Paxillin Research Articles

Semisynthetic oleanane triterpenoids inhibit migration and invasion of human breast cancer cells through downregulated expression of the ITGB1/PTK2/PXN pathway

This paper reports a study on the role of two synthetic derivatives of oleanolic acid (OA), HIMOXOL and Br-HIMOLID, in the regulation of cell migration and invasion and the underlying molecular mechanisms of breast cancer cells. The effect of the compounds on four breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-468, and T-47D) and also on noncancerous breast cells, MCF-12A, was reported. The compounds had no effect on the migration of MCF-12A cells. However, both the derivatives revealed a higher cytotoxicity than the maternal compound OA, and in sub-cytotoxic concentrations, they decreased the migration of MCF7, MDA-MB-231, and MDA-MB-468 breast cancer cells and also the invasion of MCF7 and MDA-MB-231 cells; although, the derivatives had no effect on the migration and invasion of T-47D cells. Both the derivatives of OA inhibited the cell migratory and invasive abilities of breast cancer cells by downregulating the expressions of ITGB1, PTK2, and PXN genes and by decreasing the phosphorylation status and the level of its respective proteins (integrin β1, FAK, and paxillin, respectively). This study is the first to report the antimigratory and anti-invasive activities of HIMOXOL and Br-HIMOLID in breast cancer cells.

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish

Paxillin (Pxn) is a key adapter protein and signaling regulator at sites of cell-extracellular matrix (ECM) adhesion. Here, we investigated the role of Pxn during vertebrate development using the zebrafish embryo as a model system. We have characterized two Pxn genes, pxna and pxnb, in zebrafish that are maternally supplied and expressed in multiple tissues. Gene editing and antisense gene knockdown approaches were used to uncover Pxn functions during zebrafish development. While mutation of either pxna or pxnb alone did not cause gross embryonic phenotypes, double mutants lacking maternally supplied pxna or pxnb displayed defects in cardiovascular, axial, and skeletal muscle development. Transient knockdown of Pxn proteins resulted in similar defects. Irregular myotome shape and ECM composition were observed, suggesting an “inside-out” signaling role for Paxillin genes in the development of myotendinous junctions. Inhibiting non-muscle Myosin-II during somitogenesis altered the subcellular localization of Pxn protein and phenocopied pxn gene loss-of-function. This indicates that Paxillin genes are effectors of actomyosin contractility-driven morphogenesis of trunk musculature in zebrafish. Together, these results reveal new functions for Pxn during muscle development and provide novel genetic models to elucidate Pxn functions.

Dishevelled segment polarity protein 3 (DVL3): a novel and easily applicable recurrence predictor in localised prostate adenocarcinoma.

To identify new biomarkers for biochemical recurrence (BCR) of prostate adenocarcinoma. Clinical information of 500 patients with prostate adenocarcinoma and their 152 RNA-sequencing and protein-array data from The Cancer Genome Atlas (TCGA) were separated into a discovery set and a validation set. Each dataset was analysed according to the Gleason grade groups reflecting BCR. The results obtained from the analysis using TCGA dataset were confirmed by immunohistochemistry analyses of a confirmation cohort composed of 395 patients with localised prostate adenocarcinoma. TCGA discovery set was subgrouped into lower- and higher-risk groups for recurrence-free survival (RFS) (P < 0.001). Cyclin B1 (CCNB1), dishevelled segment polarity protein 3 (DVL3), paxillin (PXN), RAF1, transferrin, X-ray repair cross complementing 5 (XRCC5) and BIM had lower expression in the lower-risk group than that in the higher-risk group (all, P < 0.05). In TCGA validation set, CCNB1, DVL3, transferrin, XRCC5 and BIM were also differently expressed between the two groups. Immunohistochemically, DVL3 positivity was associated with high prostate-specific antigen (PSA) levels, resection margin involvement, and BCR (all, P < 0.05). Ahigh Gleason score indicated a marginal relationship (P = 0.055). BIM positivity was related to high PSA levels, lymphovascular invasion, and BCR (all, P < 0.05). Both DVL3 positivity (P = 0.010) and BIM positivity (P = 0.024) were associated with shorter RFS, but statistical significance was lost when the multivariate Cox regression model included all patients. In the lower-risk group, the multivariate Cox model confirmed that DVL3 was an independent predictor for poor RFS (hazard ratio 1.80, P = 0.040), and the concordance index (C-index) was 0.805. DVL3 and BIM were expressed in patients with a higher risk of BCR. DVL3 may be a novel and easily applicable recurrence predictor of localised prostate adenocarcinoma.

Myofibroblast-Mediated Contraction.

Myofibroblast-mediated contraction is viewed as a cycle of four steps. The first step is stimulation of myofibroblasts by lysophospholipids leading to the activation of G proteins and ending with contraction of the actin-myosin complex. The next step is the transmission of the intracellular contractile force at the focal adhesions of myofibroblasts; a step that involves talin, vinculin, paxillin, Hic-5, and the integrin receptors. In the third step, fibronectin will act as the extracellular link between the integrin receptors and the extracellular collagen. Finally, "sensing" tension and the maintenance of myofibroblast activity represent the fourth step. The clinical relevance of each step is then discussed in the form of modalities to prevent excessive scarring/fibrosis.

The Percentage of Stained Cells is a More Reliable Parameter in Immunohistochemical Analysis than Scoring the Intensity of Staining: Expression of 9 Molecular Markers in Progression and Liver Metastases of Colorectal Cancer

Aims: Research for reliable molecular markers that provide prognostic and predictive information in colorectal cancer (CRC) is based on solid evidence that the staging system on its own cannot definitively predict the tumor behavior and guide clinical management of the disease. Methods and results: In this study we examined the immunohistochemical expression of 9 markers, namely membrane-bound mucin 1 (MUC1), paxillin (PAX), Focal Adhesion Kinase (FAK), G-protein coupled receptor 56 (GPR56), ORAI3 and well known markers of colon carcinoma microsatellite instability (MSI): MSH2, MSH6, MLH1 and PMS2 with respect to the percentage of stained cells and intensity score in colon carcinoma cells, both in the primary tumor and liver metastasis. We have related all of the mentioned markers with clinicopathological data, including the age of patients, grading of the primary tumor and TNM system. Western blotting assay was performed to identify the expression. Our present study showed that the evaluation of different markers with respect to intensity of staining and percentage of stained cancer cells may be considered as a prognostic marker for tumor progression and later for liver metastasis. This has been found for the percentage of stained cells particularly. Conclusions: Our results implicate that the counting percentage of stained colon cancer cells provides a more adequate method of immunohistochemical analysis than evaluation of the intensity of staining.

Overexpression of Paxillin Correlates with Tumor Progression and Predicts Poor Survival in Glioblastoma

To explore the prognostic and clinicopathological features of glioma with Paxillin (PXN) expression based on a large number of samples. RNA sequencing data of 325 glioma samples from Chinese Glioma Genome Atlas (CGGA) database were obtained as discovery set. Three additional datasets were further obtained as validation sets. The protein expression pattern of PXN in glioma was measured by IHC. Kaplan-Meier survival and multivariate Cox analysis were used to estimate the survival distributions. Moreover, the functional annotation of PXN was also analyzed. In the discovery set, PXN overexpression was significantly associated with high-grade glioma as well as the higher mortality in survival analysis (log-rank test, P < 0.01). The results of the other validation datasets showed similar findings. PXN also served as an independent prognostic biomarker in glioblastoma patients. Functional assays showed that PXN contributed to glioma cell proliferation and invasion. PXN plays as an oncogene in glioma progression and suggests a new potential biotarget for therapy.

Abstract 702: Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover

Abstract Serglycin (SRGN), a hematopoietic cell granule proteoglycan, has recently been shown to be overexpressed in several aggressive cancer types. In nasopharyngeal and hepatocellular carcinomas, elevated expression of SRGN was shown to correlate with poor prognosis. However, the molecular mechanisms underlying SRGN-mediated malignancies remain to be explored. In this study, we showed that SRGN is expressed at elevated levels in several lung cancer-derived cell lines. By gain-of-function and loss-of-function approaches, we showed that SRGN promoted NSCLC cell migration. SRGN modulated actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated Rac1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. Increased focal adhesion (FA) turnover, the process of continuous assembly and disassembly of adhesions, would lead to increased cell migration. We further showed that SRGN promoted Src activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, which were well-defined indicators of FA turnover. Taken together, these data suggested that SRGN promotes cell migration through inducing cytoskeleton reorganization and focal adhesion turnover. Citation Format: Jeou-Yuan Chen, Jing-You Guo. Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 702.

