeneCopoeia, Maryland, MD). Briefly, 5 µg of DNA and EndoFectin were diluted in 250 µl of serum-free Opti-Mem I (Invitrogen, Carlsbad, CA) and incubated for 30 mins at room temperature to form DNA-Transfection reagent complexes,and added to the 70-90 percent confluent Panc-1 cells. After 48h, transfection efficiency was checked by western blot. Cell viability Assay: To determine the effect of GEM on the viability of different pancreatic cancer cell lines, we performed crystal violet-based cell viability (Cell Biolabs, San Diego, CA, USA) in a 96-well format as described earlier by us (31,32). The cell viability was measured by means of a Spectra Max 340 microplate reader (Molecular device) at a wavelength of 600nm. The data on cell viability was either reported as an optical density (OD) value or percent viable cells of four different experiments ± standard deviation (SD). The percent cell viability was calculated according the following equation: Cell viability = AbsT/AbsC x 100. -------------------------------------------- (1) AbsT is the absorbance of GEM treated cells and AbsC is the absorbance of untreated cells. In vitro Apoptosis Assay: Apoptotic cell death was determined using cell death detection ELISA kits (Roche Diagnostic) as described previously by Maity et. al. (33). Briefly, untreated and treated cells were lysed with lysis buffer and then cytoplasmic supernatants were collected, and total protein of each sample was measured. Cell lysate was added in the streptavidin coated microplate and allowed to react with a buffer mixture containing anti-histone-biotin and antiDNA–peroxidase. Microplates were washed with incubation buffer. The ABTS (2,2¢-azino-di(3- ethylbenzthiazoline-6-sulfonic acid)) chromogen substrate was added to get a color reaction that was measured in the ELISA reader at 405 nm. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 9 Immunohistochemistry and Immunofluorescence: The immunohistochemical and immunofluorescence staining were performed according to our earlier method (21). Flowcytometry: Expression of CD133 and CXCR4 in Panc-1 cells were sorted by flow cytometry using a BD FACSAria III (BD Biosciences, San Jose, CA) equipped with blue (488 nm) and red (633 nm) lasers. Briefly, Panc-1 cells were harvested using Hank’s based enzyme free cell dissociation buffer (Gibco, Gaithersburg, MD) and 10 6 cells were stained with either pre-conjugated PE anti-human CD184 (CXCR4) or APC anti-human CD133 antibody (BioLegend, San Diego, CA) for 30 min at 2-8°C or on ice. Cells were washed several times in PBS containing 0.1% BSA or 1% fetal calf serum at 1200 RPM. Cell pellet was resuspended in 200µl wash buffer and subjected to flowcytometry analysis. Colony Formation Assay (ADG): The PDAC cells were plated in triplicate at a density of 5000 per well of six-well plate. Twenty-four hours after plating, the cells were treated with different concentrations of GEM (0, 0.1 M and 0.5 M) every alternative day for 7 days. The cells were then fixed and stained with crystal violet and solubilized with 10% acetic acid. The absorbance was determined at 600 nm using Microplate Reader (Spectra Max 340, Molecular Device, CA). Soft-agar anchorage-independent growth assay (AIG): Cells were seeded in soft agar at a concentration of 2000 in a 96-well plate per the manufacturer’s (Cell Biolabs Inc., San Diego, USA) protocol. All conditions were seeded in triplicates. The cells were treated with GEM (0.1M, and 0.5M) or vehicle every alternative day for 10 days. The cells were lysed with lysis buffer, and the plates were read in a fluorimeter using a 485/520 nm filter set. Sphere formation Assay: Panc-1 CYR61(+) and Panc-1 CYR61(-) cells were used to form the spheres according to our earlier method (33). Briefly, the cells were cultured in ultra-low attachment 6- on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 10 well plates (Corning Inc., Corning, NY, USA) at a density of 3000 cells/well. Since the half-life of gemcitabine is very short and rapidly converted into inactive metabolite (34,35), every two days after seeding the cells for 24h, the medium was 3/4 th replaced very carefully with fresh medium containing different doses of GEM. The procedure was followed by 4 days and 6 days depending on the experimental setups. Spheres were counted and area measured when the diameter reaches more than 60m. Western blot analysis and antibodies: For protein analysis, cells were harvested and subjected to Western blotting as previously described (22). Co-culture of Panc-1 CYR61(+) and Panc-1 CYR61(-) with HPaSteC: To investigate the interaction of Panc-1 CYR61(+) and Panc-1 CYR61(-) with HPaSteC, we have developed a unique co-culture in vitro experimental model. Briefly, 1% agarose gel was allowed to solidify in 12 well plate. After solidification, a round hole/ring-like structure was made in the center and periphery. Tumor cells (5000 cells) were seeded in the center of the agarose ring and HPaSteC (30,000 cells) were seeded in the outer part of agarose ring of each well and were allowed to attach for overnight. Solid agarose ring acts as barrier to avoid mixing of tumor cells and HPaSteC at the time of seeding. After attachment of cells, agarose barrier ring was taken out carefully without disturbing the cells and co-culture were allowed for 5 days. Cells were then fixed in methanol and immunofluorescence of -SMA was performed with anti-mouse -SMA, and counter stained with DAPI. Photograph was captured under Leica microscope. In vivo xenograft studies: Female- and male BALB/c nude mice (n = 5) were inoculated subcutaneously with Panc-1 CYR61(+) cells or Panc-1 CYR61(-) cells (10 6 /mouse) as per our previous method (21). Tumor size and body weight were measured two to three times per week. Once the tumor became palpable, mice were randomized according to tumor size and GEM-treatment on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 11 was started. The mice were injected with GEM (50 mg/kg in PBS) or vehicle (PBS) intraperitoneally twice per week. and the tumor growth and body weight were measured for 21 days (36,37). Tumor growth and relative tumor volume (RTV) were measured as per our established method (33) using studylog R measurement tools and software (California, USA) three times a week. Antitumor efficacy was measured as a function of tumor growth inhibition (TGI) calculated by the equation: TGI= [1-(T-T0/C-T0) x 100] ---------------------------------------------------------- (2) where T and C is the mean size of tumors in the treated (T) and control (C) groups, respectively, and T0 represents the tumor size at randomization. The treatment response was evaluated using RECIST criteria. Statistical Analysis: The statistical analysis was performed using the Graph Pad Prism 6 and PASS 15 softwares. Results are shown as mean ± SD. Means between the groups were calculated and compared among or within variants using an unpaired, two-sided Student’s t-test. P value of <0.05 was considered statistically significant. The entire studies were performed blindly by two or more investigators. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 12
Results
CYR61/CCN1 is Overexpressed in Pancreatic Ductal Adenocarcinoma and Correlates with
Drug Resistance
The CYR61/CCN1 expression has been shown to increase during the progression of PDAC, and it is needed for the proliferation, differentiation, and invasive phenotypes of pancreatic cancer cells (19). In addition, CYR61/CCN1 promotes the Sonic Hedgehog (SHh)-signaling pathway, which confers drug resistance in various cancers (38), suggesting that CYR61/CCN1 could be a key factor of drug resistance in PDAC cells (19). The current immunohistochemical analysis is consistent with our earlier findings and shows that the number of CYR61/CCN1-expressing cells was significantly elevated in PDAC samples as compared to the sections of adjacent normal cells and initial stages of the disease (PanINs) (Fig. 1A and B). Moreover, gene interaction network profiles in the Connectivity Map reference database showed links among several chemoresistant gene-signature pathways and CYR61/CCN1 overexpression (Fig. 1C). Collectively, this material supports the premise of the drug-resistance features of CYR61/CCN1. The drug resistance of pancreatic cancer cells is associated with the subpopulation of CD133 + or CD133 + /CXCR4 + -cancer stem cell-like phenotypes (39). In this study, FACS analysis found that CD133 + and CXCR4 + cells were markedly less in CYR61 ablated Panc-1 cells as compared to parental Panc-1 cells (Figure 1D). Moreover, immunofluorescence and immuneWestern blot studies also showed that CYR61/CCN1 blockade significantly reduced the production of CD133 and CXCR4 in pancreatic cancer cell lines (Fig. 1E and F). This further reinforces the concept of the drug-resistant ability of CYR61/CCN1 in pancreatic cancer cells. CYR61/CCN1 Impairs Gemcitabine Action on Pancreatic Ductal Adenocarcinoma Cells on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 13 Based on the preceding data, CYR61/CCN1 can be considered a driver of chemoresistance. Thus, in this study, we evaluated how and to what extent CYR61/CCN1-positive pancreatic cells adapt to survive with GEM. To do so, we knockdown CYR61/CCN1 in Panc-1 and AsPC-1 cells using siRNA or CRISPR/Cas-9, respectively (Fig. S2 and S3). Cells were then treated with different doses of GEM for 72 h and performed an analysis of cell viability and apoptosis. We found that lower concentrations of GEM (0.1 or 0.25 μM) significantly impaired the viability of Panc-1 CYR61(-), while no to a minimal effect was detected in Panc-1 CYR61(+) cells (Fig. 2A). In contrast, higher concentrations of GEM (0, 0.5, 1.0 or 5.0 µM) significantly blocked cell growth in both cell types with a higher effect in Panc-1 CYR61(-) (Fig. 2B). These findings were further corroborated in AsPC-1 cells in which the CYR61/CCN1 gene was ablated by CRISPR/Cas-9 (Fig. 2C). The growth inhibition of GEM in both CYR61-positive and CYR61-negative Panc-1 cells presided via apoptosis (Fig. 2D). Collectively, the studies show that CYR61/CCN1 makes pancreatic cancer cells resistant to GEM. Previously, we have shown that two sub-populations can be obtained with the Panc-1 cell line. These include side population (SP)/tumor initiating cells (TICs)/cancer stem cells (CSCs) and non-side population (NSP)/neoplastic cells (21). SP cells expressed elevated levels of CYR61/CCN1, while CYR61/CCN1 expression was undetected in NSP cells (21) (Fig. S4). To corroborate the drug resistance role of CYR61/CCN1 in pancreatic cancer cells, SP and NSP cells were exposed to different doses of GEM in the presence or absence of hrCYR61 (250 ng/ml) for 72 h, and cell viability was measured. The study showed that GEM was significantly more effective in NSP cells as compared to SP cells (Fig. 2E). Moreover, the effect of GEM on NSP cells can be reduced by treating them with hrCYR61 as compared to SP (Fig. 2E), thus on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 14 supporting the premise that CYR61/CCN1 overexpression protects pancreatic cancer cells from GEM-induced cell death. Our next goal was to investigate whether hrCYR61-treatment (250 ng/ml for 72 h) enhances the GEM-resistant phenotype in human pancreatic ductal epithelial (HPDE) cells, which lack CYR61/CCN1 expression. We found that CYR61 is unable to resist the effect of GEM in HPDE cells, suggesting a unique physiological environment is needed for CYR61 to act as a drug-resistant molecule. This environment is possibly lacking in HPDE cells (Fig. 2F). However, in order to determine this, further studies are warranted. CYR61 Ablation Enhances Inhibition of the Colony-forming Ability of Panc-1 cells by
Gemcitabine
To better understand the role of CYR61 in GEM resistance in pancreatic cancer cells, we tested the effect of GEM on anchorage-dependent growth (ADG) and anchorage-independent growth (AIG) of Panc-1 CYR61(-) and Panc-1 CYR61(+) cells. We saw that a low (0.1μM) and high (0.5 M) doses of GEM significantly impaired ADG in Panc-1 CYR61(-) as compared to Panc1 CYR61(+) (Figs. 3A and 3B). Like ADG, GEM blocked AIG in both populations. However, the CYR61-ablated cells were roughly three times less likely to undergo AIG, suggesting that GEM in a low or high dose reduces the transformation ability of Panc-1 CYR61(-) cells (Fig. 3C).
