EGF-induced Cell Migration Research Articles

Beta-Catenin Downregulation Contributes to Epidermal Growth Factor-induced Migration and Invasion of MDAMB231 Cells

We previously demonstrated that epidermal growth factor (EGF) enhances cell migration and invasion of breast cancer cells in a SMAD ubiquitination regulatory factor 1 (SMURF1)-dependent manner and that SMURF1 induces degradation of β-catenin in C2C12 cells. However, the relationship between EGF-induced SMURF1 and β-catenin expression in breast cancer cells remains unclear. So, we investigated if EGF and SMURF1 regulate β-catenin expression in MDAMB231 human breast cancer cells. When MDAMB231 cells were incubated with EGF for 24, 48, and 72 hours, EGF significantly increased expression levels of SMURF1 mRNA and protein while suppressing expression levels of β-catenin mRNA and protein. Overexpression of SMURF1 downregulated β-catenin mRNA and protein, whereas knockdown of SMURF1 increased β-catenin expression and blocked EGF-induced β-catenin downregulation. Knockdown of β-catenin enhanced cell migration and invasion of MDAMB231 cells, while β-catenin overexpression suppressed EGF-induced cell migration and invasion. Furthermore, knockdown of β-catenin enhanced vimentin expression and decreased cytokeratin expression, whereas β -catenin overexpression decreased vimentin expression and increased cytokeratin expression. These results suggest that EGF downregulates β-catenin in a SMURF1-dependent manner and that β-catenin downregulation contributes to EGF-induced cell migration and invasion in MDAMB breast cancer cells.

Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase-AKT-Rac1 axis.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

Inhibition of EGFR Signaling by N-cyclohexyl-2-(1-(phenylsulfonyl)piperidin-4-yl) acetamide.

The epidermal growth factor receptor (EGFR) is a driver oncogene and specific blockade of EGFR has been shown to be an effective therapeutic approach against multiple human cancers. Here we employed the homogeneous time-resolved fluorescence (HTRF) technology to screen new EGFR mediators. 4 hits (NDS-41107, NDS-41119, NDS-41111 and NDS-41126) were confirmed in a compound library of 8000 compounds, and the IC50 values were determined to be 15.45±2.25µM (NDS-41107), 6.16±0.88 µM (NDS-41119), 11.33±3.31 µM (NDS-41111) and 11.19±1.67µM (NDS-41126), respectively. We then showed that NDS-41119 (N-cyclohexyl-2-(1-(phenylsulfonyl) piperidin-4-yl) acetamide) significantly inhibited EGFR signaling in human lung cancer cells, as evidenced by decreased phosphorylation of EGFR、ERK and Akt. NDS-41119 also attenuated EGF-induced cell proliferation and migration in a dose-dependent manner. We finally demonstrated that NDS-41119 inhibited the T790M mutation in NCI-H1975 cells and potentiated the effect of gefitinib against resistant cells. Our results will contribute to the development of novel EGFR-targeted anti-cancer drugs.

Correction: EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration.

Volume 400(2), pages 225–234 In Figure 5 of the original paper, the authors mistakenly duplicated some panels. The authors have repeated the experiment and a new Figure 5 is shown here. Figure 5. AQP3 knockdown reduces EGF-induced AQP3 expression and cell migration in human skin fibroblasts. Cells were treated with EGF (100 ng/ml) …

