Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Epidermal Growth Factor (EGF)-enhanced Vascular Cell Adhesion Molecule-1 (VCAM-1) Expression Promotes Macrophage and Glioblastoma Cell Interaction and Tumor Cell Invasion

Activated EGF receptor (EGFR) signaling plays an instrumental role in glioblastoma (GBM) progression. However, how EGFR activation regulates the tumor microenvironment to promote GBM cell invasion remains to be clarified. Here, we demonstrate that the levels of EGFR activation in tumor cells correlated with the levels of macrophage infiltration in human GBM specimens. This was supported by our observation that EGFR activation enhanced the interaction between macrophages and GBM cells. In addition, EGF treatment induced up-regulation of vascular cell adhesion molecule-1 (VCAM-1) expression in a PKCϵ- and NF-κB-dependent manner. Depletion of VCAM-1 interrupted the binding of macrophages to GBM cells and inhibited EGF-induced and macrophage-promoted GBM cell invasion. These results demonstrate an instrumental role for EGF-induced up-regulation of VCAM-1 expression in EGFR activation-promoted macrophage-tumor cell interaction and tumor cell invasion and indicate that VCAM-1 is a potential molecular target for improving cancer therapy.     

Functional proteomic analysis reveals that fungal immunomodulatory protein reduced expressions of heat shock proteins correlates to apoptosis in lung cancer cells

BackgroundLing Zhi-8 (LZ-8) and GMI are two fungal immunomodulatory proteins (FIPs) with a similar structure and amino acid sequence and are respectively obtained from the medicinal mushroom Ganoderma lucidum and Ganoderma microsporum. They present the anti-cancer progression and metastasis. We previously demonstrated that LZ-8 reduces the tumor progression in lung cancer LLC1 cell-bearing mouse. However, it is unclear whether these FIPs induce changes in the protein expression profile in cancer cells and the mechanism for such a process is not defined.PurposeThis study determines the changes in the proteomic profile for tumor lesions of LLC1 cell-bearing mouse received with LZ-8 and the potential mechanism for FIPs in anti-lung cancer cells.MethodsThe proteomic profile of tumor lesions was determined using two-dimensional electrophoresis and a LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). The biological processes and the signaling pathway enrichment analysis were performed using Ingenuity Pathway Analysis (IPA). The differentially expressed proteins were verified by Western blot. Cell viability was determined by MTT assay. Cell morphology was characterized using electron microscopy. Migration was detected using the Transwell assay. The apoptotic response was determined using Western blot and flow cytometry.ResultsObtained results showed that 21 proteins in the tumor lesions exhibited differential (2-fold change, p < 0.05) expression between PBS and LZ-8 treatment groups. LZ-8-induced changes in the proteomic profile that may relate to protein degradation pathways. Specifically, three heat shock proteins (HSPs), HSP60, 70 and 90, were significantly downregulated in tumor lesions of LLC1-bearing mouse received with LZ-8. Both LZ-8 and GMI reduced the protein levels for these HSPs in lung cancer cells. Functional studies showed that they inhibited cell migration but effectively induced apoptotic response in LLC1 cells in vitro. In addition, the inhibitors of HSP60 and HSP70 effectively inhibited cell migration and decreased cell viability of LLC1 cells.ConclusionsLZ-8 induced changes in the proteomic profile of tumor lesions which may regulate the HSPs-related cell viability. Moreover, inhibition of HSPs may be related to the anti-lung cancer activity.     

Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells

E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12935-015-0159-3) contains supplementary material, which is available to authorized users.     

Delphinidin inhibits epidermal growth factor-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma cells

Epithelial-to-mesenchymal transition (EMT), important cellular process in metastasis of primary tumors, is characterized by loss of their cell polarity, disruption of cell-cell adhesion, and gain certain properties of mesenchymal phenotype that enable migration and invasion. Delphinidin is a member of anthocyanidin belong to flavonoid groups, known as having pharmacological and physiological effects including anti-tumorigenic, antioxidative, anti-inflammatory, and antiangiogenic effects. However, the effects of delphinidin on EMT is rarely investigated. Epidermal growth factor (EGF) is known as a crucial inducer of EMT in various cancer including hepatocellular carcinoma (HCC). To determine whether delphinidin inhibits EGF-induced EMT in HCC cells, antiproliferative effect of delphinidin on Huh7 and PLC/PRF/5 cells were measured by Cell Counting Kit-8 assay. As a result, delphinidin inhibited cell proliferation in a dose-dependent manner. Based on the result of proliferation, to measure the effects of delphinidin on EGF-induced EMT, we designated a proper concentration of delphinidin, which is not affected to cell proliferation. We found that delphinidin inhibits morphological changes from epithelial to mesenchymal phenotype by EGF. Moreover, delphinidin increased the messenger RNA and protein expression of E-cadherin and decreased those of Vimentin and Snail in EGF-induced HCC cells. Also, delphinidin prevented motility and invasiveness of EGF-induced HCC cells through suppressing activation of matrix metalloproteinase 2, EGF receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK). Taken together, our findings demonstrate that delphinidin inhibits EGF-induced EMT by inhibiting EGFR/AKT/ERK signaling pathway in HCC cells.     

