5290

PIK3CA

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. Although the exact mechanisms driving hypoxia-induced invasion and metastasis remain elusive, we hypothesized that epithelial-mesenchymal transition (EMT) may play a major role. We investigated this in vitro by treating hepatocellular carcinoma cells under 1.0% O(2). After the hypoxia treatment, the cells exhibited some morphological changes including cell elongation, cytoskeletal rearrangement, and junctional disruption. Moreover, expression of the epithelia-specific marker E-cadherin was decreased and expression of the myofibroblast-specific marker vimentin was detected in the treated cells. Cell migration and ECM gel invasion were increased. These findings were consistent with events observed during EMT. Hypoxia-induced EMT is accompanied by increased phosphorylation, activation of Akt and the downstream signaling. Hypoxia-induced EMT was blocked by PI3K inhibitor LY294002. The results suggest that the PI3K/Akt-dependent signaling pathways serve to regulate hypoxia-induced EMT of hepatocellular carcinoma cells.

Acquisition of resistance to tamoxifen is a critical therapeutic problem in breast cancer patients. epithelial-mesenchymal transition (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, is associated with invasion and motility of cancer cells. Here, we found that tamoxifen-resistant (TAMR)-MCF-7 cells had undergone EMT, as evidenced by mesenchymal-like cell shape, downregulation of basal E-cadherin expression, and overexpression of N-cadherin and vimentin, as well as increased Snail transcriptional activity and protein expression. Given the roles of glycogen synthase kinase (GSK)-3beta and nuclear factor (NF)-kappaB in Snail-mediated E-cadherin deregulation during EMT, we examined the role of these signaling pathways in the EMT of TAMR-MCF-7 cells. Both Ser9-phosphorylated GSK-3beta (inactive form) and NF-kappaB reporter activity were increased in TAMR-MCF-7 cells, as was activation of the phosphatase and tensin homolog depleted on chromosome ten (PTEN)-phosphoinositide 3 (PI3)-kinase-Akt pathway. Pin1, a peptidyl-prolyl isomerase, was overexpressed in TAMR-MCF-7 cells, and Snail transcription and the expression of EMT markers could be decreased by Pin1 siRNA treatment. These results imply that Pin1 overexpression in TAMR-MCF-7 cells is involved in the EMT process via PTEN-PI3-kinase-Akt-GSK-3beta and/or GSK-3beta-NF-kappaB-dependent Snail activation, and suggest the potential involvement of Pin1 in EMT during breast cancer development.

Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-beta1 (TGF-beta1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-beta1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-beta1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-beta1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-beta1-induced EMT of A549 cells.

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the advanced stage when the cancer cells have already migrated and invaded into other tissues and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is essential to the design of effective therapy. In this study, we investigated the function of scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration and invasion, and downregulates E-cadherin expression in ovarian cancer cells with low-Gab2 expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By expressing Gab2 wild-type and Gab2 mutants that are defective in activation of the PI3K and Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes characteristics of EMT in ovarian cancer cells.

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-beta1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-beta1 in that, unlike TGF-beta1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85alpha action, and co-immunoprecipitation of SARA and p85alpha confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85alpha knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85alpha knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle alpha-actin. Together, these data suggest that although TGF-beta1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

Chemokine (C-C motif) ligand 18 (CCL18), which is derived from tumour-associated macrophages (TAMs), plays a critical role in promoting breast cancer metastasis via its receptor, PYK2 N-terminal domain interacting receptor 1 (Nir1). However, the molecular mechanism by which Nir1 promotes breast cancer metastasis by binding to CCL18 remains elusive. In this study, Nir1 expression was associated with lymph node and distant metastasis in patients with invasive ductal carcinoma. For the first time, we report that Nir1 binding to CCL18 promotes the phosphorylation of Akt, LIN-11, Isl1 and MEC-3 protein domain kinase (LIMK), and cofilin, which is a critical step in cofilin recycling and actin polymerisation. Interestingly, Nir1 binding to CCL18 can enhance cell mesenchymal properties and induce epithelial-mesenchymal transition (EMT). Mechanistically, Nir1 binding to CCL18 stabilises Snail via the Akt/GSK3beta signalling pathway. In support of these observations, Nir1 binding to CCL18 promoted lung metastasis and LY294002 could inhibit it in vivo. In summary, our in vitro and in vivo results indicate that Nir1 binding to CCL18 plays an important role in breast cancer invasion/metastasis. This study identified both Nir1 and CCL18 as potential anti-invasion targets for therapeutic intervention in breast cancer.CI - Copyright (c) 2013 Elsevier Ltd. All rights reserved.

Breast cancer already taken the first place of incidence in Chinese female cancer patients. TRPM8 is found to be over-expressed in breast cancer, but whether it promotes breast cancer aggressiveness remains unknown. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR-75-30, while it just could be detected in MCF-10A, the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore expression status of cancer-related proteins. The Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3beta pathway, and the hypothesis had been supported by cell function tests. All the results demonstrated that TRPM8 significantly up-expressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/GSK-3beta pathway, indicating TRPM8 gets the prospects of to be developed as medication or diagnostic indicator to be applied in clinical work.