Abstract 5840: Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer

Abstract Background: KRAS mutant colorectal cancers (CRC) are resistant to many targeted therapies including cetuximab. Novel molecular targets for KRAS mutant tumors may enhance the efficacy of chemoradiation. C-MET plays important roles in treatment resistance, tumor invasion, and metastasis. In this study, we used a small molecule inhibitor of c-MET, crizotinib in cetuximab resistant mutant KRAS driven CRC cell lines and assessed radiosensitization. Methods: To test the effect of c-MET inhibition on CRC cells we used the KRAS mutant cell lines HCT116, DLD1, and LoVo. Colony formation assays were performed to assess the effects of crizotinib and cetuximab. Immunoblot analysis was used to determine the effect of crizotinib on c-MET and downstream pathways including ERK and AKT phosphorylation. We then selected doses of crizotinib below the IC50 and assessed clonogenic survival with radiation. To study potential mechanisms of radiosensitization cell cycle analysis was performed using flow cytometry. The effect of c-MET inhibition on cellular function was assessed using cell migration assays. Results: We first performed a series of clonogenic survival assays with crizotinib and cetuximab to determine the IC50 for each cell line. As expected, the KRAS mutant cell lines LoVo, HCT116, and DLD1 were resistant to cetuximab (surviving fraction greater than 95% at 100 μg/ml). These cell lines were sensitive to the c-MET inhibitor crizotinib with IC50 values of 14.4 nM for LoVo, 316 nM for HCT166, and 217 nM for DLD1. Immunoblot analysis showed that crizotinib at concentrations near the IC50 for each cell line attenuated c-MET phosphorylation and downstream signaling pathways including ERK and AKT. Crizotinib further blocked radiation induced c-MET phosphorylation and attenuated activation of downstream signaling pathways. We then performed a series of clonogenic survival assays to study the radiosensitizing potential of crizotinib. Pretreatment with crizotinib for 24 hours radiosensitized LoVo and DLD1 cell lines with enhancement ratios of 1.66 and 1.25, respectively. Interestingly, treatment with crizotinib for 1 hour prior to radiation had only modest dose enhancement, suggesting that prolonged exposure to crizotinib is required for optimal radiosensitization. Cell cycle analysis revealed minimal G1 arrest with crizotinib. Additionally, crizotinib completely blocked HGF and EGF induced cell migration. Conclusions: Inhibition of c-MET with crizotinib effectively sensitizes cetuximab resistant KRAS mutant CRC cell lines to radiation. Given the important role c-MET plays in treatment resistance, crizotinib has the potential to improve the outcome of patients with locally advanced rectal cancer undergoing radiation therapy. Citation Format: Kyle C. Cuneo, Himabindu Kurapati, Ranjit Mehta, Theodore S. Lawrence, Mukesh K. Nyati. Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5840. doi:10.1158/1538-7445.AM2017-5840

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration

BackgroundPenetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process.MethodsFor our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development.ResultsOur results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland.ConclusionTogether, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

Chitosan oligosaccharide inhibits EGF-induced cell growth possibly through blockade of epidermal growth factor receptor/mitogen-activated protein kinase pathway

Chitosan oligosaccharide (COS) has been shown to regulate various cellular and biological functions. Epidermal growth factor (EGF) plays a significant role in tumorigenesis and invasiveness of some cancers. The aim of this study was to evaluate the effects of COS on EGF-induced cell growth and the further mechanisms. The results demonstrated that COS could inhibit EGF-induced epithelial GE11 cells proliferation in a dose dependent manner. In addition, EGF stimulated the epithelial cells to undergo morphological alteration, exhibiting mesenchymal cells higher metastatic and invasive potential, however, COS could partly suppress aforementioned morphological change. Signal transduction studies indicated COS repressed epidermal growth factor receptor (EGFR) phosphorylation and mitogen-activated protein kinase (MAPK) activation, but not Grb2, Ras, and Raf. Taken together, chitosan oligosaccharide inhibited EGF-induced cell growth and migration through blockade of the EGFR/MAPK signal transduction pathway.

TGF-β Effects on Prostate Cancer Cell Migration and Invasion Require FosB.

Activator Protein-1 (AP-1) family (cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2) plays a central role in the transcriptional regulation of many genes that are associated with cell proliferation, differentiation, migration, metastasis, and survival. Many oncogenic signaling pathways converge at the AP-1 transcription complex. Transforming growth factor beta (TGF-β) is a multifunctional regulatory cytokine that regulates many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. This study investigated, the role of FOS proteins in TGF-β signaling in prostate cancer cell proliferation, migration, and invasion. Steady state expression levels of FOS mRNA and proteins were determined using RT-PCR and western blotting analyses. DU145 and PC3 prostate cancer cells were exposed to TGF-β1 at varying time and dosage, RT-PCR, western blot, and immunofluorescence analyses were used to determine TGF-β1 effect on FOS mRNA and protein expression levels as well as FosB subcellular localization. Transient silencing of FosB protein was used to determine its role in cell proliferation, migration, and invasion. Our data show that FOS mRNA and proteins were differentially expressed in human prostate epithelial (RWPE-1) and prostate cancer cell lines (LNCaP, DU145, and PC3). TGF-β1 induced the expression of FosB at both the mRNA and protein levels in DU145 and PC3 cells, whereas cFos and Fra1 were unaffected. Immunofluorescence analysis showed an increase in the accumulation of FosB protein in the nucleus of PC3 cells after treatment with exogenous TGF-β1. Selective knockdown of endogenous FosB by specific siRNA did not have any effect on cell proliferation in PC3 and DU145 cells. However, basal and TGF-β1- and EGF-induced cell migration was significantly reduced in DU145 and PC3 cells lacking endogenous FosB. TGF-β1- and EGF-induced cell invasion were also significantly decreased after FosB knockdown in PC3 cells. Our data suggest that FosB is required for migration and invasion in prostate cancer cells. We also conclude that TGF-β1 effect on prostate cancer cell migration and invasion may be mediated through the induction of FosB. Prostate 77:72-81, 2017. © 2016 Wiley Periodicals, Inc.

