Mutations in the PIK3CA gene, which encodes the p110A catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been reported in human cancers, including colorectal cancer. Most of the mutations cluster at hotspots within the helical and kinase domains. Whereas H1047R, one of the hotspot mutants, is reported to have elevated lipid kinase activity, the functional consequences of other mutations have not been examined. In this study, we examined the effects of colon cancer-associated PIK3CA mutations on the lipid kinase activity in vitro, activation of the downstream targets Akt and p70S6K in vivo and NIH 3T3-transforming ability. Of eight mutations examined, all showed increased lipid kinase activity compared with wild-type p110A. All the mutants strongly activated Akt and p70S6K compared with wild-type p110A as determined by immunoblotting using phospho-specific antibodies. These mutants also induced morphologic changes, loss of contact inhibition, and anchorage-independent growth of NIH 3T3 cells. The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities. These results show that almost all the colon cancer-associated PIK3CA mutations are functionally active so that they are likely to be involved in carcinogenesis. (Cancer Res 2005; 65(11): 4562-7)

The interaction between the chemokine receptor CXCR4 and its specific ligand, stromal cell-derived factor-1 (SDF-1/ CXCL12), mediates several cellular functions. In cancer, SDF-1-positive or CXCR4-positive cells of various lineages are detected within tumor tissues. Recent intensive research has indicated the possibility that blocking CXCR4 could reduce the metastatic potential of cancer cells. Here, we show that the inhibition of the SDF-1/CXCR4 axis decreases the growth of s.c. gastrointestinal tumors through the suppression of tumor neoangiogenesis. The neutralization of CXCR4 suppressed the growth in vivo of tumors derived from mouse Colon38 and PancO2 cells, whereas it did not affect the growth of Colon38 and PancO2 cells in vitro. This attenuation of tumor growth was found to be independent of the expression of CXCR4 by the cancer cells themselves, because CXCR4 knocked-down Colon38 cells grew similarly to control cells. Furthermore, CD31-positive tumor capillaries were reduced to 45% (P < 0.001) and intratumor blood flows were decreased to 65% (P < 0.01) by blockade of CXCR4. The vascular endothelial growth factor (VEGF) concentration in the tumors was not affected by the neutralization of CXCR4. Taken together with the detection of CXCR4-positive endothelial cells in the tumor tissues, the findings suggest that the antiangiogenic effects of the blockade of CXCR4 are related to a reduction of the establishment of tumor endothelium independently of VEGF inhibition. Our data indicate that the SDF-1/CXCR4 pathway might be a general target for anticancer strategies and that blocking this system could be cooperatively effective in combination with other antiangiogenic therapies, such as blockade of VEGF. (Cancer Res 2005; 65(13): 5864-71)

Circulatory antigens transit through the small intestine via the fenestrated capillaries in the lamina propria prior to entering into the draining lymphatics. But whether or how this process controls mucosal immune responses remains unknown. Here we demonstrate that dendritic cells (DCs) of the lamina propria can sample and process both circulatory and luminal antigens. Surprisingly, antigen cross-presentation by resident CX3CR1+ DCs induced differentiation of precursor cells into CD8+ T cells that expressed interleukin-10 (IL-10), IL-13 and IL-9 and could migrate into adjacent compartments. We conclude that lamina propria CX3CR1+ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally-absorbed-antigens, leading to the induction of CD8+ T cells, that partake in the control of T cell activation during mucosal immune responses.

Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

The transforming growth factor-b (TGF-b)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-b-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-b and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-b. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-b stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-b. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-b was inhibited in the S4KD cells, which might be associated with a different regulation of integrin b7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

The activation of Hedgehog (Hh) signaling has been implicated in the growth of various tumor types, including gastric carcinoma. However, the precise mechanisms of Hh activation and suppression of tumor growth by the blockade of Hh signaling in gastric carcinoma cells remain unknown. The aim of this study was to elucidate the mechanism of abnormal Hh signaling and the key molecules contributing to dysregulated growth of gastric carcinoma. The Sonic hedgehog (Shh) ligand and its receptor Patched were expressed in all five gastric carcinoma cell lines examined (MKN1, MKN7, MKN45, MKN74, and AGS cells). The blockade of Hh signaling with anti-Shh antibody inhibited the growth of all five gastric carcinoma cell lines. Shh was overexpressed (mean, 12.8-fold) in 8 of 14 (57.0%) cancerous tissue samples from patients with gastric carcinoma as compared with expression in the surrounding noncancerous tissues. The disruption of glioma-associated oncogene 1 (Gli1) by small interfering RNA induced an increase in p21/cyclin-dependent kinase-interacting protein 1 (CIP1), interfered with the G 1 -S transition, and suppressed cell proliferation. The stimulation or inhibition of Hh signaling did not affect p53 activity and the induction of p21/CIP1 expression and the G 1 arrest by inhibition of Hh signaling were not affected by the p53 status. These findings suggest that the overexpression of Shh contributes to constitutive Hh activation and that this signaling pathway negatively regulates p21/CIP1 through a Gli1-dependent and p53-independent mechanism in gastric carcinoma cells. (Cancer Res 2005; 65(23): 10822-9)

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