TC-N19, a novel dual inhibitor of EGFR and cMET, efficiently overcomes EGFR-TKI resistance in non-small-cell lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) show a clinical benefit when used to treat patients with EGFR-mutated non-small-cell lung cancer (NSCLC), but this treatment unfortunately fails in patients with TKI-resistant tumors. We here provide evidence that TC-N19 (N19), a novel dual inhibitor of EGFR and cMET, efficiently overcomes the EGFR-TKI resistance in EGFR-mutated NSCLC cells via simultaneous degradation of both proteins by ubiquitin proteasomes. Comparison with HSP90 inhibitor treatment and knockdown of EGFR and cMET by small hairpin RNAs reveal that the reduction of EGFR and cMET expression by N19 is responsible for overcoming the intrinsic TKI resistance mediated by paxillin (PXN) in high PXN-expressing cells, PXN-overexpressing PC9 cells (PC9-PXN), the EGFR-T790M-mediated TKI resistance in H1975 and CL97 cells, and the acquired resistance to gefitinib in gefitinib-resistant PC9 cells (PC9GR). Annexin V-PI staining assay showed that the induction of apoptosis in NSCLC cells by N19 depended on the reduction in levels of both proteins. Xenograft tumor formation in nude mice induced by a PC9-PXN-stable clone and by PC9GR cells was nearly completely suppressed by N19 treatment, with no changes in animal body weight. MTT assays of normal lung cells and reticulocytes showed no cytotoxicity responses to N19. In summary, N19 may act as a novel dual inhibitor of EGFR and cMET that induces apoptosis in TKI-resistant EGFR-mutated NSCLC cells and suppresses xenograft tumor formation. We suggest that N19 may be a potential new-generation TKI or HSP90 inhibitor used for treatment of NSCLC patients who show resistance to current TKI-targeting therapies.

Molecular mechanism of fluoroquinolones modulation on corneal fibroblast motility

Topical fluoroquinolones are widely used to prevent ocular infections after ophthalmic surgery. However, they have been shown to affect the corneal cell motility, whose mechanism remains indefinite. The purpose of this study was to investigate how fluoroquinolones affect corneal stromal cell motility. Human corneal fibroblasts (HCFs) were incubated in ciprofloxacin (CIP), levofloxacin (LEV), or moxifloxacin (MOX) at 0, 10, 50, and 100 μg/ml for up to 3 days. Effect of CIP, LEV, or MOX on HCF migration was monitored using migration assay. HCF viability was determined by WST-1 assay. Expression of focal adhesion kinase (FAK), paxillin (PXN), and their phosphorylated forms were analyzed by immunoblotting. Binding affinity between FAK and PXN was determined by co-immunoprecipitation. Our results revealed that CIP and MOX, but not LEV, noticeably retarded HCF migration. HCF proliferation was significantly reduced by CIP (38.2%), LEV (29.5%), and MOX (21.3%), respectively (p = 0.002). CIP and MOX suppressed the phosphorylation of PXN at tyrosines (10.2 ± 4.3%, p < 0.001; 11.7 ± 2.4%, p < 0.001, respectively), including tyrosine 118 (33.3 ± 5.2%, p < 0.001; 34.0 ± 4.4%, p < 0.001, respectively). CIP and MOX diminished the binding affinity between FAK and PXN (8.2 ± 1.8%, p < 0.001; 9.0 ± 4.5%, p < 0.001, respectively). Nevertheless, tyrosine dephosphorylation and FAK dissociation of PXN were not found in LEV-treated HCFs. None of these fluoroquinolones affect phosphorylation of FAK-Y397. We conclude that CIP and MOX, but not LEV, might delay corneal fibroblast migration via interfering with recruitment of PXN to focal adhesions and dephosphorylation of PXN at the tyrosines.

Paxillin is positively correlated with the clinicopathological factors of colorectal cancer, and knockdown of Paxillin improves sensitivity to cetuximab in colorectal cancer cells.