Deguelin attenuates non-small cell lung cancer cell metastasis through inhibiting the CtsZ/FAK signaling pathway

Lung cancer is the leading cause of cancer-related death among both men and women every year, mainly due to metastasis. Although natural compound deguelin has been reported to inhibited cell migration and invasion in various cancer cells, the details of this regulation progress remain to be fully elucidated. In this study, we investigated the underlying mechanism of deguelin-suppressed metastasis of non-small cell lung cancer (NSCLC) cells. Our results demonstrate that deguelin inhibits NSCLC cell migration, invasion, and metastasis both in vitro and in vivo. These inhibitory effects of deguelin were mediated by suppressing of Cathepsin Z (CtsZ) expression and interrupting the interaction of CtsZ with integrin β3. Moreover, deguelin inhibits the activation of CtsZ downstream FAK/Src/Paxillin signaling. Knockdown of CtsZ mimicked the effect of deguelin on NSCLC cells migration and invasion. Our study reveals that deguelin exerts its anti-metastatic effect both in vitro and in vivo is partly dependent on the suppression of CtsZ signaling. Deguelin would be a potential anti-metastasis agent against NSCLC.     

Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.     

Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src,NADPH Oxidase,PI3K,and Akt in Human Colon Cancer Cells

Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47^phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.     

AKT2 contributes to increase ovarian cancer cell migration and invasion through the AKT2-PKM2-STAT3/NF-κB axis

Multiple studies have shown that protein kinase Bβ (AKT2) is involved in the development and progression of ovarian cancer, however, its precise role remains unclear. Here we explored the underlying molecular mechanisms how AKT2 promotes ovarian cancer progression. We examined the effects of AKT2 in vitro in two ovarian cancer cell lines (SKOV3 and HEY), and in vivo by metastasis assay in nude mice. The migration and invasion ability of SKOV3 and HEY cells was determined by transwell assay. Overexpression and knockdown (with shRNA) experiments were carried out to unravel the underlying signaling mechanisms induced by AKT2. Overexpression of AKT2 led to increased expression of pyruvate kinase (PKM2) in ovarian cancer cells and in lung metastatic foci from nude mice. Elevated AKT2/PKM2 expression induced cell migration and invasion in vitro, as well as lung metastasis in vivo; silencing AKT2 blocked these effects. Meanwhile, PKM2 overexpression was unable to increase AKT2 expression. The expressions of p-PI3K, p-AKT2, and PKM2 were increased when stimulated by epidermal growth factor (EGF); however, these expressions were blocked when inhibited the PI3K by LY294002. STAT3 expression was elevated and NF-κB p65 nuclear translocation was activated both in vitro and in vivo when either AKT2 or PKM2 was overexpressed; and these effects were inhibited when silencing AKT2 expression. Taken together, AKT2 increases the migration and invasion of ovarian cancer cells in vitro and promotes lung metastasis in nude mice in vivo through PKM2-mediated elevation of STAT3 expression and NF-κB activation. In conclusion, we highlight a novel mechanism of the AKT2-PKM2-STAT3/NF-κB axis in the regulation of ovarian cancer progression, and our work suggested that both AKT2 and PKM2 may be potential targets for the treatment of ovarian cancer.     

The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.     

AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program.     

GMI,an immunomodulatory protein from Ganoderma microsporum,induces autophagy in non-small cell lung cancer cells

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.     

Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium

The ovarian surface epithelium (OSE) is the precursor of common epithelial ovarian carcinomas. In the present study, we examined the molecular mechanisms and possible physiological basis for the propensity of OSE cells to undergo epithelio-mesenchymal transition (EMT) in response to environmental influences. We hypothesized that EMT may be a homeostatic mechanism that permits displaced OSE to assume a stromal phenotype within the ovarian cortex. We report that EGF in conjunction with hydrocortisone is the EMT-inducing factor of OSE as shown by changes to a fibroblast-like morphology and growth pattern. EGF increased cell motility, enhanced the activities of secreted pro-matrix metalloproteinase (MMP)-2 and -9, and enhanced expression and activation of Erk and integrin-linked kinase (ILK). Increased ILK expression correlated with the activation of PKB/Akt, the phosphorylation of GSK-3, and the increased expression of cyclin E and cdk2 kinase. EGF withdrawal resulted in a more epithelial morphology and reversal of the EGF-induced activation of signaling pathways and pro-MMP activity. In contrast, treatment of EGF-treated cells with specific inhibitors of phosphatidylinositol 3-kinase, Mek, or ILK inhibited the inhibitor-specific pathways. The inhibitors caused suppression of EGF-induced migration and pro-MMP-2/-9 activities but did not lead to any change in EGF-induced mesenchymal morphology. ILK small interfering RNA inhibited Akt phosphorylation and reduced pro-MMP-2/-9 activities but had no effect on Erk activation or cell morphology. These results indicate that the EGF-induced morphological and functional changes in OSE cells are controlled by distinct signaling mechanisms working in concert. EMT of OSE cells displaced by ovulation likely permits their survival and integration with a fibroblast-like identity within the stroma. Failure to do so may lead to the formation of epithelium-derived inclusion cysts, which are known preferential sites of malignant transformation. epidermal growth factor; migration; invasion     

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.     

Combination treatment of Src inhibitor Saracatinib with GMI,a Ganoderma microsporum immunomodulatory protein,induce synthetic lethality via autophagy and apoptosis in lung cancer cells

Saracatinib is an oral Src‐kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed‐resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase‐7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B‐II in A400 cells. ATG5 silencing abolished the caspase‐7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib.