A novel role for carcinoembryonic antigen-related cell adhesion molecule 6 as a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.     

Overexpression of CEACAM6 promotes insulin-like growth factor I-induced pancreatic adenocarcinoma cellular invasiveness

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-linked immunoglobulin superfamily member that is overexpressed in a variety of human cancers. We have recently reported that suppression of CEACAM6 expression impairs pancreatic adenocarcinoma progression in vivo. In order to characterize the mechanisms through which CEACAM6 influences the malignant phenotype, CEACAM6-overexpressing Capan2 pancreatic adenocarcinoma cells were established by stable transfection. We determined the effect of CEACAM6 overexpression on cellular invasiveness towards insulin-like growth factor I (IGF-I), a peptide of critical importance in pancreatic cancer malignant cellular behavior and tumor progression. IGF-I-induced cellular invasiveness and IGF-IR expression were significantly increased in clones overexpressing CEACAM6. Using inhibitory anti-IGF-IR antibody, a requirement for IGF-IR signaling in the enhanced invasiveness towards IGF-I induced by CEACAM6 overexpression was confirmed. CEACAM6-overexpressing clones exhibited increased Akt and c-Src kinase activities, as well as higher levels of matrix metalloproteinase-2 (MMP-2) expression and activity in the presence of IGF-I. While Akt kinase is both necessary and sufficient to induce IGF-IR upregulation, c-Src kinase activity is necessary, but alone is insufficient to upregulate IGF-IR expression. CEACAM6 is an important determinant of pancreatic adenocarcinoma malignant cellular behavior and, together with its downstream targets, warrants further investigation as a therapeutic target in this disease.     

EphA2: a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human cancers. We sought to characterize the role of EphA2 in pancreatic adenocarcinoma and, using RNA interference (RNAi) mediated by small interfering RNA (siRNA), we determined the effects of suppressing EphA2 expression in vitro and in vivo. EphA2 expression in PANC1, MIAPaCa2, BxPC3 and Capan2 cells was assessed by Northern and Western blot. We artificially overexpressed EphA2 by transient transfection and suppressed EphA2 expression using RNAi. Cellular invasiveness was quantified by modified Boyden chamber assay. Anoikis was induced by anchorage-independent polyHEMA culture and caspase 3 activity was quantified fluorometrically. Focal adhesion kinase (FAK) phosphorylation was assessed by immunoprecipitation. EphA2 siRNA treatment was assessed in a nude mouse xenograft model. Pancreatic adenocarcinoma cells differentially express EphA2. Inherent and induced EphA2 overexpression is associated with increased cellular invasiveness and anoikis resistance. EphA2 siRNA suppresses EphA2 expression, cellular invasiveness, anoikis resistance and FAK phosphorylation in vitro and retards tumor growth and inhibits metastasis in vivo. EphA2 is both a determinant of malignant cellular behavior and a potential therapeutic target in pancreatic adenocarcinoma.     

CEACAM6 gene silencing impairs anoikis resistance and in vivo metastatic ability of pancreatic adenocarcinoma cells

Anoikis is the apoptotic response induced in normal cells by inadequate or inappropriate adhesion to substrate. It is postulated that resistance to anoikis facilitates tumorigenesis and metastasis. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member overexpressed in a number of human cancers and implicated in anoikis resistance. We tested the effect of CEACAM6 gene silencing on anoikis in pancreatic adenocarcinoma cell lines. Anoikis was induced in PANC1, Capan2, MiaPaCa2 and Mia(AR) (a MiaPaCa2-derived anoikis-resistant subline) by culture in poly-2-hydroxyethylmethacrylate-coated wells. Anoikis was quantified by YO-PRO-1/propidium iodide staining and flow cytometry. The role of caspase activation was determined using fluorometric profiling and the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk). CEACAM6 expression was suppressed by RNA interference. Using a nude mouse orthotopic xenograft model, we assessed the effect of this treatment on in vivo metastatic ability. Anoikis resistance was associated with increased CEACAM6 expression. CEACAM6-specific short interfering ribonucleic acid (siRNA), but not control siRNA, increased susceptibility to caspase-mediated anoikis, an effect abrogated by Z-VAD-fmk, and decreased Akt phosphorylation (Ser-473) under anchorage-independent conditions. CEACAM6 gene silencing reversed the acquired anoikis resistance of Mia(AR) and inhibited its in vivo metastatic ability. CEACAM6 warrants further investigation as a novel therapeutic target for the treatment of pancreatic adenocarcinoma.     

Gemcitabine‐induced epithelial‐mesenchymal transition‐like changes sustain chemoresistance of pancreatic cancer cells of mesenchymal‐like phenotype

Growing body of evidence suggests that epithelial‐mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT‐like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC‐3, Capan‐2, Panc‐1, and MiaPaca‐2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine‐resistant Panc‐1 and MiaPaca‐2 cells of mesenchymal‐like phenotype undergo further EMT‐like molecular changes mediated by ERK‐ZEB‐1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB‐1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine‐resistant BxPC‐3 and Capan‐2 cells of epithelial‐like phenotype did not show such typical EMT‐like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB‐1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT‐like changes that sustain invasion and chemoresistance in PC cells.     

Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation,migration, invasion and angiogenesis of pancreatic cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC₅₀s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5 μM, 8 μM and 12 nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6.     

Context-dependent function of the deubiquitinating enzyme USP9X in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly malignancies. Recently, the deubiquitinating protease USP9X has been shown to behave as an oncogene in a number of neoplasms, including those of breast, brain, colon, esophagus and lung, as well as KRAS wild-type PDAC. However, other studies suggest that USP9X may function as a tumor-suppressor in a murine PDAC model when USP9X expression is depleted during early pancreatic development. To address the conflicting findings surrounding the role of USP9X in PDAC, we examined the effects of knocking down USP9X in five human PDAC cell lines (BxPC3, Capan1, CD18, Hs766T, and S2-013). We demonstrate that knocking down USP9X in each of the PDAC cell lines reduces their anchorage-dependent growth. Using an inducible shRNA system to knock down USP9X in both BxPC3 and Capan1 cells, we also determined that USP9X is necessary for the anchorage-independent growth. In addition, knockdown of USP9X alters the cell cycle profile of BxPC3 cells and increases their invasive capacity. Finally, we show that an inhibitor of deubiquitinating proteases, WP1130, induces significant cytotoxicity in each of the five PDAC cell lines tested. Overall, our work and the work of others indicate that the function and role of USP9X is highly context-dependent. Although USP9X may function as a tumor-suppressor during the establishment of PDAC, data presented here argue that USP9X promotes cell growth in advanced PDAC cells when PDAC is typically diagnosed. Hence, USP9X may be a promising therapeutic target for the treatment of advanced PDAC.     

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6 pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6 pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6 pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX-2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6 pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.     

Expression and activity of SRC regulate interleukin-8 expression in pancreatic adenocarcinoma cells: implications for angiogenesis

Interleukin-8 (IL-8) is an angiogenic factor that promotes growth of pancreatic tumors. The purpose of this study was to determine if c-Src, a protein tyrosine kinase frequently overexpressed in pancreatic cancer, regulated IL-8 expression and to elucidate the Src-mediated signaling pathways that contribute to angiogenesis in pancreatic adenocarcinoma cells. In a panel of pancreatic cancer cell lines, expression of total and activated Src correlated with IL-8 production. Furthermore, ectopic expression of activated Src in PANC-1 cells with low endogenous Src activity significantly increased IL-8 production (P < 0.005). In contrast, pharmacologic inhibition of endogenous c-Src kinase activity or small interfering RNA-mediated "knockdown" of c-Src expression in L3.6pl cells with high Src expression and activity caused significant decreases in IL-8 production (P < 0.005). Inhibition of c-Src activity resulted in decreased phosphorylation of Akt, p38, and extracellular signal-regulated kinase (Erk)-1/2. Significant (P < 0.005) dose-dependent decreases were observed in IL-8 expression by inhibiting Src-dependent signaling molecules Erk-1/2 and p38 but not phosphatidylinositol 3-kinase. To assess the relevance of Src inhibition to angiogenesis, in vivo gelfoam assays were done. Robust infiltration of vessels was observed in gelfoam saturated with conditioned medium from pancreatic carcinoma cells. This angiogenesis was nearly abrogated in gelfoams saturated with conditioned medium from cells treated with the Src family kinase inhibitor, PP2 (P < 0.001). Thus, c-Src regulates critical "downstream" signaling pathways that contribute to expression of IL-8 in human pancreatic tumor cells, suggesting c-Src may be a target for therapeutic intervention in pancreatic adenocarcinoma.     

Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma

The expression of the Wilms' tumor gene WT1 was examined by immunohistochemistry in 40 cases of pancreatic ductal adenocarcinoma. WT1 protein was expressed in 30 (75%) of the 40 pancreatic ductal adenocarcinomas, but not in the remaining 10 (25%). In normal pancreatic ductal cells, WT1 protein was undetectable. No correlations between WT1 expression and clinicopathological parameters such as age, sex, T or N stage, tumor location, and tumor differentiation were observed. Treatment with WT1 antisense oligomers significantly inhibited the growth of five human pancreatic cancer cell lines, PSN1, MiaPaCa2, ASPC1, BxPC3, and PCI6, expressing the WT1 gene. These results indicate an important role of the WT1 gene in the tumorigenesis of pancreatic ductal adenocarcinoma expressing WT1 and provide a rationale for new treatment strategies to treat pancreatic ductal adenocarcinoma by targeting the WT1 gene and its product.     

JDP2 inhibits the epithelial-to-mesenchymal transition in pancreatic cancer BxPC3 cells

Pancreatic carcinoma is one of the most malignant and aggressive cancers. Increased motility and invasiveness of pancreatic cancer cells are believed to be associated with epithelial-to-mesenchymal transition (EMT). However, the molecular basis of EMT in pancreatic cancer cells is poorly understood. In this study, we examined the relationship between Jun dimerization protein 2 (JDP2), which is an AP-1 inhibitor, and EMT in human pancreatic carcinoma cells. We demonstrated that transforming growth factor-??1 (TGF-??1) promoted epidermal growth factor (EGF)-induced EMT in co-treated human pancreatic BxPC3 cells and that JDP2 overexpression reversed the EMT that was induced by co-treatment with TGF-??1 and EGF. These results suggest that EGF plays a principal role in EMT through its association with TGF-??1 in human pancreatic BxPC3 cells and that JDP2 may be a molecular target for pancreatic carcinoma intervention.     

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer

Even with current promising antitumor antibodies, their antitumor effects on stroma‐rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa‐2; moderate, BxPC‐3; and abundant, Capan‐1 and Ope‐xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra‐abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa‐2 model, moderate (1063) in the BxPC‐3 model, and negative in the Capan‐1 and Ope‐xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8‐fold (2980, 3015) in the BxPC‐3 model, 2.5‐ or 4.8‐fold (1881, 3615) in the Capan‐1 model, and 3.2‐ or 4.2‐fold (1469, 1922) in the Ope‐xeno model, respectively. Cetuximab was effective in treating even stroma‐rich and k‐ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.     

Overexpression of CEACAM6 promotes migration and invasion of oestrogen-deprived breast cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast and lung and is associated with tumourigenesis, tumour cell adhesion, invasion and metastasis. In this study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERalpha)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation, and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6-15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced phosphorylated Akt and c-Src expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen-deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.     

PKCα-induced drug resistance in pancreatic cancer cells is associated with transforming growth factor-β1

Background

Drug resistance remains a great challenge in the treatment of pancreatic cancer. The goal of this study was to determine whether TGF-β1 is associated with drug resistance in pancreatic cancer.

Methods

Pancreatic cancer BxPC3 cells were stably transfected with TGF-β1 cDNA. Cellular morphology and cell cycle were determined and the suppressive subtracted hybridization (SSH) assay was performed to identify differentially expressed genes induced by TGF-β1. Western blotting and immunohistochemistry were used to detect expression of TGF-β1-related genes in the cells and tissue samples. After that, the cells were further treated with an anti-cancer drug (e.g., cisplatin) after pre-incubated with the recombinant TGF-β1 plus PKCα inhibitor Gö6976. TGF-β1 type II receptor, TβRII was also knocked down using TβRII siRNA to assess the effects of these drugs in the cells. Cell viability was assessed by MTT assay.

Results

Overexpression of TGF-β1 leads to a markedly increased invasion potential but a reduced growth rate in BxPC3 cells. Recombinant TGF-β1 protein increases expression of PKCα in BxPC3 cells, a result that we confirmed by SSH. Moreover, TGF-β1 reduced the sensitivity of BxPC3 cells to cisplatin treatment, and this was mediated by upregulation of PKCα. However, blockage of PKCα with Gö6976 and TβRII with siRNA reversed the resistance of BxPC3 cells to gemcitabine, even in the presence of TGF-β1. Immunohistochemical data show that pancreatic cancers overexpress TGF-β1 and P-gp relative to normal tissues. In addition, TGF-β1 expression is associated with P-gp and membranous PKCα expression in pancreatic cancer.

Conclusions

TGF-β1-induced drug resistance in pancreatic cancer cells was associated with PKCα expression. The PKCα inhibitor Gö6976 could be a promising agent to sensitize pancreatic cancer cells to chemotherapy.     

Expression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in breast cancers

Carcino-embryonic antigen-related cell adhesion molecule 6 (CEACAM6), one of the members of human carcino-embryonic antigens, is a multifunctional regulatory protein involved in various cellular processes in cancers. Its role in malignant transformation and the clinical significance has been extensively studied in colonic and pancreatic cancers. However, relatively few studies have been done on breast cancers. In the current study, CEACAM6 expression in two independent cohorts of invasive breast cancers were evaluated immunohistochemically and correlated with clinico-pathological features, biomarker profiles and patient survival. In the primary cohort, CEACAM6 expression was detected in 37.1 % (312/840) of primary invasive cancers. It was positively correlated with HER2 (p < 0.001). Concordantly, HER2-OE subtype showed the highest CEACAM6 expression (62.7 %) among all molecular subtypes; whereas, other subtypes also showed substantial CEACAM6 expression (21.8–37.5 %). Interestingly, a significantly worse overall survival was found in high pN stage HER2 positive cancers with CEACAM6 positivity (log-rank = 4.452, p = 0.035) and this could be validated in an independent cohort. Additionally, HER2 signaling was found to induce SMAD3 phosphorylation and CEACAM6 expression in a cell line model. Likewise, in the primary tumors, a positive association was found between HER2 and SMAD3 phosphorylation in CEACAM6 positive cancers (p = 0.012). Overall, CEACAM6 was widely expressed in different molecular subtypes, but highest and significantly in HER2-OE breast cancer. Within this group, CEACAM6 was associated with adverse high nodal stage patient outcome. Given the wide expression of CEACAM6 in all breast cancers, its roles as prognostic marker and therapeutic target warrant further evaluation.