Paxillin (PXN) encodes a 68-kDa focal adhesion-associated protein and plays an important role in signal transduction, regulation of cell morphology, migration, proliferation and apoptosis. The aim of the present study was to evaluate the relationship between PXN and clinicopathological factors in colorectal cancer, the role of PXN in cetuximab resistance, and whether knockdown of PXN expression could improve the sensitivity to cetuximab in colorectal cancer cells. In the present study, immunohistochemical staining in 148 colorectal carcinoma and 126 normal adjacent tissues was performed, which showed that the positive rate of PXN was significantly higher in the colorectal adenocarcinoma samples than that in the normal colorectal mucosa samples (P<0.001). Moreover, PXN presence was also positively correlated with TNM stage (P=0.023), distant metastasis (P=0.014), recurrence (P=0.032) and reduced survival (P=0.004). In vitro, PXN expression was positively correlated with the proliferation rate in colorectal cells insensitive to cetuximab. Inhibition of PXN expression by PXN-siRNA clearly increased apoptosis by downregulating the phosphorylation of extracellular signal regulated kinase (p-Erk) level, and overexpression of PXN by PXN-cDNA decreased apoptosis by upregulating the p-Erk level. This suggests that overexpression of PXN could be one of the reasons for cetuximab resistance, and downregulation of PXN plays an important role in improving sensitivity to cetuximab by suppressing the activitation of p-Erk in colorectal cancer cells. Above all, knockdown of PXN could represent a rational therapeutic strategy for increasing the sensitivity or overcoming cetuximab-resistance in patients with colorectal cancer.

Paxillin confers resistance to tyrosine kinase inhibitors in EGFR-mutant lung cancers via modulating BIM and Mcl-1 protein stability.

Tyrosine kinase inhibitors (TKIs) have been documented to have substantial clinical benefits to non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutation. TKI resistance occurs in nearly all patients who receive TKI-targeting therapy, resulting in a modest overall survival benefit. Therefore, establishing a biomarker for early prediction and exploring the mechanism of primary TKI resistance is essential for improving the therapeutic efficacy in non-small cell lung cancer patients. In this study, we provide evidence indicating that paxillin (PXN) overexpression may confer TKI resistance in EGFR-mutant lung cancer cells. Mechanistically, PXN-mediated extracellular signal-regulated kinases (ERK) activation is responsible for TKI resistance via decreased Bcl2-interacting mediator of cell death (BIM) and increased Mcl-1 expression due to modulating their protein stabilities by phosphorylation of BIM at serine 69 and Mcl-1 at threonine 163. The mechanistic action in the cell model was further confirmed by the observation of xenograft tumors in nude mice, revealing that the PXN-mediated TKI resistance was conquered by ERK inhibitor (AZD6244) and Bcl-2 family inhibitor (obatoclax), but the TKI resistance overcome by AZD6244 is more effective than that of obatoclax. Therefore, we suggest that PXN expression may be useful in predicting primary TKI resistance, and combining TKI with ERK inhibitors may clinically benefit EGFR-mutant non-small cell lung cancer patients whose tumors exhibit high PXN expression.

α-Actinin-4 enhances colorectal cancer cell invasion by suppressing focal adhesion maturation.

α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.

Paxillin promotes colorectal tumor invasion and poor patient outcomes via ERK-mediated stabilization of Bcl-2 protein by phosphorylation at Serine 87.

Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

Paxillin-dependent regulation of IGF2 and H19 gene cluster expression.

ABSTRACTPaxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation. Here, we introduce paxillin as a transcriptional regulator of IGF2 and H19 genes. It does not affect the allelic expression of the two genes; rather, it regulates long-range chromosomal interactions between the IGF2 or H19 promoter and a shared distal enhancer on an active allele. Specifically, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, thus activating IGF2 gene transcription, whereas it restrains the interaction between the enhancer and the H19 promoter, downregulating the H19 gene. We found that paxillin interacts with cohesin and the mediator complex, which have been shown to mediate long-range chromosomal looping. We propose that these interactions occur at the IGF2 and H19 gene cluster and are involved in the formation of loops between the IGF2 and H19 promoters and the enhancer, and thus the expression of the corresponding genes. These observations contribute to a mechanistic explanation of the role of paxillin in proliferation and fetal development.