CYR61/CCN1 Ablation Sensitizes the Gemcitabine Action on Sphere Formation
Our previous studies revealed that CYR61 promotes EMT followed by cancer stemness in pancreatic cancer cells (21,26). Several studies have shown that cancer stem-like cells are able to proliferate and expand unlimitedly in vitro as tumor sphere formations, and thus, we wanted to investigate whether GEM, in the absence of CYR61, was able to significantly destroy the sphereforming ability of pancreatic cancer cells. We found that Panc-1 CYR61(+) and Panc-1 CYR61(-) cells on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 15 could form the spheres in presence or absence of GEM (Fig. S5). However, the number and the size of spheres are significantly reduced in GEM-exposed Panc-1 CYR61(-) cells as compared to GEM-exposed Panc-1 CYR61(+) cells in both concentrations (0.1 and 0.5 µM) (Fig. S5 and Fig 3D), indicating CYR61 ablation makes pancreatic cancer stem cells sensitive to GEM.
CYR61/CCN1 Suppresses Deoxycytidine Kinase (dCK) Expression in Pancreatic Cancer
Cells
To produce the cytotoxic effect of GEM, it needs to be activated by complex intracellular phosphorylations to yield GEM diphosphate (dFdCDP) and triphosphate (dFdCTP). Deoxycytidine kinase (dCK) plays a vital role in initiating this sequential process (Fig. 4A). Lack of expression of this enzyme, which is a common event in PDAC, diminishes the functional efficiency of GEM. We found that the expression level of CYR61/CCN1 is inversely proportional to the expression level of dCK. The PDAC cell line BxPC-3, which is not very GEM-resistant as compared to other cell lines (40)(Fig. S6), overexpressed dCK and had almost no CYR61/CCN1 expression. In contrast, the dCK expression is downregulated in CYR61/CCN1-overexpressing and GEM-resistant Panc-1 cells (Fig. 4B-C). Moreover, we found that the expression level of dCK can be restored in parental and GEM-induced over resistant Panc-1 cells by knocking down CYR61/CCN1 (Fig. 4D) or by reducing dCK levels by treating BxPC-3 with hrCYR61 (Fig. 4E). Collectively, this study suggests that CYR61/CCN1-induced chemoresistance could be mediated through the down-regulation of dCK. To corroborate the notion, we transfected Panc-1 cells with the expression vectors containing dCK gene or vector alone in Panc-1 cells. The dCK overexpressing and under-expressing Panc-1 cells (Fig. 5A) were then treated with GEM (5 μM) for 72h and cell viability was measured. We found that the on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 16 cell viability was significantly reduced in dCK-over expressing Panc-1 cells as compared to vector-alone-transfected cells (Fig. 5B).
CYR61/CCN1 Regulates CTGF in PDAC Cells and Stellate Cells for Desmoplastic
Reactions
Desmoplasia in PDAC plays a critical role in the intrinsic and extrinsic resistance to GEM (15). A desmoplastic reaction in PDAC is marked by an intense increase in the proliferation of α-smooth muscle actin (α-SMA)-positive fibroblasts (41). Stromal fibroblasts in PDAC are recognized as activated pancreatic stellate cells (PSCs) (42). In a normal pancreas, PSCs are quiescent. But in response to a pancreatic injury or neoplasm, PSCs transform from a quiescent to “activated” state, which is also called a “myofibroblastic state” (42). Activated PSCs/myofibroblasts are characterized by α-SMA expression. Consistent with earlier work, we found that a PSCs (hPaSteC, human pancreatic stellate cells) cell line is always in an active state under culture conditions and achieve a myofibroblast-like phenotype with high expression of αSMA (Fig. 6A). PSCs do not or minimally express CYR61 under a regular culture setup (Fig. 6A). Given the importance of PSCs/myofibroblast cells in desmoplasia during PDAC development, we first investigated the effect of CYR61/CCN1 on α-SMA in stellate cells. We found the level of α-SMA significantly increased in CYR61 recombinant protein (250 ng/ml)treated stellate cells as compared to untreated cells (Fig. S7). Next, we examined the effect of pancreatic cancer cell-secreted CYR61 on fibroblast cells’ activity and growth. To do so, we cocultured Panc-1 CYR61(+) or Panc-1 CYR61(-) and HPaSteC for 7 days in our unique experimental settings (Fig. 6B, left panel). We found that α-SMA + HPaSteC were significantly reduced when they interact with CYR61/CCN1-depleted pancreatic tumor cells as compared to Panc1CYR61(+) where abundant α-SMA + PSCs were detected (Fig. 6B, middle and bottom panels). Finally, we on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 17 investigated whether CYR61 has any role in the viability of stellate cells. To do so, HPaSteC cells were grown in condition media (CM) of Panc-1 CYR61(+) or Panc-1 CYR61(-) for 72h, and cell viability was measured. The results show that the viability of HPaSteC cells was significantly decreased when they were grown in CM of Panc-1 CYR61(-) cells as compared to the CM of parental CYR61 positive Panc-1 cells (Fig. 6B, bottom right panel bar graph). Collectively, these studies suggest that CYR61 plays a vital role in survival and activation of stellate cells that needed for desmoplasia. CTGF/CCN2, a profibrotic secretory growth factor, plays a vital role in tumor-stromal interaction and desmoplasia in PDAC (20,43-45). CTGF/CCN2 is overexpressed in PDAC samples, various PDAC cells and reactive fibroblast/stellate cells (43,44). We found CTGF was highly expressed in AsPC-1 and Panc-1 cell lines as compared to BxPC-3 