Thiruvengadam Arumugam

Cancer-Associated Stromal Fibroblasts Promote Pancreatic Tumor Progression

Pancreatic adenocarcinoma is characterized by a dense background of tumor associated stroma originating from abundant pancreatic stellate cells. The aim of this study was to determine the effect of human pancreatic stellate cells (HPSC) on pancreatic tumor progression. HPSCs were isolated from resected pancreatic adenocarcinoma samples and immortalized with telomerase and SV40 large T antigen. Effects of HPSC conditioned medium (HPSC-CM) on in vitro proliferation, migration, invasion, soft-agar colony formation, and survival in the presence of gemcitabine or radiation therapy were measured in two pancreatic cancer cell lines. The effects of HPSCs on tumors were examined in an orthotopic murine model of pancreatic cancer by co-injecting them with cancer cells and analyzing growth and metastasis. HPSC-CM dose-dependently increased BxPC3 and Panc1 tumor cell proliferation, migration, invasion, and colony formation. Furthermore, gemcitabine and radiation therapy were less effective in tumor cells treated with HPSC-CM. HPSC-CM activated the mitogen-activated protein kinase and Akt pathways in tumor cells. Co-injection of tumor cells with HPSCs in an orthotopic model resulted in increased primary tumor incidence, size, and metastasis, which corresponded with the proportion of HPSCs. HPSCs produce soluble factors that stimulate signaling pathways related to proliferation and survival of pancreatic cancer cells, and the presence of HPSCs in tumors increases the growth and metastasis of these cells. These data indicate that stellate cells have an important role in supporting and promoting pancreatic cancer. Identification of HPSC-derived factors may lead to novel stroma-targeted therapies for pancreatic cancer.

S100P Promotes Pancreatic Cancer Growth, Survival, and Invasion

Purpose: In the current study, we examined the functional significance and mechanism of action of S100P in pancreatic cancer cells. Experimental Design: S100P levels were increased in Panc-1 cells, which do not express S100P, by transfection with an S100P cDNA and S100P levels were reduced in BxPC3 cells, which express high levels of S100P, by small interfering RNA gene silencing. Effects of these manipulations on cell proliferation, resistance to apoptotic insults, cell migration, and invasion were estimated in vitro using standard assays. The influences of S100P on tumor growth in vivo were studied using xenograft mouse models. To identify the mechanisms involved in these responses, coimmunoprecipitation studies were conducted with S100P with receptor for advanced glycation end products (RAGE) and the effects of inhibiting RAGE using an antagonistic peptide were analyzed. Results: S100P levels correlated with the rates of cell proliferation, survival, migration, and invasion in both cell models in vitro. In vivo, increased S100P levels increased the growth of tumors in mice with s.c.-implanted Panc-1 cells and decreased S100P levels decreased tumor growth after orthotopic implantation of BxPC-3 cells. A direct interaction between S100P and RAGE was indicated by coimmunoprecipitation of these molecules from pancreatic cancer cells. A RAGE antagonist peptide inhibited this interaction and also inhibited the biological effects of S100P on these cells in vitro. Conclusions: These data suggest that S100P plays a major role in the aggressiveness of pancreatic cancer that is likely mediated by its ability to activate RAGE. Thus, interference with S100P may provide a novel approach for treatment of pancreatic cancer.

RAGE and RAGE Ligands in Cancer

The receptor for advanced glycation end-products (RAGE) is a multifunctional receptor with multiple ligands that is known to play a key role in several diseases, including diabetes, arthritis, and Alzheimers disease. Recent evidence indicates that this receptor also has an important role in cancer. RAGE ligands, which include the S100/calgranulins and high-mobility group box 1 (HMGB1) ligands, are expressed and secreted by cancer cells and are associated with increased metastasis and poorer outcomes in a wide variety of tumors. These ligands can interact in an autocrine manner to directly activate cancer cells and stimulate proliferation, invasion, chemoresistance, and metastasis. RAGE ligands derived from cancer cells can also influence a variety of important cell types within the tumor microenvironment, including fibroblasts, leukocytes, and vascular cells, leading to increased fibrosis, inflammation, and angiogenesis. Several of the cells in the tumor microenvironment also produce RAGE ligands. Most of the cancer-promoting effects of RAGE ligands are the result of their interaction with RAGE. However, these ligands also often have separate intracellular roles, and some may interact with other extracellular targets, so it is not currently possible to assign all of their effects to RAGE activation. Despite these complications, the bulk of the evidence supports the premise that the ligand-RAGE axis is an important target for therapeutic intervention in cancer.