Abstract 2: Paxillin enhances angiogenesis through transcriptional regulation of Src in ovarian cancer

Abstract Purpose: Paxillin (PXN) expression is increased in various cancer types, but its biological and clinical impact is not well understood. Here, we examined the biological effects of paxillin in ovarian cancer. Methods: The in vivo (HeyA8 and HeyA8-MDR orthotopic mouse models of ovarian cancer) and in vitro (tube formation assays, reverse phase protein array (RPPA) analysis, RT-PCR chromatin-immunoprecipitation (ChIP) assay) effects of paxillin were examined in ovarian cancer. Results: Paxillin silencing using siRNA incorporated in DOPC nanoliposomes resulted in 55% reduction in tumor growth in a HeyA8 orthotopic ovarian cancer mouse model (p&amp;lt;0.05). There was an unexpected effect of marked reduction in vascular density (67%, p&amp;lt;0.01). In addition, tumor proliferation was decreased by 60% (p&amp;lt;0.01) in PXN-siRNA treatment group. This anti-proliferative effect of siPXN was not observed in vitro. In contrast, a significant anti-angiogenic effect was observed in vitro using a tube formation assay, where condition media collected from ovarian cancer cells treated with siPXN compared to siControl resulted in a prominent (60%,p&amp;lt;0.01) reduction in tube formation ability of endothelial cells. RPPA analyses pointed to Src protein expression being significantly decreased following PXN silencing in HeyA8 cells. Using RT-PCR and western blotting techniques, we observed that Src mRNA and protein levels were significantly decreased in siPXN-treated cells. Moreover, p-Src protein level was also found to be decreased following PXN silencing. ChIP analysis showed direct PXN binding to the Src promoter. Conclusion PXN exerts indirect effects on tumor angiogenesis, mediated through Src. These findings provide a new understanding of the role of PXN in malignant biology. Citation Format: Hyun Jin Choi, Seung-Wook Kim, Sunila Pradeep, Heather J. Dalton, Rajesha Rupaimoole, Selanere Mangala, Gabriel Lopez, Sood K. Anil. Paxillin enhances angiogenesis through transcriptional regulation of Src in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2014-2

Interactions between E6, FAK, and GIT1 at paxillin LD4 are necessary for transformation by bovine papillomavirus 1 E6.

Bovine papillomavirus 1 E6 interacts with two similar proteins that regulate cell attachment and cell migration called paxillin (PXN) and HIC-5 (also known as HIC5, ARA55, HIC-5, TSC-5, and TGFB1I1). Despite the similarity between HIC-5 and paxillin, paxillin is required for E6 to transform mouse embryo fibroblasts while HIC-5 is not. Using mutants of paxillin, we found that dynamic competitive interactions between E6, focal adhesion kinase, and the GIT1 ARF-GAP protein for binding to paxillin are required but not sufficient for transformation by E6. Using mutants of paxillin and chimeric proteins between HIC-5 and paxillin, we demonstrate that a critical difference between HIC-5 and paxillin is within the LIM domains of paxillin that do not directly interact with E6. Mutational analysis indicates that at least six distinct domains of paxillin are required for E6 transformation. Papillomaviruses cause epitheliomas in vertebrates through the actions of virus-encoded oncoproteins. Despite the immense diversity of papillomavirus types, our understanding of the mechanisms by which the virus-encoded E6 oncoproteins contribute to cell transformation is restricted to human papillomavirus types that are associated with cancer. Bovine papillomavirus 1 (BPV-1) E6 has served as a model system for studies of E6 structure and function. This study examines the mechanisms by which BPV-1 E6 association with the cellular focal adhesion adapter protein paxillin contributes to cell transformation and extends our knowledge of the diverse mechanisms by which papillomaviruses transform host cells.

Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.

To enable new blood vessel growth, endothelial cells (ECs) express neuropilin 1 (NRP1), and NRP1 associates with the receptor tyrosine kinase VEGFR2 after binding the vascular endothelial growth factor A (VEGF) to enhance arteriogenesis. We report that NRP1 contributes to angiogenesis through a novel mechanism. In human and mouse ECs, the integrin ligand fibronectin (FN) stimulated actin remodeling and phosphorylation of the focal adhesion component paxillin (PXN) in a VEGF/VEGFR2-independent but NRP1-dependent manner. NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling. This complex promoted EC motility in vitro and during angiogenesis on FN substrates in vivo. Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins. The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth.

Paxillin promotes tumor progression and predicts survival and relapse in oral cavity squamous cell carcinoma by microRNA-218 targeting.