JWA loss promotes cell migration and cytoskeletal rearrangement by affecting HER2 expression and identifies a high-risk subgroup of HER2-positive gastric carcinoma patients.

Background and AimsJWA, a microtubule-associated protein (MAP) involved in apoptosis, has been identified as a suppressor of metastasis, and it affects cell migration in melanoma and its downregulation in tumor is an idependent negative prognostic factor in resectable gastric cancer. HER2 overexpression has been observed in gastric cancer (GC) cells and implicated in the metastatic phenotype. However, the biological role of JWA in migration and its clinical value in HER2-positive GC remain elusive.ResultsJWA suppresses EGF-induced cell migration and actin cytoskeletal rearrangement by abrogating HER2 expression and downstream PI3K/AKT signaling in HER2-overexpressing GC cell lines. The modulation of HER2 by JWA is dependent on ERK activation and consequent PEA3 upregulation and activation. Reduced JWA expression is associated with high HER2 expression and with poor survival in patients with AGC, whereas HER2 expression alone is not associated with survival. However, concomitant low JWA and high HER2 expression is associated with unfavorable outcomes. Additionally, when patients were stratified by JWA expression, those with higher HER2 expression in the low JWA expression subgroup exhibited worse survival.MethodsThe impact of JWA on the EGF-induced migration of HER2-positive GC cells was studied using transwell assays and G-LISA assays. Western blotting, real-time PCR, electrophoretic mobility shift assays and luciferase assays were utilized to investigate the mechanisms by which JWA affects HER2. The association of JWA with HER2 and its clinical value were further analyzed by IHC in 128 pairs of advanced gastric cancer (AGC) and adjacent normal tissue samples.ConclusionsThis study characterizes a novel mechanism for regulating cell motility in HER2-overexpressing GC cells involving JWA-mediated MEK/ERK/PEA3 signaling activation and HER2 downregulation. Furthermore, JWA may be a useful prognostic indicator for advanced GC and may help stratify HER2-positive patient subgroups to better identify unfavorable outcomes.

MIG6 Is MEK Regulated and Affects EGF-Induced Migration in Mutant NRAS Melanoma

Activating mutations in NRAS are frequent driver events in cutaneous melanoma. NRAS is a GTP-binding protein, whose most well-characterized downstream effector is RAF leading to activation of MEK-ERK1/2 signaling. While there are no FDA-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we utilized a high-throughput reverse-phase protein array (RPPA) platform to identify signaling alterations. RPPA analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in AKT signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR/ERBB receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of EGF-induced signaling and cell migration and invasion. In MEK inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma especially in response to EGF stimulation.

Lysine-specific demethylase 1 mediates epidermal growth factor signaling to promote cell migration in ovarian cancer cells.

Epigenetic abnormalities play a vital role in the progression of ovarian cancer. Lysine-specific demethylase 1 (LSD1/KDM1A) acts as an epigenetic regulator and is overexpressed in ovarian tumors. However, the upstream regulator of LSD1 expression in this cancer remains elusive. Here, we show that epidermal growth factor (EGF) signaling upregulates LSD1 protein levels in SKOV3 and HO8910 ovarian cancer cells overexpressing both LSD1 and the EGF receptor. This effect is correlated with a decrease in the dimethylation of H3K4, a major substrate of LSD1, in an LSD1-dependent manner. We also show that inhibition of PI3K/AKT, but not MEK, abolishes the EGF-induced upregulation of LSD1 and cell migration, indicating that the PI3K/PDK1/AKT pathway mediates the EGF-induced expression of LSD1 and cell migration. Significantly, LSD1 knockdown or inhibition of LSD1 activity impairs both intrinsic and EGF-induced cell migration in SKOV3 and HO8910 cells. These results highlight a novel mechanism regulating LSD1 expression and identify LSD1 as a promising therapeutic target for treating metastatic ovarian cancer driven by EGF signaling.

