Hiromitsu Takeyama

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.

Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction.

Background:Interleukin-6 (IL-6) has an important role in cancer progression, and high levels of plasma IL-6 are correlated with a poor prognosis in a variety of cancers. It has also been reported that tumour stromal fibroblasts are necessary for steps in cancer progression, such as angiogenesis. There have been few reports of a correlation between fibroblast actions and IL-6 levels. In this study, we examined the correlation between cancer stromal fibroblasts and IL-6 and the utility of IL-6 as a therapeutic target in human colon cancer.Methods:The expression levels of IL-6 and VEGF of fibroblasts and cancer cell lines were evaluated using real-time PCR and ELISA. The anti-angiogenic effect of inhibiting IL-6 signalling was measured in an angiogenesis model and animal experiment.Results:We demonstrate that stromal fibroblasts isolated from colon cancer produced significant amounts of IL-6 and that colon cancer cells enhanced IL-6 production by stromal fibroblasts. Moreover, IL-6 enhanced VEGF production by fibroblasts, thereby inducing angiogenesis. In vivo, anti-IL6 receptor antibody targeting stromal tissue showed greater anti-tumour activity than did anti-IL6 receptor antibody targeting xenografted cancer cells.Conclusion:Cancer stromal fibroblasts were an important source of IL-6 in colon cancer. IL-6 produced by activated fibroblasts induced tumour angiogenesis by stimulating adjacent stromal fibroblasts. The relationship between IL-6 and stromal fibroblasts offers new approaches to cancer therapy.

Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth

Cancer tissues are composed of cancer cells and the surrounding stromal cells (e.g., fibroblasts, vascular endothelial cells, and immune cells), in addition to the extracellular matrix. Most studies investigating carcinogenesis and the progression, invasion, metastasis, and angiogenesis of cancer have focused on alterations in cancer cells, including genetic and epigenetic changes. Recently, interactions between cancer cells and the stroma have attracted considerable attention, and increasing evidence has accumulated on this. Several researchers have gradually clarified the origins, features, and roles of cancer-associated fibroblasts (CAFs), a major component of the cancer stroma. CAFs function in a similar manner to myofibroblasts during wound healing. We previously reported the relationship between CAFs and angiogenesis. Interleukin-6 (IL-6), a multifunctional cytokine, plays a central role in regulating inflammatory and immune responses, and important roles in the progression, including proliferation, migration, and angiogenesis, of several cancers. We showed that CAFs are an important IL-6 source and that anti-IL-6 receptor antibody suppressed angiogenesis and inhibited tumor-stroma interactions. Furthermore, CAFs contribute to drug-resistance acquisition in cancer cells. The interaction between cancer cells and the stroma could be a potential target for anti-cancer therapy.

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.

Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation

BackgroundInteraction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1α and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.ResultsIn immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and β1 integrin. Enhancement of FAK protein association with β1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1α. Preincubation with anti-β1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with β1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1α of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1α stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1α-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, β1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1α-induced enhancement of adhesion and invasion in pancreatic cancer cells.ConclusionOur results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.

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.

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.

Experimental implication of celiac ganglionotropic invasion of pancreatic-cancer cells bearing c-ret proto-oncogene with reference to glial-cell-line-derived neurotrophic factor (GDNF)

Perineural invasion is a prominent clinical feature of pancreatic cancer which causes difficulty in curative resection. In the present study, the human pancreatic cancer cell lines, PaCa‐2, AsPC‐1, SW1990 and Capan‐2, were all found to express abundant c‐ret proto‐oncogene mRNA and RET protein, a member of the receptor‐tyrosine‐kinase superfamily, identified as being a receptor for glial‐cell‐line‐derived neurotrophic factor (GDNF). In an invasion assay, the migration of pancreatic cancer cells was markedly induced by co‐cultivation with human glioma cells, T98G or A172, capable of producing and secreting GDNF. Anti‐GDNF antibody in conditioned media of glioma cells suppressed much of the migratory activity. Checkerboard analysis of the migration showed both chemotactic and chemokinetic activity of GDNF. There was no detectable expression of another GDNF receptor component, a glycosyl‐phosphatidylinositol‐linked receptor (GFRα‐1), in pancreatic‐cancer cell lines, suggesting that the neural invasion of pancreatic‐cancer cells spreads along a concentration gradient of GDNF produced from peripheral ganglions through direct interaction of GDNF with its receptor, the c‐ret proto‐oncogene product. Immunochemical localization of GDNF in human celiac ganglionic tissue supported this contention. Int. J. Cancer 81:67–73, 1999.

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.