Therapeutic Targeting of Neuropilin-2 on Colorectal Carcinoma Cells Implanted in the Murine Liver

BACKGROUND Neuropilin-2 (NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor (VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers. METHODS Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immunoblotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice (groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA (siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided. RESULTS NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs (at 28 days after implantation, mean control siRNAs = 420 mm3, mean NRP2 siRNAs = 36 mm3, NRP2 vs control: difference = 385 mm3, 95% confidence interval = 174 mm3 to 595 mm3, P = .005). CONCLUSION NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.

Nuclear factor-κB maintains TRAIL resistance in human pancreatic cancer cells

Although it displays promising activity in other tumor models, the effects of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on human pancreatic cancer cells have not been comprehensively explored. We report that a majority of human pancreatic cancer cell lines (seven of nine) underwent apoptosis when they were exposed to recombinant human TRAIL in vitro. Characterization of surface TRAIL receptors by fluorescence-activated cell sorting showed that TRAIL-resistant cells (Panc-1 and HS766T) expressed lower levels of DR4 and DR5 than did TRAIL-sensitive cells. The proteasome inhibitor bortezomib (PS-341, Velcade) further increased TRAIL responsiveness in the TRAIL-sensitive cells and synergized with TRAIL to reverse resistance in Panc-1 and HS776T cells. The effects of bortezomib were mimicked by transfection with a small interfering RNA construct specific for the p65 subunit of nuclear factor-κB (NF-κB) or exposure to a selective chemical inhibitor of IKK (PS-1145). Silencing IκBα prevented TRAIL sensitization by PS-1145, confirming that IκBα mediated the effects of PS-1145. NF-κB inhibition resulted in down-regulation of BCL-XL and XIAP, and silencing either restored TRAIL sensitivity in TRAIL-resistant cells. Finally, therapy with TRAIL plus PS-1145 reversed TRAIL resistance in vivo to produce synergistic growth inhibition in orthotopic Panc-1 tumors. Together, our results show that NF-κB inhibits TRAIL-induced apoptosis in human pancreatic cancer cells and suggest that combination therapy with TRAIL and NF-κB inhibitors, such as bortezomib, PS-1145, or curcumin, should be considered as a possible treatment strategy in patients with pancreatic cancer. [Mol Cancer Ther 2006;5(9):2251–60]

RAGE Activation by S100P in Colon Cancer Stimulates Growth, Migration, and Cell Signaling Pathways

PurposeColon cancer is the third most prevalent cancer in the United States. However, the molecular mechanisms involved in the development and progression of colon cancer are incompletely understood. This study was initiated to explore the potential role of the receptor for advanced glycation end-products and S100P in modulation of key properties of human colon cancer cells.MethodsWestern blot, reverse transcription-polymerase chain reaction, and quantitative polymerase chain reaction were performed for detection of the receptor for advanced glycation end-products and S100P in colon cancer and matched normal colon. The influence of exogenously added S100P was analyzed on SW480 colon cancer cell line proliferation, migration, phosphorylation of mitogen activated protein kinases, and NFκB activation. To identify the mechanisms involved in these responses, coimmunoprecipitation examining the S100P/Receptor for advanced glycation end-products interaction and the effects of receptor for advanced glycation end-products inhibition in this interaction were analyzed.ResultsAlthough the receptor for advanced glycation end-products was present in normal and malignant colon specimens, only the malignant specimens expressed S100P. Treatment of SW480 cells with S100P increased proliferation and cell migration. Addition of exogenous S100P stimulated both ERK1/2 phosphorylation and NFκB activity. The interaction between S100P and the receptor for advanced glycation end-products was demonstrated by coimmunoprecipitation of these molecules from SW480 cells. Antagonism of the receptor for advanced glycation end-products blocked this interaction and the biologic effects of S100P on these cells.ConclusionsThese data indicate that S100P is expressed at greater levels in colon cancer than matched normal tissue and that S100P stimulates colon cancer cell growth, migration, Erk phosphorylation, and NFκB activation in vitro, suggesting that this ligand/receptor pair may be targeted for the development of new therapies.