and Capan-1 cell lines where CTGF/CCN2 expression was undetected or minimally detected (Fig. 6C). We then analyzed CTGF expression levels during the blockade of CYR61/CCN1 expression by shRNA or function by specific antibody-treatment in PDAC cells. We found that CTGF production was significantly impaired in CYR61/CCN1 ablated Panc-1 and AsPC-1 cells (Fig. 6D and 6E). Finally, we determined the CTGF status in human fibroblasts (FEF3)(46), and human pancreatic stellate cells (HPaSteC). We observed CTGF expression was undetected in HPaSteC (Fig. 6F). However, treatment of hrCYR61/CCN1 protein (250 ng/ml for 48 h) enhanced the production of CTGF in HPaSteC (Fig. 6G). Collectively, these results suggest an autocrineparacrine pathway is involved in CYR61/CCN1-induced CTGF/CCN2 expression in pancreatic cancer cells and pancreatic stellate cells (Fig. 6H). CYR61 Prevents Gemcitabine Action on Pancreatic Cancer Cell Growth in Vivo on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 18 Finally, we tested the role of CYR61/CCN1 in a pancreatic cancer cells-subcutaneous-transplantxenograft model. Weekly measurements of tumor volumes revealed that tumors in the Panc1 CYR61(+) group (n = 5) continued to increase significantly throughout a 21-day period in the presence of GEM (50 mg/Kg/twice a week by injection) as compared to Panc-1 CYR61(-) tumors without having significant difference in body weight (Fig. 7A), supporting the concept that CYR61/CCN1 is a prime driving force toward PDAC cells’ resistance to GEM. Since we found in this study that CYR61/CCN1 controls stellate cells’ activity and proliferation (Fig. 6), we next sought to address whether CYR61/CCN1 is linked with desmoplastic reaction in vivo. The studies showed that the desmoplastic reaction was markedly higher in Panc-1 CYR61(+) tumors as compared to Panc-1 CYR61(-) tumors where desmoplasia was minimal or undetected (Fig. 7B). The non-significant effect of GEM on desmoplastic reaction was observed in Panc-1 CYR61(+) tumors. Finally, we tested whether a CYR61/CCN1 deficiency along with GEM can alter the expression level of -SMA, CTGF, and dCK. We found that -SMA and CTGF are markedly down-regulated while the levels of dCK were significantly up-regulated in the cell lysates of CYR61-deficient Panc-1 cells and along with GEM treatment using immuno-western blotting (Fig. 7C). on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 19
Discussion
While GEM is still considered as a first-line therapy and is given alone or combined with other agents to attack locally advanced or metastatic pancreatic cancers, the success is very limited and disappointing (5,6,8,47,48). The failure of GEM to attack aggressive pancreatic cancers cells is due to intrinsic (metabolic malfunction) or extrinsic resistance (drug delivery/desmoplasia) or both to GEM (9,15,47). Thus, multiple studies have been carried out to understand the mechanism, and they gave new insights into the GEM resistance of aggressive cancer cells having cancer stem cell-like properties (47,49-51). These studies, collectively, suggested that simultaneously targeting intrinsic- and extrinsic-resistant events may increase the toxic effects of GEM and increase patient survival. Our studies show that depleting CYR61/CCN1 impairs cancer stemness behaviors, while concomitantly suppressing intrinsic and extrinsic resistant phenotypes of GEM in aggressive PDAC cells via the regulation of dCK and CTGF. Therefore, targeting CYR61/CCN1 could be an ideal strategy to promote the effects of GEM in killing pancreatic cancer cells. In PDAC, CYR61/CCN1 needs to maintain cancer stem cell/tumor initiating behaviors that are critical for invasive progression and drug resistance (19). In this study, we gave three evidences that broaden our understanding of the involvement of CYR61/CCN1 in chemoresistance to PDAC cells. First, we show that compared to normal, and early-stages of the disease such as PanIN1, CYR61/CCN1 was found to be overexpressed in invasive pancreatic cancers (Fig. 1A and B). Second, we showed through the interaction network profile analysis that CYR61/CCN1-signaling is necessary to make cancer cells chemoresistant (Fig. 1C). Third and lastly, we showed that CYR61/CCN1 depletion in pancreatic cancer cells significantly reduces the number of CD133 and CXCR4 producing cells (Fig. 1D & 1E), which have the capacity to on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 20 resist chemotherapy (51). Taken together, our current studies validate the drug-resistance function of CYR61/CCN1 in pancreatic cancer cells. Given the importance of the role of CYR61/CCN1 in chemoresistance, we investigated the definite role of CYR61/CCN1 in resistance to GEM. We showed that the depletion of CYR61/CCN1 in PDAC cell lines significantly enhances the growth-inhibition efficacy of GEM via enhancing apoptotic cell death (Fig. 2). This finding was further supported by an analysis of side-populations (SP and NSP) of pancreatic cancer cells (Fig. 2E). The studies revealed that SP cells in which CYR61/CCN1 is overexpressed are more resistant to GEM as compared to NSP cells having minimal or no expression of CYR61/CCN1. We found that NSP cells were less likely to survive than SP cells after exposing to them to GEM. Interestingly, the growth inhibitory effect of GEM could be reduced significantly when the NSP cells were exposed conjointly with hrCYR61. Collectively, these studies suggest that CYR61/CCN1 overexpression