Bone morphogenetic protein -4 and -5 in pancreatic cancer--novel bidirectional players

Bone morphogenetic proteins (BMPs) are multifunctional signaling molecules that have gained increasing interest in cancer research. To obtain a systematic view on BMP signaling in pancreatic cancer we first determined the mRNA expression levels of seven BMP ligands (BMP2–BMP8) and six BMP specific receptors in pancreatic cancer cell lines and normal pancreatic tissue. BMP receptor expression was seen in all cancer and normal samples. Low expression levels of BMP5 and BMP8 were detected in cancer cells compared to the normal samples, whereas BMP4 expression was elevated in 25% of the cases. The impact of BMP4 and BMP5 signaling on cell phenotype was then evaluated in five pancreatic cancer cell lines. Both ligands suppressed the growth of three cell lines (up to 79% decrease in BMP4-treated PANC-1 cells), mainly due to cell cycle changes. BMP4 and BMP5 concurrently increased cell migration and invasion (maximally a 10.8-fold increase in invaded BMP4-treated PANC-1 cells). The phenotypic changes were typically associated with the activation of the canonical SMAD pathway, although such activation was not observed in the PANC-1 cells. Taken together, BMP4 and BMP5 simultaneously inhibit the growth and promote migration and invasion of the same pancreatic cells and thus exhibit a biphasic role with both detrimental and beneficial functions in pancreatic cancer progression.     

Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.     

Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Promotion of cell-invasive activity through the induction of LPA receptor-1 in pancreatic cancer cells

Abstract

Lysophosphatidic acid (LPA) is a simple biological lipid and mediates several biological functions with LPA receptors (LPA₁ to LPA₆). In the present study, to assess whether LPA receptors promote cell-invasive activity of pancreatic cancer cells, highly invasion PANC-R9 cells were established from PANC-1 cells, using Matrigel-coated Cell Culture Insert. The cell-invasive activity of PANC-R9 cells was shown to be approximately 15 times higher than that of PANC-1 cells. LPAR1 expression level was markedly elevated in PANC-R9 cells in comparison with PANC-1 cells, while LPAR3 expression level was reduced. The cell-invasive activity of PANC-R9 cells was enhanced by LPA, but LPA had no impact on PANC-1 cell invasion. Before initiation of the cell invasion assay, PANC-R9 cells were pretreated with dioctanoylglycerol pyrophosphate (DGPP), an antagonist of LPA₁/LPA₃. The invasive activity of PANC-R9 cells was markedly suppressed by DGPP. Autotaxin (ATX) is a key enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to LPA. ATX expression level was elevated in PANC-R9 cells compared with PANC-1 cells. In the presence of LPC, the cell motile activity of PANC-R9 cells was markedly stimulated. In contrast, LPC did not affect the cell motile activity of PANC-1 cells. PANC-R9 cell motility was inhibited by an ATX inhibitor, PF-8380. These results suggest that LPA signaling via LPA₁ is a potent molecular target for the regulation of tumor progression in PANC-1 cells.     

MUC6 mucin expression inhibits tumor cell invasion

The MUC6 mucin has a critical protective function in the normal stomach, pancreas and duodenum and is aberrantly expressed during the progression of some gastrointestinal cancers. Our aim was to determine whether MUC6 contributes to the etiology or progression of pancreatic cancer and elucidate the molecular basis of its involvement. Expression of MUC6 glycoprotein was examined in pancreatic cancer tissues by immunofluorescence and loss of MUC6 was observed. Next, to determine whether MUC6 inhibits tumor growth and metastasis by altering cell adhesion and invasion, recombinant MUC6 cDNA and separate MUC6 N-terminal and C-terminal domains were transfected into pancreatic, colorectal and breast cancer cell lines. The recombinant N- and C-terminal proteins were each seen to oligomerize under non-reducing conditions. Overexpression of both domains of the MUC6 glycoprotein significantly inhibited cell adhesion to matrix proteins (collagen I, collagen IV, fibronectin and laminin) in LS 180 but not in PANC-1 cells. Moreover, the N- and C-terminal domains of MUC6 inhibited invasion of both LS 180 and PANC-1 cells by 40% and 70%, respectively, in comparison with controls. These results suggest that MUC6 may inhibit invasion of tumor cells through the basement membrane of the pancreatic duct and slow the development of infiltrating carcinoma.     