High-risk human papillomavirus (HPV) 16-infected oral cavity squamous cell carcinoma (OCSCC) differs significantly from non-HPV-infected OCSCC. However, the molecular pathogenesis of HPV-infected OCSCC remains unclear. Paxillin (PXN) has been reported to promote lung tumor progression by miR-218 targeting. In addition, expression of miR-218 has been shown to be reduced by HPV16 E6 in cervical cancer. We thus asked whether PXN can promote tumor progression by E6-reduced miR-218 in OCSCC, especially in HPV-infected OCSCC. Mechanistic studies demonstrated that PXN expression increased markedly upon E6-mediated reductions in miR-218, resulting in increased colony formation and invasion capabilities in HPV-infected OCSCC cells. Among tumor specimens, HPV16/18 infection was negatively associated with miR-218 expression and positively associated with PXN expression. Kaplan-Meier and Cox regression models demonstrated that patients with low-miR-218 tumors or high-PXN tumors exhibited shorter overall survival (OS) and relapse-free survival (RFS) than those with high-miR-218 tumors or low-PXN tumors. Interestingly, HPV-infected patients with low-miR-218, high-PXN tumors and both combinations exhibited the worst OS and RFS compared with patients in their counterparts. These observations in patients were consistent with the findings from the cell model. Therefore, we suggest that PXN might be targeted to suppress tumor progression and consequently to improve outcomes in OCSCC, especially in HPV-infected OCSCC.

MiR-137 impairs the proliferative and migratory capacity of human non-small cell lung cancer cells by targeting paxillin

Human lung cancer is the leading cause of cancer motility worldwide, with nearly 1.4 million deaths each year, among which non-small cell lung cancer (NSCLC) accounts for almost 85% of this disease. The discovery of microRNAs (miRNAs) provides a new avenue for NSCLC diagnostic and treatment regiments. Currently, a large number of miRNAs have been reported to be associated with the progression of NSCLC, among which serum miR-137 has been examined to be down-regulated in NSCLC patients. However, the function of miR-137 on NSCLC cells migration and invasion and the relative mechanisms were less known. Here, we found that ectopic expression of miR-137 could inhibit cell proliferation, induce cell apoptosis, and suppress cell migration and invasion in NSCLC cell line A549. Moreover, we found that paxillin (PXN) was a target gene of miR-137 in NSCLC cells and restored expression of PXN abolished the miR-137-mediated suppression of cell migration and invasion. Taken together, our results showed that miR-137 acted as a tumor suppressor in NSCLC by targeting PXN, and it may provide novel diagnostic and therapeutic options for human NSCLC clinical operation in future.

Phosphorylation of paxillin confers cisplatin resistance in non-small cell lung cancer via activating ERK-mediated Bcl-2 expression

Paxillin (PXN) is required for receptor tyrosine kinase-mediated ERK activation, and the activation of the Raf/MEK/ERK cascade has been linked with Bcl-2 expression. We hypothesized that phosphorylation of PXN by the EGFR/Src pathway might contribute to cisplatin resistance via increased Bcl-2 expression. We show that cisplatin resistance was dependent on PXN expression, as evidenced by PXN overexpression in TL-13 and TL-10 cells and PXN knockdown in H23 and CL1-5 cells. Specific inhibitors of signaling pathways indicated that the phosphorylation of PXN at Y118 and Y31 via the Src pathway was responsible for cisplatin resistance. We further demonstrated that ERK activation was also dependent on this PXN phosphorylation. Bcl-2 transcription was upregulated by phosphorylated PXN-mediated ERK activation via increased binding of phosphorylated CREB to the Bcl-2 promoter. A subsequent increase in Bcl-2 levels by a PXN/ERK axis was responsible for the resistance to cisplatin. Animal models further confirmed the findings of in vitro cells indicating that xenograft tumors induced by TL-13-overexpressing cells were successfully suppressed by cisplatin combined with Src or ERK inhibitor compared with treatment of cisplatin, Src inhibitor or ERK inhibitor alone. A positive correlation of phosphorylated PXN with phosphorylated ERK and Bcl-2 was observed in lung tumors from NSCLC patients. Patients with tumors positive for PXN, phosphorylated PXN, phosphorylated ERK and Bcl-2 more commonly showed a poorer response to cisplatin-based chemotherapy than did patients with negative tumors. Collectively, PXN phosphorylation might contribute to cisplatin resistance via activating ERK-mediated Bcl-2 transcription. Therefore, we suggest that Src or ERK inhibitor might be helpful to improve the sensitivity for cisplatin-based chemotherapy in NSCLC patients with PXN-positive tumors.