in aggressive pancreatic cancer cells is a driving force for GEM-resistant phenotypes. Ironically, the above concept was not pertinent in the non-transformed human pancreas cell line (HPDE) lacking CYR61/CCN1 expression (Fig. 2F), suggesting a unique physiological environment is needed for CYR61 to act as a drug-resistant molecule, which is possibly lacking in HPDE cells. GEM-resistant pancreatic cancers cells have increased anchorage-dependent, and independent colony formation, migration, invasion, and sphere-forming ability (52). These aggressive features were weakly affected by GEM treatment. The current findings reveal that CYR61/CCN1 depletion makes aggressive pancreatic cancer cells sensitive to GEM even at a low concentration and significantly reduces the transforming ability (Fig. 3A-C), and sphereforming skill of PDAC cells, implicating that CYR61/CCN1 ablation reverses the cancer stem on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 21 cell properties and they could be mediated by reprogramming the mesenchymal-epithelial transition (21,53). The acquisition of chemoresistance against GEM is complex in its chemistry and pathobiology. Classically, GEM is activated inside the cells after sequential phosphorylations, and the first step of phosphorylation is carried out by an enzyme deoxycytidine kinase (dCK) (Fig. 4A) (13,54). In GEM-resistant pancreatic cancer cells, the expression of dCK is remarkably less, and upregulation of dCK makes these cells sensitive to GEM. Thus, the inactivation of dCK is considered as a critical intrinsic pathway to make cancer cells resistant to GEM (13,54). Our studies find that dCK expression in PDAC cells can be suppressed by CYR61/CCN1 overexpression or treatment. Further, induced expression of dCK in CYR61-overexpressing PDAC cells promotes sensitive of gemcitabine in gemcitabine-resistance cells (Fig. 5). Therefore, we suggest that CYR61/CCN1 overexpression in pancreatic cancer cells co-opt the resistant phenotype of these cells via suppressing dCK (Figs. 4 and 5). A recent study has shown that pancreatic stromal cell-secreted CYR61, which is induced by the TGF--ALK5- Smad pathway, has been involved in GEM-resistance (50). Although we did not find CYR61/CCN1 expression in the stroma of human PDAC or mouse model, we can now speculate that the CYR61/CCN1 from both sources could be involved in regulation of GEM-resistance to PDAC cells. Despite some controversies (55), many PDAC patients are resistant to GEM because of desmoplasia, which is about 90 percent of the tumor volume and help in promoting intrinsic and extrinsic resistance to GEM (47,49). One of the primary cellular compartments of desmoplasia is cancer cell-associated fibroblasts (CAFs). During the progression of the PDAC, CAFs are generated from quiescent fibroblasts to active myofibroblast-like phenotypes/ stellate cells on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 22 (PSCs) characterized by the expression of α-SMA (41,42,47). However, the mechanism to kickstart this transformation process is largely unknown. The current studies with a unique in vitro model show that pancreatic cancer cell secreted CYR61/CCN1 plays a vital role in the transformation of CAFs from quiescent fibroblasts and their proliferation (Fig. 6A &B). CTGF/CCN2 plays a vital role in tumor-stromal interaction and desmoplasia in PDAC (20,43- 45). Recent studies have shown that tumor cell-derived CTGF is a prerequisite signal for fibroblast activation (56). Since our current studies found that CTGF expression in pancreatic cancer cells and fibroblast cells is regulated by CYR61/CCN5 (Fig. 6C-H), we suggest that CTGF could be an intermediate signaling molecule necessary for fibroblast activation. However, further studies are warranted. Finally, to validate the in vitro data, we tested the impact of GEM in a pre-clinical mouse model. We observed that GEM significantly suppressed the growth of subcutaneous tumors that developed by CYR61/CCN1-depleted Panc-1 cells (Fig. 7A). Moreover, in CYR61/CCN1 depleted tumors, CAFs transformation and CTGF production were significantly decreased and at the same time dCK production was markedly elevated (Fig. 7B). These studies collectively suggest that pancreatic cancer’s patients’ intrinsic and extrinsic resistance of GEM are CYR61/CCN1-dependent, and therefore, blocking CYR61/CCN1 activity would be an ideal strategy to kill cancer cells as well as destroy desmoplastic growth in PDAC by GEM.
Authors’ Contributions
Conception, design and manuscript preparation: G. Maity, V. Gupta, A. Ghosh, S. Banerjee and S.K. Banerjee Development of method: G. Maity, I. Haque, A. Ghosh, S. Sarkar, V. Gupta. K. Dhar and S. Banerjee Acquisition of data: G. Maity, A. Ghosh, I. Haque, V. Gupta. A. Das, S. Sarkar, K. Dhar and S. Banerjee on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 23 Bioinformatics and statistics: S.S. Gunewardena and S.K. Banerjee Writing, review and/or revision of the manuscript: Gargi Maity, Arnab Ghosh, D. VonHoff, S. Kambhampati, S. Mallik, S. Bhavanasi, S. Banerjee and S.K. Banerjee Adminstrative, technical, or material support: Gargi Maity, A. Ghosh, S. Sarkar, S.Banerjee Funding support: S. Banerjee, S. Kambhampati and S.K. Banerjee Study supervision: S. Banerjee, Arnab Ghosh and S.K. Banerjee
Acknowledgements
We thank Archana De for technical help, VA Research office and Midwest Biomedical Research Foundation for administrative and secreterial supports.