Smad4 is essential for down-regulation of E-cadherin induced by TGF-beta in pancreatic cancer cell line PANC-1

Smad4 is a tumour suppressor gene frequently deleted in pancreatic cancer. To investigate the roles of Smad4 deficiency in invasive and matastatic capabilities of pancreatic cancer, we examined the effects of Smad4 deficiency on regulation of the invasion suppressor E-cadherin in pancreatic cancer cell line PANC-1. TGF-beta decreased expression of E-cadherin and beta-catenin proteins at the plasma membrane, increased Snail and Slug mRNA expression, and induced fibroblastoid morphology in PANC-1 cells. These effects of TGF-beta were abrogated in Smad4-knocked-down PANC-1 cells. We also found that TGF-beta-induced down-regulation of E-cadherin expression was partially inhibited in Snail- and Slug-knocked-down PANC-1 cells. Thus, Smad4 mediates down-regulation of E-cadherin induced by TGF-beta in PANC-1 cells, at least in part, through Snail and Slug induction. These results suggest that Smad4 deficiency observed in invasive and metastatic pancreatic cancer might not be linked to the loss of E-cadherin.     

Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells

Oncogenic KRAS, an important target for antitumor therapy, contributes to pancreatic cancer tumorigenesis, progression and maintenance. However, intracellular compensation regulation can help cells to resist the targeted therapy. Gene knockdown method such as RNAi may help to understand this intracellular regulatory system and discover novel therapeutic approach. In this study, two stable transfected cell lines were constructed through lentivirus-mediated shRNA targeting KRAS of PANC-1 cells, to investigate cell response to the knockdown of KRAS. Human whole genome microarray was then used to compare the gene expression profile. As a result, ribosomal proteins L26 and L29 (RPL26 and RPL29) were dramatically upregulated by KRAS-shRNA specifically. To identify whether RPL26 or RPL29 was critical for PANC-1 cells, siRNAs against RPL26 and RPL29 were designed and transfected in vitro. The results showed that knockdown of RPL26 or RPL29 expression significantly suppressed cell proliferation, induced cell arrest at G0/G1 phase and enhanced cell apoptosis. Reactive oxygen species (ROS) assay indicated that silencing of RPL26 or RPL29 markedly decreased the intracellular ROS generation. Our findings imply that siRNA interference against RPL26 and RPL29 is of potential value for intervention of pancreatic cancer.     

Cancer spheroids derived exosomes reveal more molecular features relevant to progressed cancer

Cancer cell spheroids have been shown to be more physiologically relevant to native tumor tissue than monolayer 2D culture cells. Due to enhanced intercellular communications among cells in spheroids, spheroid secreted exosomes which account for transcellular transportation should exceed those from 2D cell culture and may display a different expression pattern of miRNA or protein. To test this, we employed a widely used pancreatic cancer cell line, PANC-1, to create 3D spheroids and compared exosomes generated by both 2D cell culture and 3D PANC-1 spheroids. We further measured and compared exosomal miRNA and GPC-1 protein expression with qRT-PCR and enzyme-linked immunosorbent assay, respectively. It showed that PANC-1 cells cultured in 3D spheroids can produce significantly more exosomes than PANC-1 2D cells and exosomal miRNA and GPC-1 expression derived from spheroids show more features relevant to the progression of pancreatic cancer. These findings point to the potential importance of using spheroids as in vitro model to study cancer development and progression.     