Abstract 506: TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival

Abstract Glioblastoma (GBM) is a highly lethal CNS malignancy in adults with a median survival of about 15 months. Effective therapeutic control of GBM is thwarted by the invasive nature of the tumor, which prevents complete surgical removal and results in tumor recurrence. Thus, there is a dire need to develop innovative approaches to target the invasive tumor cells for improved treatment of this disease. Expression of TROY, a member of the TNFR family, increases with glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. We have demonstrated that TROY forms a novel complex with EGFR and modulates EGFR survival signaling. IHC analysis of GBM specimens showed that tumors with elevated TROY expression had a statistically positive correlation with increased EGFR expression. TROY expression enhanced EGFR phosphorylation, stabilized EGFR surface expression in the presence of ligand, and significantly increased EGF-stimulated GBM cell invasion. Consistent with invasion, co-expression of TROY with EGFR increased cell survival to TMZ. Moreover, we have shown that in cells treated with EGF, the TROY and ErbB4 receptor are recruited to the EGFR complex. Although the role of ErbB4 in GBM is not well defined, inhibition of ErbB4 expression suppressed tumor growth and invasion in multiple tumor subtypes, decreased EGFR surface retention, and limited EGF-induced cell migration. These data suggest that the TROY-EGFR-ErbB4 complex may represent an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance. Citation Format: Alison Roos, Zachary Mayo, Jean Kloss, Serdar Tuncali, Harshil Dhruv, Michael E. Berens, Joseph C. Loftus, Nhan L. Tran. TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2015-506

Phosphorylation of phosphatidylinositol 4-phosphate 5-kinase γ by Akt regulates its interaction with talin and focal adhesion dynamics

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members and their lipid product, phosphatidylinositol 4,5-bisphosphate (PIP2) are important regulators of actin cytoskeleton. PIP5Kγ 90kDa (PIP5Kγ90), an isoform of PIP5K, localizes to focal adhesions (FAs) and is activated via its interaction with the cytoskeletal protein, talin. Currently, regulatory signaling pathways of talin–PIP5Kγ90 interaction related to FA dynamics and cell motility are not well understood. Considering the presence of Akt consensus motifs in PIP5Kγ90, we examined a potential link of Akt activation to talin–PIP5Kγ90 interaction. We found that Akt phosphorylated PIP5Kγ90 specifically at serine 555 (S555) in vitro and in epidermal growth factor (EGF)-treated cells phosphoinositide 3-kinase-dependently. EGF treatment suppressed talin–PIP5Kγ90 interaction and PIP2 levels. Similarly, a phosphomimetic mutant (S555D), but not non-phosphorylatable mutant (S555A), of PIP5Kγ90 had reduced talin binding affinity, lowered PIP2 levels, and was dislocated from FAs. The S555D mutant also caused decreases in actin stress fibers and vinculin-positive FAs. Moreover, assembly and disassembly of FAs were enhanced by S555D expression and EGF-induced cell migration was relatively low in S555A-expressing cells compared to wild-type-expressing cells. PIP5Kγ87, a PIP5Kγ splice variant lacking the talin binding motif, was phosphorylated by Akt, which, however, hardly affected PIP2 levels. Taken together, our results suggested that Akt-mediated PIP5Kγ90 S555 phosphorylation is a novel regulatory point for talin binding to control PIP2 level at the FAs, thereby modulating FA dynamics and cell motility.

Kindlin-2 interacts with and stabilizes EGFR and is required for EGF-induced breast cancer cell migration

Epidermal growth factor receptor (EGFR) mediates multiple signaling pathways that regulate cell proliferation, migration and tumor invasion. Kindlin-2 has been known as a focal adhesion molecule that binds to integrin to control cell migration and invasion. However, molecular mechanisms underlying the role of Kindlin-2 in breast cancer progression remain elusive. Here we report that Kindlin-2 interacts with EGFR and mediates EGF-induced breast cancer cell migration. We found that EGF treatment dramatically increases Kindlin-2 expression at both mRNA and protein levels in a variety of cancer cells. Inhibitors specific for EGFR or PI3K blocked Kindlin-2 induction by EGF. Importantly, Kindlin-2 interacted with EGFR kinase domain, which was independent of Kindlin-2 binding to integrin cytoplasmic domain. Intriguingly, Kindlin-2 stabilized EGFR protein by blocking its ubiquitination and degradation. Depletion of Kindlin-2 impaired EGF-induced cell migration. Our results demonstrated that Kindlin-2 participates in EGFR signaling and regulates breast cancer progression.

