Nobuo Ochi

CXCL8/IL-8 and CXCL12/SDF-1α Co-operatively Promote Invasiveness and Angiogenesis in Pancreatic Cancer

CXC‐chemokines are involved in the chemotaxis of neutrophils, lymphocytes and monocytes. However, role of these chemokines in tumorigenesis, especially with regard to interaction between tumor and its microenvironment, has not been clearly elucidated. The purpose of this study was to analyze the co‐operative role of CXCL8 and CXCL12 in the tumor‐stromal interaction in pancreatic cancer (PaCa). Using enzyme‐linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT‐PCR), we initially confirmed the expression of ligands and receptors, respectively, of CXC‐chemokines in PaCa and stromal cells. We examined the co‐operative role of CXCL8 and CXCL12 in proliferation/invasion of PaCa and human umbilical vein endothelial cells (HUVECs), and in HUVEC tube‐formations through tumor‐stromal interaction by MTS, Matrigel invasion, and angiogenesis assays, respectively. We detected expression of CXCR4, but not CXCR2, in all PaCa cells and fibroblasts. PaCa cells secreted CXCL8, and fibroblast cells secreted CXCL12. CXCL8 production in PaCa was significantly enhanced by CXCL12, and CXCL12 production in fibroblasts was significantly enhanced by co‐culturing with PaCa. CXCL8 enhanced proliferation/invasion of HUVECs but did not promote proliferation/invasion of PaCa. Both recombinant and PaCa‐derived CXCL8 enhanced tube formation of HUVECs that were co‐cultured with fibroblast cells. CXCL12 enhanced the proliferation/invasion of HUVECs and the invasion of PaCa cells but had no effect on tube formation of HUVEC. We showed that PaCa‐derived CXCL8 and fibroblast‐derived CXCL12 cooperatively induced angiogenesis in vitro by promoting HUVEC proliferation, invasion, and tube formation. Thus, corresponding receptors CXCR2 and CXCR4 are potential antiangiogenic and antimetastatic therapeutic targets in PaCa.

Loss of PTEN expression is associated with colorectal cancer liver metastasis and poor patient survival

BackgroundThe tumour suppressor phosphatase and tensin homolog (PTEN) is an important negative regulator of cell-survival signaling. To evaluate the correlation between PTEN expression and clinicopathological characteristics of colorectal cancer patients with and without liver metastases, we investigated PTEN expression in primary colorectal cancer and colorectal cancer liver metastases.MethodsSixty-nine pairs of primary colorectal cancer and corresponding liver metastasis specimens were analyzed immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. Seventy primary colorectal cancer specimens from patients without liver metastases were used as controls.ResultsPTEN was strongly expressed in 44 (62.9%) colorectal cancer specimens from patients without liver metastases. In contrast, PTEN was weakly expressed in 52 (75.4%) primary colorectal cancer specimens from patients with liver metastases, and was absent in liver metastases. Weak PTEN expression in colorectal cancer tissues was significantly associated with advanced TNM stage (p < 0.01) and lymph node metastasis (p < 0.05). PTEN expression was significantly stronger in primary colorectal cancer specimens from patients without liver metastases. Furthermore, among colorectal cancer patients with liver metastases, the 5-year survival rate was significantly higher in patients with positive PTEN expression compared to those with negative PTEN expression (p = 0.012).ConclusionOur results suggest that loss of PTEN expression is involved with colorectal cancer aggressive capacity and that diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for colorectal cancer patients.

Diagnostic accuracy of EUS in differentiating mucosal versus submucosal invasion of superficial esophageal cancers: a systematic review and meta-analysis