In vivo and in vitro degradation of heparan sulfate (HS) proteoglycans by HPR1 in pancreatic adenocarcinomas. Loss of cell surface HS suppresses fibroblast growth factor 2-mediated cell signaling and proliferation

Heparan sulfate proteoglycans (HSPGs) function as a co-receptor for heparin-binding growth factors, such as fibroblast growth factors (FGFs) and heparin-bound epidermal growth factor (HB-EGF). The HS side chain of HSPGs can be cleaved by HPR1 (heparanase-1), an endoglycosidase that is overexpressed in many types of malignancies. In the present study, we demonstrated that HPR1 expression in pancreatic adenocarcinomas inversely correlated with the presence of heparan sulfate (HS) in the basement membrane. In vitro cell culture study revealed that cell surface HS levels inversely correlated with HPR1 activity in five pancreatic cancer cell lysates and their conditioned media. Heparin and PI-88, two HPR1 inhibitors, were able to increase cell surface HS levels in PANC-1 cells in a dose-dependent manner. The ability of HPR1 to degrade cell surface HS was confirmed by showing that cell surface HS levels were increased in HT1080 cells stably transfected with the HPR1 antisense gene but was decreased in the cells overexpressing HPR1. Further studies showed that PI-88 and heparin were able to stimulate PANC-1 cell proliferation in the absence or presence of exogenous FGF2, whereas exogenous HPR1 was able to inhibit PANC-1 cell proliferation in a dose-dependent manner. Modulation of PANC-1 cell proliferation by HPR1 or HPR1 inhibitors corresponded with the inhibition or activation of the mitogen-activated protein kinase. Our results suggest that HPR1 expressed in pancreatic adenocarcinomas can suppress the proliferation of pancreatic tumor cells in response to the growth factors that require HSPGs as their co-receptors.     

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

SDF-1α upregulation of MMP-2 is mediated by p38 MAPK signaling in pancreatic cancer cell lines

Pancreatic cancer is highly invasive and is currently the fourth leading cause of cancer death worldwide. CXC chemokine receptor-4 (CXCR4) is a G protein-coupled receptor for CXC chemokine ligand 12/stromal cell-derived factor-1α (SDF-1α), a member of a large family of small, structurally related, heparin-binding chemokine proteins. SDF-1α/CXCR4 plays an important role in tumor growth, invasion, metastasis, and angiogenesis. SDF-1α and CXCR4 are upregulated in many tumors, including pancreatic cancer tissues, and preliminary data indicate that the SDF-1/CXCR4 axis plays an important role in tumor invasion. However, their precise role and the mechanism through which they function remain largely unknown. In this study, analysis of SDF-1α, CXCR4 and MMP-2 expression in pancreatic cancer and adjacent tissue samples from ten patients revealed that all three proteins are overexpressed in human pancreatic cancer. SDF-1α induced MMP-2 and MMP-9 upregulation in PANC-1 and SW-1990 cells, which was associated with increased pancreatic cancer cell proliferation and invasion. Furthermore, SDF-1α induced p38 phosphorylation and p38 inhibition reduced both the level of SDF-1α-stimulated MMP-2 expression and PANC-1 cell invasion. Overall, our results demonstrate that SDF-1α/CXCR4 upregulates MMP-2 expression and induces pancreatic cancer cell invasion in PANC-1 and SW-1990 cell lines by activating p38 MAPK.     

Effect of NeuroD Gene Silencing on the Migration and Invasion of Human Pancreatic Cancer Cells PANC-1

The aim of this study is to investigate the influence of Lenti-EGFP-NeuroD-miR, RNAi lentiviral expression vector, on the expression level of NeuroD and migration, and invasion of PANC-1 cell line. PANC-1 cells were cultured and cotransfected with Lenti-EGFP-NeuroD-miR and Lenti-GFP. The infection rate of lentivirus was determined by fluorescence. The interfering effection by the expression of NeuroD mRNA in PANC-1 cells was analyzed by real-time PCR after transfected. Biological behavior of PANC-1 cells transinfected was observed, and the migration and invasion were studied by transwell assay. Intrapancreatic allografts model in nude mice was established to observe the effects of NeuroD on tumorigenesis, tumor growth, and invasion in vivo. The expression of NeuroD mRNA decreased significantly after RNAi lentivirus transinfecting PANC-1 cell. The cell’s migration and invasion ability decreased obviously as soon as down regulate of NeuroD in PANC-1 cells. Comparing with control group, the tumors were smaller in size and the invasiveness was inhibited after 8 weeks intrapancreatic allografts in nude mice. Lenti-EGFP-NeuroD-miR transfected into PANC-1 cells shows a stable, effective, and especial blocking expression of NeuroD in mRNA level. The RNAi of lentiviral vector target NeuroD can reduce the migration and invasion abilities of PANC-1 cells.     

SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐induced epithelial‐mesenchymal transition

Metastasis is a crucial impediment to the successful treatment for gastric cancer. SPOCK1 has been demonstrated to facilitate cancer metastasis in certain types of cancers; however, the role of SPOCK1 in the invasion and metastasis of gastric cancer remains elusive. SPOCK1 and epithelial‐mesenchymal transition (EMT)‐related biomarkers were detected by immunohistochemistry and Western blot in gastric cancer specimens. Other methods including stably transfected against SPOCK1 into gastric cancer cells, Western blot, migration and invasion assays in vitro and metastasis assay in vivo were also performed. The elevated expression of SPOCK1 correlates with EMT‐related markers in human gastric cancer tissue, clinical metastasis and a poor prognosis in patients with gastric cancer. In addition, knockdown of SPOCK1 expression significantly inhibits the invasion and metastasis of gastric cancer cells in vitro and in vivo, inversely, SPOCK1 overexpression results in the opposite effect. Interestingly, SPOCK1 expression has no effect on cell proliferation in vitro and in vivo. Regarding the mechanism(s) of SPOCK1‐induced cells invasion and metastasis, we prove that Slug‐induced EMT is involved in SPOCK1‐facilitating gastric cancer cells invasion and metastasis. The elevated SPOCK1 expression is closely correlated with cancer metastasis and patient survival, and SPOCK1 promotes the invasion and metastasis of gastric cancer through Slug‐mediated EMT, thereby possibly providing a novel therapeutic target for gastric cancer.     

miR-223在结肠癌组织中的表达及促进结肠癌细胞迁移侵袭的机制研究

目的:探讨微小RNA-223 (mi R-223)在结肠癌组织中的表达及对结肠癌HT-29细胞侵袭、迁移能力的影响及机制。方法:检测mi R-223在结肠癌组织与癌旁组织中的表达。通过脂质体转染法将mi R-223模拟物(mi R-223 mimics,mi R-223 mimics组)及microRNA无关序列(mi R-223 NC,NC组)转染入结肠癌HT-29细胞。采用Real-time PCR检测转染后细胞中mi R-223和TWIST的表达,Western blot检测TWIST的蛋白表达,Tranwell检测细胞的迁移与侵袭能力。双荧光素酶报告基因检测mi R-223对TWIST基因启动子活性的影响。采用Transwell迁移与侵袭实验检测mi R-223 mimic及Twist si RNA共转染后人结肠癌细胞系HT-29迁移与侵袭能力的变化。结果:与癌旁结肠组织比较,mi R-223在结肠癌组织中呈现明显高表达(P0.05);与空白对照组和mi R-223 NC组比较,转染mi R-223 mimics后的HT-29细胞中的mi R-223表达显著增加(P0.05)。与阴性对照组和空载转染组相比较,mi R-223 mimics转染组穿透的细胞数目明显增加(P0.05),且mi R-223 mimics转染组的细胞侵袭能力显著增强(P0.05)。与mi R-223 NC组和空白对照组比较,转染mi R-223 mimics的HT-29细胞的TWIST基因m RNA和蛋白表达均显著增加(P0.05)。双荧光素酶检验结果显示TWIST为mi R-223的下游靶基因。共转染TWIST si RNA和mi R-223 mimics的结肠癌HT-29细胞的迁移与侵袭能力较单独转染mi R-223 mimics的HT-29细胞显著减弱(P0.05)。结论:mi R-223可能通过上调下游靶基因TWIST水平促进结肠癌HT-29细胞的迁移与侵袭。     

Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo

Background

Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo.

Methods

Cryo transmission electron microscopy (TEM) examined ApoA-II’s influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively.

Results

ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold.

Conclusion

Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.     

Interleukin-32 Expression in the Pancreas

Interleukin (IL)-32 is a recently described proinflammatory cytokine characterized by the induction of nuclear factor (NF)-κB activation. We studied IL-32 expression in human pancreatic tissue and pancreatic cancer cell lines. Tissue samples were obtained surgically. IL-32 expression was evaluated by standard immunohistochemical procedures. IL-32 mRNA expression was analyzed by Northern blotting and real time PCR analyses. IL-32 was weakly immunoexpressed by pancreatic duct cells. In the inflamed lesions of chronic pancreas, the ductal expression of IL-32 was markedly increased. A strong expression of IL-32α was detected in the pancreatic cancer cells. In pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3 cells), the expression of IL-32 mRNA and protein was enhanced by IL-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. An inhibitor of phosphatidylinositol 3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) significantly suppressed the IL-1β-, IFN-γ- and TNF-α-induced IL-32 mRNA expression. The blockade of NF-κB and activated protein-1 activation markedly suppressed the IL-1β-, IFN-γ-, and/or TNF-α-induced IL-32 mRNA expression. Furthermore, IL-32-specific small interfering RNA significantly decreased the uptake of [³H]thymidine and increased the annexin V-positive population (apoptotic cells) in PANC-1 cells. IL-32 knockdown also suppressed the mRNA expression of antiapoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1). Pancreatic duct cells are the local source of IL-32, and IL-32 may play an important role in inflammatory responses and pancreatic cancer growth.     

Delivery of BR2‐SOX17 fusion protein can inhibit cell survival,proliferation, and invasion in gastric cancer cells through regulating Klotho gene expression

The prognosis of advanced gastric cancer is poor and understanding the biology and subsequent development of new targeting therapy is still an urgent need. This study was conducted to explore the effect of BR2 (a 17‐amino acid peptide)‐SOX17 (human sex determining region Y (SRY)‐related high‐mobility group (HMG) box protein family member 17) fusion protein on Klotho gene expression in gastric cancer cells. The regulatory effects of SOX17 on Klotho gene in gastric cancer cells were tested using dual‐luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. The therapeutic effects of BR2‐SOX17 were evaluated by proliferation, colony formation, invasion assay, and cell apoptosis analysis. Results showed that SOX17 enhanced Klotho gene expression in gastric adenocarcinoma cells through binding to the promoter of Klotho gene. BR2‐SOX17 fusion protein was effective in delivering SOX17 into gastric cancer cells and subsequently inhibited the cell proliferation, colony formation, and invasion, increased E‐cadherin protein expression, decreased vimentin protein expression, as well as induced apoptosis. Our findings suggested SOX17 can bind to the promoter of Klotho gene to enhance Klotho gene expression in gastric cancer cells. The fused BR2‐SOX17 protein is an effective agent for targeting therapy of gastric cancer.     

Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model

Chondroitin sulfate E (CS-E), a highly sulfated glycosaminoglycan, is known to promote tumor invasion and metastasis. Because the presence of CS-E is detected in both tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC), multistage involvement of CS-E in the development of PDAC has been considered. However, its involvement in the early stage of PDAC progression is still not fully understood. In this study, to clarify the direct role of CS-E in tumor, but not stromal, cells of PDAC, we focused on carbohydrate sulfotransferase 15 (CHST15), a specific enzyme that biosynthesizes CS-E, and investigated the effects of the CHST15 siRNA on tumor cell proliferation in vitro and growth in vivo. CHST15 mRNA is highly expressed in the human pancreatic cancer cell lines PANC-1, MIA PaCa-2, Capan-1 and Capan-2. CHST15 siRNA significantly inhibited the expression of CHST15 mRNA in these four cells in vitro. Silencing of the CHST15 gene in the cells was associated with significant reduction of proliferation and up-regulation of the cell cycle inhibitor-related gene p21^CIP1/WAF1. In a subcutaneous xenograft tumor model of PANC-1 in nude mice, a single intratumoral injection of CHST15 siRNA almost completely suppressed tumor growth. Reduced CHST15 protein signals associated with tumor necrosis were observed with the treatment with CHST15 siRNA. These results provide evidence of the direct action of CHST15 on the proliferation of pancreatic tumor cells partly through the p21^CIP1/WAF1 pathway. Thus, CHST15-CS-E axis-mediated tumor cell proliferation could be a novel therapeutic target in the early stage of PDAC progression.