Grant Support
The work is supported by Merit review grant from Department of Veterans Affairs (Sushanta K. Banerjee, 5I01BX001989-04 and Snigdha Banerjee, I01BX001002-05), KUMC Lied Basic Science Grant Program (SKB), and Grace Hortense Greenley Trust, directed by The Research Foundation in memory of Eva Lee Caldwell (SB and SK). on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 24 References 1. Hruban RH, Goggins M, Parsons J, Kern SE. Progression model for pancreatic cancer. Clin Cancer Res 2000;6(8):2969-72. 2. Lennon AM, Wolfgang CL, Canto MI, Klein AP, Herman JM, Goggins M, et al. The Early Detection of Pancreatic Cancer: What Will It Take to Diagnose and Treat Curable Pancreatic Neoplasia? Cancer Res 2014;74(13):3381-9 doi 10.1158/0008-5472.CAN-140734. 3. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res 2014;74(11):2913-21 doi 10.1158/0008-5472.CAN-14-0155. 4. Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA. Genetics and biology of pancreatic ductal adenocarcinoma. Genes & development 2006;20(10):1218-49 doi 10.1101/gad.1415606. 5. Gnanamony M, Gondi CS. Chemoresistance in pancreatic cancer: Emerging concepts. Oncology letters 2017;13(4):2507-13 doi 10.3892/ol.2017.5777. 6. Carmichael J, Fink U, Russell RC, Spittle MF, Harris AL, Spiessi G, et al. Phase II study of gemcitabine in patients with advanced pancreatic cancer. British journal of cancer 1996;73(1):101-5. 7. Garrido-Laguna I, Hidalgo M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies. Nature reviews Clinical oncology 2015;12(6):319-34 doi 10.1038/nrclinonc.2015.53. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 25 8. de Sousa Cavalcante L, Monteiro G. Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. European journal of pharmacology 2014;741:8-16 doi 10.1016/j.ejphar.2014.07.041. 9. Koay EJ, Truty MJ, Cristini V, Thomas RM, Chen R, Chatterjee D, et al. Transport properties of pancreatic cancer describe gemcitabine delivery and response. The Journal of clinical investigation 2014;124(4):1525-36 doi 10.1172/JCI73455. 10. van Haperen VW, Veerman G, Vermorken JB, Pinedo HM, Peters G. Regulation of phosphorylation of deoxycytidine and 2',2'-difluorodeoxycytidine (gemcitabine); effects of cytidine 5'-triphosphate and uridine 5'-triphosphate in relation to chemosensitivity for 2',2'-difluorodeoxycytidine. Biochemical pharmacology 1996;51(7):911-8. 11. Ohhashi S, Ohuchida K, Mizumoto K, Fujita H, Egami T, Yu J, et al. Down-regulation of deoxycytidine kinase enhances acquired resistance to gemcitabine in pancreatic cancer. Anticancer Res 2008;28(4B):2205-12. 12. Galmarini CM, Clarke ML, Jordheim L, Santos CL, Cros E, Mackey JR, et al. Resistance to gemcitabine in a human follicular lymphoma cell line is due to partial deletion of the deoxycytidine kinase gene. BMC pharmacology 2004;4:8 doi 10.1186/1471-2210-4-8. 13. Nakano T, Saiki Y, Kudo C, Hirayama A, Mizuguchi Y, Fujiwara S, et al. Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK. Biochemical and biophysical research communications 2015;464(4):1084-9 doi 10.1016/j.bbrc.2015.07.080. 14. Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. The New England journal of medicine 2013;369(18):1691-703 doi 10.1056/NEJMoa1304369. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 26 15. Mahadevan D, Von Hoff DD. Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Molecular cancer therapeutics 2007;6(4):1186-97 doi 10.1158/1535- 7163.MCT-06-0686. 16. Merika EE, Syrigos KN, Saif MW. Desmoplasia in pancreatic cancer. Can we fight it? Gastroenterology research and practice 2012;2012:781765 doi 10.1155/2012/781765. 17. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646-74 doi 10.1016/j.cell.2011.02.013. 18. Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, et al. Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res 2009;69(14):5820-8 doi 10.1158/0008-5472.CAN-08-2819. 19. Banerjee SK, Maity G, Haque I, Ghosh A, Sarkar S, Gupta V, et al. Human pancreatic cancer progression: an anarchy among CCN-siblings. Journal of cell communication and signaling 2016;10(3):207-16 doi 10.1007/s12079-016-0343-9. 20. Neesse A, Frese KK, Bapiro TE, Nakagawa T, Sternlicht MD, Seeley TW, et al. CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer. Proceedings of the National Academy of Sciences of the United States of America 2013;110(30):12325-30 doi 10.1073/pnas.1300415110. 21. Haque I, Mehta S, Majumder M, Dhar K, De A, McGregor D, et al. Cyr61/CCN1 signaling is critical for epithelial-mesenchymal transition and stemness and promotes pancreatic carcinogenesis. Molecular cancer 2011;10:8 doi 10.1186/1476-4598-10-8. 22. Maity G, Haque I, Ghosh A, Dhar G, Gupta V, Sarkar S, et al. The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 27 cancer cell invasion via CRAF-ERK signaling. The Journal of biological chemistry 2018;293(12):4334-49 doi 10.1074/jbc.RA117.000333. 23. Holloway SE, Beck AW, Girard L, Jaber MR, Barnett CC, Jr., Brekken RA, et al. Increased expression of Cyr61 (CCN1) identified in peritoneal metastases from human pancreatic cancer. Journal of the American College of Surgeons 2005;200(3):371-7 doi 10.1016/j.jamcollsurg.2004.10.005. 24. Leask A. CCN1: a novel target for pancreatic cancer. Journal of cell communication and signaling 2011;5(2):123-4 doi 10.1007/s12079-011-0127-1. 25. Shi W, Zhang C, Chen Z, Chen H, Liu L, Meng Z. Cyr61-positive cancer stem-like cells enhances distal metastases of pancreatic cancer. Oncotarget 2016;7(45):73160-70 doi 10.18632/oncotarget.12248. 26. Haque I, De A, Majumder M, Mehta S, McGregor D, Banerjee SK, et al. The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis. The Journal of biological chemistry 2012;287(46):38569-79 doi 10.1074/jbc.M112.389064. 27. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nature reviews Cancer 2005;5(4):275-84 doi 10.1038/nrc1590. 28. Donnenberg VS, Donnenberg AD. Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis. JClinPharmacol 2005;45(8):872-7 doi 45/8/872 [pii];10.1177/0091270005276905 [doi]. 29. Mallini P, Lennard T, Kirby J, Meeson A. Epithelial-to-mesenchymal transition: what is the impact on breast cancer stem cells and drug resistance. Cancer treatment reviews 2014;40(3):341-8 doi 10.1016/j.ctrv.2013.09.008. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 28 30. Nurwidya F, Takahashi F, Murakami A, Takahashi K. Epithelial mesenchymal transition in drug resistance and metastasis of lung cancer. Cancer research and treatment : official journal of Korean Cancer Association 2012;44(3):151-6 doi 10.4143/crt.2012.44.3.151. 31. Haque I, Ghosh A, Acup S, Banerjee S, Dhar K, Ray A, et al. Leptin-induced ER-alpha- positive breast cancer cell viability and migration is mediated by suppressing CCN5signaling via activating JAK/AKT/STAT-pathway. BMC cancer 2018;18(1):99 doi 10.1186/s12885-018-3993-6. 32. Kambhampati S, Banerjee S, Dhar K, Mehta S, Haque I, Dhar G, et al. 2- methoxyestradiol inhibits Barrett's esophageal adenocarcinoma growth and differentiation through differential regulation of the beta-catenin-E-cadherin axis. MolCancer Ther 2010;9(3):523-34 doi 1535-7163.MCT-09-0845 [pii];10.1158/15357163.MCT-09-0845 [doi]. 33. Maity G, De A, Das A, Banerjee S, Sarkar S, Banerjee SK. Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition. Lab Invest 2015 doi 10.1038/labinvest.2015.49. 34. Moog R, Burger AM, Brandl M, Schuler J, Schubert R, Unger C, et al. Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels. Cancer chemotherapy and pharmacology 2002;49(5):356-66 doi 10.1007/s00280-002-0428-4. 35. Abbruzzese JL, Grunewald R, Weeks EA, Gravel D, Adams T, Nowak B, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol 1991;9(3):491-8. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 29 36. Yip-Schneider MT, Wu H, Stantz K, Agaram N, Crooks PA, Schmidt CM. Dimethylaminoparthenolide and gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer. BMC cancer 2013;13:194 doi 10.1186/1471-2407-13-194. 37. Garcia-Cremades M, Pitou C, Iversen PW, Troconiz IF. Characterizing Gemcitabine Effects Administered as Single Agent or Combined with Carboplatin in Mice Pancreatic and Ovarian Cancer Xenografts: A Semimechanistic Pharmacokinetic/Pharmacodynamics Tumor Growth-Response Model. The Journal of pharmacology and experimental therapeutics 2017;360(3):445-56 doi 10.1124/jpet.116.237610. 38. Du J, Chen W, Yang L, Dai J, Guo J, Wu Y, et al. Disruption of SHH signaling cascade by SBE attenuates lung cancer progression and sensitizes DDP treatment. Scientific reports 2017;7(1):1899 doi 10.1038/s41598-017-02063-x. 39. Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proceedings of the National Academy of Sciences of the United States of America 2009;106(38):16281-6 doi 10.1073/pnas.0905653106. 40. Awasthi N, Zhang C, Schwarz AM, Hinz S, Wang C, Williams NS, et al. Comparative benefits of Nab-paclitaxel over gemcitabine or polysorbate-based docetaxel in experimental pancreatic cancer. Carcinogenesis 2013;34(10):2361-9 doi 10.1093/carcin/bgt227. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 30 41. Whatcott CJ, Han H, Posner RG, Hostetter G, Von Hoff DD. Targeting the tumor microenvironment in cancer: why hyaluronidase deserves a second look. Cancer discovery 2011;1(4):291-6 doi 10.1158/2159-8290.CD-11-0136. 42. Omary MB, Lugea A, Lowe AW, Pandol SJ. The pancreatic stellate cell: a star on the rise in pancreatic diseases. The Journal of clinical investigation 2007;117(1):50-9 doi 10.1172/JCI30082. 43. Wells JE, Howlett M, Cole CH, Kees UR. Deregulated expression of connective tissue growth factor (CTGF/CCN2) is linked to poor outcome in human cancer. International journal of cancer Journal international du cancer 2015;137(3):504-11 doi 10.1002/ijc.28972. 44. Korc M. Pancreatic cancer-associated stroma production. American journal of surgery 2007;194(4 Suppl):S84-6 doi 10.1016/j.amjsurg.2007.05.004. 45. Kubota S, Takigawa M. Cellular and molecular actions of CCN2/CTGF and its role under physiological and pathological conditions. Clinical science 2015;128(3):181-96 doi 10.1042/CS20140264. 46. Noma K, Smalley KS, Lioni M, Naomoto Y, Tanaka N, El-Deiry W, et al. The essential role of fibroblasts in esophageal squamous cell carcinoma-induced angiogenesis. Gastroenterology 2008;134(7):1981-93 doi 10.1053/j.gastro.2008.02.061. 47. Liang C, Shi S, Meng Q, Liang D, Ji S, Zhang B, et al. Complex roles of the stroma in the intrinsic resistance to gemcitabine in pancreatic cancer: where we are and where we are going. Experimental & molecular medicine 2017;49(12):e406 doi 10.1038/emm.2017.255. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 31 48. Liang C, Shi S, Meng Q, Liang D, Ji S, Zhang B, et al. Do anti-stroma therapies improve extrinsic resistance to increase the efficacy of gemcitabine in pancreatic cancer? Cellular and molecular life sciences : CMLS 2018;75(6):1001-12 doi 10.1007/s00018-017-26787. 49. Heinemann V, Schulz L, Issels RD, Plunkett W. Gemcitabine: a modulator of intracellular nucleotide and deoxynucleotide metabolism. Seminars in oncology 1995;22(4 Suppl 11):11-8. 50. Hesler RA, Huang JJ, Starr MD, Treboschi VM, Bernanke AG, Nixon AB, et al. TGF- beta-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3. Carcinogenesis 2016 doi 10.1093/carcin/bgw093. 51. Hu G, Li F, Ouyang K, Xie F, Tang X, Wang K, et al. Intrinsic gemcitabine resistance in a novel pancreatic cancer cell line is associated with cancer stem cell-like phenotype. International journal of oncology 2012;40(3):798-806 doi 10.3892/ijo.2011.1254. 52. Wang R, Cheng L, Xia J, Wang Z, Wu Q, Wang Z. Gemcitabine resistance is associated with epithelial-mesenchymal transition and induction of HIF-1alpha in pancreatic cancer cells. Curr Cancer Drug Targets 2014;14(4):407-17. 53. Haque I, De A, Majumder M, Mehta S, McGregor D, Banerjee SK, et al. The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis. JBiolChem 2012;287(46):38569-79 doi M112.389064 [pii];10.1074/jbc.M112.389064 [doi]. 54. Saiki Y, Yoshino Y, Fujimura H, Manabe T, Kudo Y, Shimada M, et al. DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells. Biochemical on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 32 and biophysical research communications 2012;421(1):98-104 doi 10.1016/j.bbrc.2012.03.122. 55. Gore J, Korc M. Pancreatic cancer stroma: friend or foe? Cancer Cell 2014;25(6):711-2 doi 10.1016/j.ccr.2014.05.026. 56. Wu W, Zaal EA, Berkers CR, Lemeer S, Heck AJR. CTGF/VEGFA-activated fibroblasts promote tumor migration through micro-environmental modulation. Molecular & cellular proteomics : MCP 2018 doi 10.1074/mcp.RA118.000708. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from 33
Figure Legends
Figure 1: Distribution pattern of Cyr61/CCN1 in human pancreatic cancer patient in relation to stemness and drug resistance A. Representative `H&E staining of a normal duct (a) and PDAC area (b), and immunohistochemical staining of Cyr61/CCN1 in normal duct (c) and PDAC area (d). B. Labeling intensity score of Cyr61/CCN1 in tissue array sections of adjacent normal and pancreatic cancer patients’ samples. AN, adjacent normal; PanINs, pancreatic intraepithelial neoplasia; and PT, primary tumor samples. C. The Connectivity Map represents the network of functional connection of the drugresistant genes with CYR61/CCN1 in pancreatic cancer patient samples. The network was designed in IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity- pathway-analysis), using its knowledge base of known, experimentally validated, molecular interactions. The network reflects the potential pathways by which CYR61 interacts with drug resistance genes. The molecules, TP73, SNAI1, forskolin and doxorubicin are up-stream of CYR61. The rest of the molecules are down-stream of CYR61. Solid lines in the network represent direct interactions. Perforated lines represent indirect interactions. D. FACS analysis of CD133 (upper panel) and CXCR4 (lower panel) in in parental Panc1 Cyr61(+) and Panc-1 Cyr61(-) cell lines. E. A representative photograph of immunofluorescence of CD133 (Upper panel) and CXCR4 (Lower panel) protein in Panc-1 Cyr61(+) and Panc-1 Cyr61(-) cells, respectively. The intensity of the fluorescently labeled cells was measured using flow cytometry. Arrows (yellow) indicate cells with low intensity. Data represent the mean ± SD. Scale, 100 m. on February 24, 2019. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from F. Quantification of the intensity of CD133 (upper panel) and CXCR4 (lower panel) in parental Panc-1 Cyr61(+) and Panc-1 Cyr61(-) cell lines. Data (bar graphs) represent the mean ± SD of three independent experiments. Figure 2: Pancreatic cancer cell resistant to GEM is mediated through Cyr61/CCN1 overexpression. A& B. The dose-dependent effect of GEM on Panc-1 Cyr61(+) and Panc-1 Cyr61(-) cells viability after 72 h treatment. Cell viability was measured by crystal violet assay. Data represent the mean ± SD of values of four samples. C. Dose-dependent effect of GEM on the viability of AsPC-1Cyr61(+) and AsPC-1Cyr61(-) cells after 72 h treatment. Data represent the mean ± SD of values of four samples. D. Dose-dependent effect of GEM on apoptosis of Panc-1Cyr61(+) and Panc-1Cyr61(-) cells after 72 h of treatment. Data represent the mean ± SD of values of four samples. *p<0.01; **p<0.001 E. Dose-dependent effect of GEM alone or in combination with hrCyr61 (250 ng/ml) protein on SP and NSP population of Panc-1 cells viability after 72 h treatment. Data represent the mean ± SD of values of six samples. F. Combined effect of GEM (0.5M) and hrCYR61 (250 ng/ml) protein on HPDE cells viability after 72 h of treatment. Data represent the mean ± SD of values of eight samples. B. The bar graphs represent the quantification of the number of colonies of three independent experiments. Data represent the mean ± SD of values of three samples. *p<0.001 and *
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Bioz is the world’s most comprehensive AI search engine for scientific experimentation. The Bioz search engine offers researchers billions of data-driven product, technique, and protocol recommendations. Bioz taps into the latest advances in Artificial Intelligence (AI) – including Natural Language Processing (NLP), Machine Learning (ML) and Generative AI to mine and structure hundreds of millions of pages of complex and unstructured scientific papers. By harnessing the power of AI, Bioz provides researchers with an unprecedented amount of summarized scientific experimentation knowledge right at their fingertips, ultimately helping to speed up drug discovery and the rate of success in finding cures for diseases. Bioz is used by over 15 Million researchers from over 17,000 different universities and companies in 196 countries.