BACKGROUND The prognosis of esophageal cancer (EC) depends on the depth of tumor invasion and lymph node metastasis. EC limited to the mucosa (T1a) can be treated effectively with minimally invasive endoscopic therapy, whereas submucosal (T1b) EC carries relatively high risk of lymph node metastasis and requires surgical resection. OBJECTIVE To determine the diagnostic accuracy of EUS in differentiating T1a EC from T1b EC. DESIGN We performed a comprehensive search of MEDLINE, SCOPUS, Cochrane, and CINAHL Plus databases to identify studies in which results of EUS-based staging of EC were compared with the results of histopathology of EMR or surgically resected esophageal lesions. DerSimonian-Laird random-effects model was used to estimate the pooled sensitivity, specificity, and likelihood ratio, and a summary receiver operating characteristic (SROC) curve was created. SETTING Meta-analysis of 19 international studies. PATIENTS Total of 1019 patients with superficial EC (SEC). INTERVENTIONS EUS and EMR or surgical resection of SEC. MAIN OUTCOME MEASUREMENTS Sensitivity and specificity of EUS in accurately staging SEC. RESULTS The pooled sensitivity, specificity, and positive and negative likelihood ratio of EUS for T1a staging were 0.85 (95% CI, 0.82-0.88), 0.87 (95% CI, 0.84-0.90), 6.62 (95% CI, 3.61-12.12), and 0.20 (95% CI, 0.14-0.30), respectively. For T1b staging, these results were 0.86 (95% CI, 0.82-0.89), 0.86 (95% CI, 0.83-0.89), 5.13 (95% CI, 3.36-7.82), and 0.17 (95% CI, 0.09-0.30), respectively. The area under the curve was at least 0.93 for both mucosal and submucosal lesions. LIMITATIONS Heterogeneity was present among the studies. CONCLUSION Overall EUS has good accuracy (area under the curve ≥0.93) in staging SECs. Heterogeneity among the included studies suggests that multiple factors including the location and type of lesion, method and frequency of EUS probe, and the experience of the endosonographer can affect the diagnostic accuracy of EUS.

PTEN regulate angiogenesis through PI3K/Akt/VEGF signaling pathway in human pancreatic cancer cells

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.

A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth In vitro and In vivo

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-κB activation; and abrogated the expression of NF-κB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 μmol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67–positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive apoptotic cells (P < 0.05), and abrogated the expression of NF-κB–dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136–46. ©2010 AACR.

IGF-1 Mediates PTEN Suppression and Enhances Cell Invasion and Proliferation via Activation of the IGF-1/PI3K/Akt Signaling Pathway in Pancreatic Cancer Cells

BACKGROUND Type-1 insulin-like growth factor (IGF-1) up-regulates cell proliferation and invasiveness through activation of PI3K/Akt signaling pathway. IGF-1 also down-regulates the tumor suppressor chromosome 10 (PTEN). We investigated the mechanism by which IGF-1 affects cell proliferation and invasion by suppression of PTEN phosphorylation and interaction with PI3K/PTEN/Akt/NF-small ka, CyrillicB signaling pathway in pancreatic cancer. MATERIALS AND METHODS The expression of IGF-1 receptor (IGF-1R) and PTEN in five pancreatic cancer cell lines was determined by RT-PCR and Western blot. Proliferation and invasion were investigated by WST-1 assay and Matrigel-double chamber assay. Pancreatic cancer cells were transfected with PTEN siRNA to investigate which signaling pathway correlates in regulation of cancer cell proliferation and invasion. RESULTS Five pancreatic cancer cell lines expressed PTEN and IGF-1R in mRNA and protein levels. Suppression of PTEN phosphorylation strongly enhanced cell proliferation and invasion stimulated with IGF-1 via activation of PI3K/Akt/NF-small ka, CyrillicB signaling pathway. In addition, knockdown of PTEN by siRNA transfection also enhanced activation of PI3K/Akt/NF-small ka, CyrillicB pathway, subsequently up-regulating cell invasiveness and proliferation. CONCLUSIONS The IGF-1/PI3K/PTEN/Akt/NF-small ka, CyrillicB cascade may be a key pathway stimulating metastasis of pancreatic cancer cells. We suggest that interfering with the functions of IGF-1/PI3K/Akt/NF-small ka, CyrillicB might be a novel therapeutic approach to inhibit aggressive spread of pancreatic cancer.

The stem cell factor/c- kit receptor pathway enhances proliferation and invasion of pancreatic cancer cells

BackgroundThe transmembrane protein c-kit is a receptor tyrosine kinase (KIT) and KIT is expressed in solid tumors and hematological malignancies such as gastrointestinal stromal tumor (GIST), small-cell lung cancer and chronic myelogenous leukemia (CML). KIT plays a critical role in cell proliferation and differentiation and represents a logical therapeutic target in GIST and CML. In pancreatic cancer, c-kit expression has been observed by immunohistochemical techniques. In this study, we examined the influence of c-kit expression on proliferation and invasion using five pancreatic cancer cell lines. In addition, the inhibitory effect of imatinib mesylate on stem cell factor (SCF)-induced proliferation and invasion was evaluated. Finally, we also analyzed KIT and SCF expression in pancreatic cancer tissues using immunohistochemistry and correlated the results with clinical features.ResultsRT-PCR revealed that two pancreatic cancer cell lines, PANC-1 and SW1990, expressed c-kit mRNA. By Western blot analysis, c-kit protein was also present in those lines. In KIT-positive pancreatic cancer cell lines, proliferation and invasion were significantly enhanced by addition of SCF. In contrast, SCF did not enhance proliferation and invasion in the three KIT-negative lines (BxPC-3, Capan-2 and MIA PaCa-2). 5 μM imatinib mesylate significantly inhibited SCF-enhanced proliferation to the same extent compared with the control. Similarly, SCF-enhanced invasive ability was significantly inhibited by 5 μM imatinib mesylate. KIT was expressed in 16 of 42 clinical specimens by immunohistochemistry, and KIT expression was significantly related to venous system invasion. Furthermore, patients expressing both KIT and SCF had a somewhat lower survival.ConclusionOur results demonstrated that the SCF-KIT pathway enhanced the proliferation and invasiveness in KIT-positive pancreatic cancer cell lines and that the enhanced proliferation and invasion were inhibited by imatinib mesylate. We propose that inhibitors of c-kit tyrosine kinase receptor have the potential to slow the progression of KIT-positive pancreatic cancers.

Stem Cell Factor/c-kit Receptor Signaling Enhances the Proliferation and Invasion of Colorectal Cancer Cells Through the PI3K/Akt Pathway

In this study, we examined the role of c-kit receptor (KIT) signal transduction on the proliferation and invasion of colorectal cancer cells. We found that c-kit was expressed in 2 colorectal cancer cell lines as determined by RT-PCR, Western blot, and flow cytometry. In KIT-positive lines, KIT was activated by stem cell factor (SCF). SCF enhanced cellular proliferation of positive lines as demonstrated by the WST-1 proliferation assay. Furthermore, SCF enhanced the invasive ability of KIT-positive cell lines. SCF stimulation upregulated p44/42 mitogen-activated protein kinase (MAPK) and Akt as shown by Western blot. We examined the roles played by p44/42 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways in proliferation and invasion. PI3K/Akt activity strongly correlated with proliferation and invasion and p44/42 MAPK was correlated with only invasion. In conclusion, the SCF-enhanced proliferation and invasion of KIT-positive colorectal cancer cells is achieved mainly through the PI3K/Akt pathway.

IL‐1α secreted by colon cancer cells enhances angiogenesis: The relationship between IL‐1α release and tumor cells' potential for liver metastasis

Interleukin (IL)‐1α plays an important role in colon cancer progression and angiogenesis. We here asked whether IL‐1α derived from cancer cells modulates vascular endothelial cell growth, migration and tubule formation.

Protein kinase D1 promotes anchorage-independent growth, invasion, and angiogenesis by human pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases. Novel molecularly targeted therapies are urgently needed. Here, we extended our studies on the role of protein kinase D1 (PKD1) in PDAC cell lines. Given that Panc‐1 express moderate levels of PKD1, we used retroviral‐mediated gene transfer to create a Panc‐1 derivative that stably over‐expresses PKD1 (Panc‐1‐PKD1). Reciprocally, we used shRNA targeting PKD1 in Panc‐28 to produce a PKD1 under‐expressing Panc‐28 derivative (Panc‐28‐shPKD1). Our results demonstrate that Panc‐1‐PKD1 cells exhibit significantly increased anchorage‐independent growth in soft agar and increased in vitro invasion compared with Panc‐1‐mock. Reciprocally, Panc‐28‐shPKD1 cells show a significant decrease in anchorage‐independent growth and invasiveness, as compared with Panc‐28‐mock cells. The selective PKD family inhibitor CRT0066101 markedly decreased colony‐forming ability and invasiveness by either Panc‐1‐PKD1 or Panc‐28‐mock cells. Secretion of the pro‐angiogenic factors vascular endothelial growth factor (VEGF) and CXC chemokines (CXCL8) was significantly elevated by PKD1 over‐expression in Panc‐1 cells and reduced either by depletion of PKD1 via shRNA in Panc‐28 cells or by addition of CRT0066101 to either Panc‐1‐PKD1 or Panc‐28‐mock cells. Furthermore, human umbilical vein endothelial cell (HUVEC) tube formation was significantly enhanced by co‐culture with Panc‐1‐PKD1 compared with Panc‐1‐mock in an angiogenesis assay in vitro. Conversely, PKD1 depletion in Panc‐28 cells decreased their ability to induce endotube formation by HUVECs. PDAC‐induced angiogenesis in vitro and in vivo was markedly inhibited by CRT0066101. Our results lend further support to the hypothesis that PKD family members provide a novel target for PDAC therapy. J. Cell. Physiol. 226: 1074–1085, 2011.