The zinc finger protein 143 (ZNF143) is a transcription factor, which regulates many cell cycle-associated genes. ZNF143 expressed strongly in multiple solid tumors. However, the influence of ZNF143 on gastric cancer (GC) remains largely unknown. In this study, we investigated the ZNF143 mRNA level in GC tissues and cells by quantitative real-time PCR (qRT-PCR). The protein expression of ZNF143 in GC cells, and the signaling pathway proteins were detected by Western blotting. Transwell assay and wound healing assay were performed to explore the effects of ZNF143 for the migration ability of GC cells in vitro. We also performed the tail vein injection in nude mice with GC cells to explore the impact of ZNF143 on GC metastasis in vivo. ZNF143 was overexpressed in specimens of GC compared with adjacent normal tissues and increased more significantly in GC tissues of patients who had lymph node metastasis. Ectopic overexpression of ZNF143 enhanced GC migration, whereas ZNF143 knockdown suppressed this effect in vitro. In vivo, ZNF143 knockdown reduced distant metastasis of GC cells in nude mice. In addition, overexpression of ZNF143 reduced the expression of epithelial cell marker (E-cadherin) and induced the expression of mesenchymal cell marker (N-cadherin,Vimentin), Snail and Slug. We also found that ZNF143 enhanced GC cell migration by promoting the process of EMT through PI3K/AKT signaling pathway. In general, our findings show that ZNF143 expressed strongly in GC and enhanced migration of GC cells in vitro and in vivo. It is conceivable that ZNF143 could be a therapeutic genetic target for GC treatment.

Colorectal cancer(CRC) is one of the most commonly diagnosed cancers in human beings and metastasis is the main death reason. Recently, Gli1 has been reported to be a key regulator of various cancer biologies and genes expressions. However, the detailed molecular mechanism of Gli1 in CRC metastasis remains largely unknown. In this study, we aimed to investigate the role of Gli1 in CRC metastasis. We used qRT-PCR, Immunohistochemistry and Western blot to test the expression levels of Gli1, Foxm1 and other target genes in the tissues and cells; Lentivirus stable transfection to change the expression levels of Gli1 and Foxm1; Wound-healing, cell invasion, migration assays and tail vein metastatic assay to test the role of Gli1 in CRC metastasis in vitro and vivo. We demonstrated that Gli1 was significantly overexpressed in colorectal cancer tissues and cells. Foxm1 level had a positive correlation with Gli1. Furthermore, we found that Gli1 promotes colorectal cancer cells metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling. Thus, we proved that Gli1 plays important role in CRC metastasis and provided a new visual field on the therapy of CRC metastasis.

G proteincoupled receptor 56 (GPR56), a member of the orphan GPCR family, has been reported to be an oncogene in various malignancies. However, little is known regarding the detailed molecular mechanism of GPR56 in colorectal cancer (CRC). The present study aimed to detect the expression level and biological function of GPR56 in CRC. We examined the expression of GPR56 in CRC tissues and cell lines by quantitative real time (qRT)PCR, immunohistochemistry, and western blot analysis. The prognostic significance of GPR56 in CRC patients was evaluated by KaplanMeier survival analysis. The influence of GPR56 on tumor cell proliferation (via Cell Counting Kit8, and a tumor formation assay in mice), apoptosis (flow cytometry), cell cycle distribution (flow cytometry) and migration (Transwell assay) was explored. We also investigated the underlying mechanism of GPR56 by western blot analysis. We found GPR56 expression was significantly upregulated in CRC tissues and cell lines compared to corresponding normal controls. Higher GPR56 expression in patients predicted poorer prognosis. Depletion of GPR56 markedly suppressed cell proliferation, migration, and invasion. GPR56 overexpression promoted CRC cell metastasis by expediting epithelialmesenchymal transition by activating PI3K/AKT signaling. In conclusion, GPR56 played an important role in CRC progression and may represent a new therapeutic target to reduce CRC metastasis.

Melanoma is one of the most aggressive skin cancers and is well known for its high metastatic rate. Studies have shown that epithelial mesenchymal transition (EMT) is essential for melanoma cell metastasis. However, the molecular mechanisms underlying EMT are still not fully understood. We have shown that IRGM1, a member of immunity-related GTPase family that regulates immune cell motility, is highly expressed by melanoma cells. The current study aimed to explore whether and how IRGM1 may regulate melanoma cell metastasis. To test this, we modified IRGM1 expression in B16 melanoma cells. We found that over-expression of IRGM1 substantially enhanced pulmonary metastasis in vivo. In keeping with that, knocking-in IRGM1 strongly enhanced while knocking-down IRGM1 impaired B16 cell migration and invasion ability in vitro. Interestingly, we observed that IRGM1 enhanced F-actin polymerization and triggers epithelial mesenchymal transition (EMT) through a mechanism involved in PIK3CA mediated Rac1 activation. Together, these data reveals a novel molecular mechanism that involved in melanoma metastasis.