CEACAM1, a novel serum biomarker for pancreatic cancer

Objectives: Serum biomarkers for early diagnosis of pancreatic adenocarcinoma are not currently available. We recently observed elevated expression of CEACAM1 in pancreatic adenocarcinomas and sought to determine whether serum CEACAM1 levels were elevated in pancreatic cancer patients. Methods: CEACAM1 messenger RNA levels were measured in pancreatic tissue samples using quantitative reverse transcription-polymerase chain reaction. CEACAM1 was localized by immunohistochemistry in adenocarcinomas and in pancreatic intraductal neoplasia lesions. CEACAM1 serum levels were assessed by a double determinant enzyme-linked immunosorbent assay and compared with serum levels of CA19-9. Results: CEACAM1 had higher expression levels in pancreatic adenocarcinomas compared with noncancerous pancreas (P < 0.0001) and was localized to neoplastic cells (95% (45/47) of adenocarcinomas and 85% (17/20) of pancreatic intraductal neoplasia 3 lesions. CEACAM1 was expressed in the sera of 91% (74/81) of pancreatic cancer patients, 24% (15/61) of normal patients, and 66% (35/53) of patients with chronic pancreatitis, with a sensitivity and specificity superior to CA19-9. The combination of CEACAM1 and CA19-9 had significantly higher diagnostic accuracy than CA19-9. Conclusions: CEACAM1 is expressed in pancreatic adenocarcinoma, and serum levels of CEACAM1 serve as a useful indicator for the presence of pancreatic cancer. Additional validation studies on the use of serum CEACAM1 as a diagnostic marker in pancreatic cancer are warranted.Abbreviations: AUC - area under the curve, ROC - receiver operator characteristics

Anterior gradient 2 is expressed and secreted during the development of pancreatic cancer and promotes cancer cell survival

Pancreatic cancer is a major oncological challenge due to its aggressive growth and metastasis. In the current study, we investigated the role of anterior gradient 2 (AGR2) in these processes. AGR2 mRNA, as assessed by quantitative real-time reverse transcription-PCR (Q-RT-PCR), was 14-fold higher in pancreatic cancer compared with normal and pancreatitis tissues. Immunohistochemistry revealed high expression of AGR2 in neoplastic cells with 98% (56 of 57) positivity on pancreatic cancer and minimal staining in normal and pancreatitis tissues. AGR2 was also expressed in early pancreatic intraepithelial neoplastic lesions. RT-PCR and Western blotting showed elevated AGR2 expression in seven of nine pancreatic cancer cell lines. AGR2, as detected in conditioned media from cancer cells, indicated that it was secreted. The influence of AGR2 on pancreatic cancer cells was evaluated by silencing with small interfering RNA and short hairpin RNA. Silencing of AGR2 significantly reduced cell proliferation (MTS assay) and invasion (Boyden chamber assay) and improved gemcitabine sensitivity (fluorescence-activated cell sorting analysis). Conditioned media from cells in which AGR2 was silenced had a reduced ability to stimulate proliferation of pancreatic cancer cells, suggesting that secreted AGR2 was active. In vivo, silencing of AGR2 in MPanc-96 cells led to a significant reduction of tumor growth and increased the effectiveness of gemcitabine treatments in orthotopic tumor models evaluated by noninvasive bioluminescence imaging. In summary, AGR2 is expressed and secreted during pancreatic cancer development and plays an important role in cancer cell growth and survival. These observations suggest that AGR2 may be a useful molecular target in pancreatic cancer.

Hematogenous Metastasis of Ovarian Cancer: Rethinking Mode of Spread

Ovarian cancer has a clear predilection for metastasis to the omentum, but the underlying mechanisms involved in ovarian cancer spread are not well understood. Here, we used a parabiosis model that demonstrates preferential hematogenous metastasis of ovarian cancer to the omentum. Our studies revealed that the ErbB3-neuregulin 1 (NRG1) axis is a dominant pathway responsible for hematogenous omental metastasis. Elevated levels of ErbB3 in ovarian cancer cells and NRG1 in the omentum allowed for tumor cell localization and growth in the omentum. Depletion of ErbB3 in ovarian cancer impaired omental metastasis. Our results highlight hematogenous metastasis as an important mode of ovarian cancer metastasis. These findings have implications for designing alternative strategies aimed at preventing and treating ovarian cancer metastasis.

Nuclear Factor-κB p65/relA Silencing Induces Apoptosis and Increases Gemcitabine Effectiveness in a Subset of Pancreatic Cancer Cells

Purpose: Nuclear factor κB (NFκB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFκB activity may be prognostic, and inhibition of NFκB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFκB activity and the effects of inhibiting NFκB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine. Experimental Design: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFκB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFκB activity were determined in vitro and in vivo. NFκB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity. Results: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFκB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFκB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo. Conclusions: NFκB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFκB may be a useful therapeutic target for a subset of pancreatic cancers.