Involvement of cysteine-rich protein 61 in the epidermal growth factor-induced migration of human anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.

Hsp27 regulates EGF/β-catenin mediated epithelial to mesenchymal transition in prostate cancer.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3β phosphorylation and β-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of β-catenin on tyrosine 142 and 654, enhances β-catenin ubiquitination and degradation, prevents β-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the β-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

CS-22 * MIXED LINEAGE KINASE SIGNALING IS REQUIRED FOR GLIOBLASTOMA CELL MIGRATION AND INVASION

Glioblastoma multiforme (GBM) tumors are the most deadly of brain tumors due to their diffuse infiltrative nature and tremendous heterogeneity. EGFR signaling is aberrantly activated in most GBM tumors, and clearly contributes to malignancy. However, targeting EGFR directly has been largely unsuccessful, for multiple reasons, including compensatory upregulation of other receptor tyrosine kinases (RTKs). Identifying and targeting key common signaling nodes downstream of multiple RTKs that drive invasion may be an effective therapeutic strategy. The Mixed Lineage Kinases (MLKs) are a family of MAP3Ks that activate multiple MAPK pathways, including c-Jun N-terminal kinase (JNK). Binding of GTP-bound Rac/Cdc42 activates MLK3. We investigated the role of MLK signaling on GBM cell migration and 3D spheroid invasion using an ATP-competitive pan-MLK inhibitor, as well as siRNA-mediated silencing of MLK3. Our data demonstrate that both MLK inhibition and MLK3 silencing dramatically reduce GBM migration and 3D invasion. EGF-induced GBM cell migration, as well as JNK activation, is blocked by a pan-MLK inhibitor and by MLK3 silencing. The Rac GEF, Dock180, is critical for GBM invasion. Based upon Drosophila genetics, the Dock180 homolog (MBC), a Rac GEF, acts upstream of Rac, the MLK homolog (slipper) and JNK. Therefore we hypothesized that Dock180 might be an upstream activator of MLKs. Our data show that Dock180 complexes with MLK3; and that ectopic expression of Dock180 activates both MLK3 and JNK in a Rac-dependent manner. Furthermore, silencing of either Dock180 or MLK3 leads to a similar reduction in EGF-induced JNK activation. Finally, ectopic expression of MLK3 (which leads to its auto-activation) is sufficient to rescue the defect in migration due to Dock180 silencing. Based upon these data, we propose that an EGFR-Dock180-Rac-MLK-JNK signaling axis drives GBM migration and 3D invasion, and that targeting MLKs may be a useful therapeutic approach for treating patients with GBM.

Aquaporin 8 involvement in human cervical cancer SiHa migration via the EGFR-Erk1/2 pathway.

Overexpression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases 1/2 (Erk1/2) are associated with tumorigenesis and cancer progression and may upregulate AQP expression. In this study, we demonstrated that EGF (epidermal growth factor) induces SiHa cells migration and AQP8 expression. Wound healing results showed that cell migration was increased by 2.79-1.50-fold at 24 h and 48 h after EGF treatment. AQP8 expression was significantly increased (3.33-fold) at 48 h after EGF treatment in SiHa cells. An EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration and AQP8 expression was decreased from 1.59-fold (EGF-treated) to 0.43-fold (PD153035-treated) in SiHa. Furthermore, the MEK (MAPK (mitogen-activated protein kinase)/Erk (extracellular signal regulated kinase)/Erk inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 1.21-fold (EGF-treated) to 0.43-fold (U0126-treated). Immunofluorescence microscopy further confirmed the results. Collectively, our findings show that EGF induces AQP8 expression and cell migration in human cervical cancer SiHa cells via the EGFR/Erk1/2 signal transduction pathway.

5‐Lipoxygenase and cysteinyl leukotriene receptor 1 regulate epidermal growth factor‐induced cell migration through Tiam1 upregulation and Rac1 activation

Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF-induced Rac1 activation was required for EGF-induced cell migration, however, the spatiotemporal regulation of the second wave of EGF-induced Rac1 activation remains largely unclear. In this study, we found that 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4) produced by 5-LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4-mediated CysLT1 signaling regulates the second EGF-induced wave of Rac1 activation. We also found that 5-LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF-induced T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5-LOX/LTC4/CysLT1 signaling pathway regulates EGF-induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent.