Quantitative acetylome and phosphorylome analysis reveals Girdin affects pancreatic cancer progression through regulating Cortactin.

The actin-binding protein Girdin is involved in a variety of cellular processes, including pancreatic cancer. The objective of this study is to explore the role and the mechanism of Girdin in pancreatic cancer by quantitative acetylome and phosphorylome analysis. We firstly found that Girdin was overexpressed in pancreatic cancer tissue and increased expression of Girdin was associated with tumor size and stage of patients with pancreatic cancer. We established the shRNA knockdown of Girdin in PANC-1 and Aspc-1 cells, and we found that shGirdin inhibited proliferation, migration and invasion, and promoted apoptosis. Subsequently, we identified and quantified 5,338 phosphorylated sites in 2,263 proteins that changed in response to Girdin knockdown, and identified a similar set of Girdin-responsive acetylome data as well. Additional data revealed that down-regulation of Girdin affected Cortactin phosphorylation and acetylation, suggesting Cortactin as an important regulatory target of Girdin. Moreover, we found that overexpression of Cortactin could rescue the effect of shGirdin on proliferation, apoptosism, migration and invasion of pancreatic cancer cells. In general, our results provided new insights into the mechanisms of Girdin function including cell proliferation, migration and invasion, and offer biomarker candidates for clinical evaluation of Girdin.

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

Invadopodia proteins, cortactin, N-WASP and WIP differentially promote local invasiveness in ameloblastoma.

BACKGROUND: Cell migration and invasion through interstitial tissues are dependent upon several specialized characteristics of the migratory cell notably generation of proteolytic membranous protrusions or invadopodia. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behaviour. Cortactin and MMT1-MMP are two invadopodia proteins implicated in its local invasiveness. Other invadopodia regulators, namely N-WASP, WIP and Src kinase remain unclarified. This study addresses their roles in ameloblastoma. MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters. RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations. CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.

Expression and clinical significance of cortactin protein in ovarian neoplasms.

OBJECTIVE: To examine the expression of cortactin in epithelial ovarian cancer, and discuss the relationship between the expression of cortactin and the clinical pathology characteristics in epithelial ovarian cancer, as well as clinical significance. METHODS: The expression of cortactin was detected using real-time fluorescence quantitative PCR and immunohistochemical SP method in epithelial ovarian cancer. RESULTS: (1) The relative content of cortactin mRNA in epithelial ovarian cancer tissue was higher than that in benign control tissue, and expression was related to histological classification and FIGO stage. (2) Cortactin protein was localized in the cytoplasm and membrane of tumor cells. The positive rate of cortactin was 73.3 % in epithelial ovarian cancer, and the rate of cortactin expression was related to histological classification. (3) The average survival period of epithelial ovarian cancer patients with positive expression of cortactin was 19.5 ± 1.2 months (95 % CI 14.6-21.4 months), compared with 34.5 ± 4.3 months in the negative expression group (95 % CI 22.1-25.9 months). Univariate survival analysis showed that: negative expression of cortactin had a significant survival advantage (χ (2) = 5.739, P = 0.017). A cox regression model for multivariate analysis revealed that cortactin was an independent prognostic factor for epithelial ovarian cancer (P = 0.001; RR = 6.452, 95 % CI 2.289-7.112). CONCLUSIONS: Negative expression of cortactin was an independent prognostic factor and had a survival advantage. This suggested that cells with poor differentiation showed increasing motility. Cortactin is closely related to poor prognosis.

Fra-1 is upregulated in gastric cancer tissues and affects the PI3K/Akt and p53 signaling pathway in gastric cancer.

Gastric cancer is an aggressive disease that continues to have a daunting impact on global health. Fra-1 (FOSL1) plays important roles in oncogenesis in various malignancies. We investigated the expression of Fra-1 in gastric cancer (GC) tissues by qPCR, immunohistochemistry (IHC) and western blot technologies. The results showed that Fra-1 was overexpressed in gastric cancer tissues compared with the adjacent non­cancerous tissues. To explore the possible mechanism of Fra-1 in GC, we elucidated the effect of Fra-1 in the apoptosis and cell cycle of gastric cancer cells, AGS, and found that a considerable decrease in apoptotic cells and increase of S phase rate were observed for AGS cells with Fra-1 overexpession. We identified and confirmed that Fra-1 affected the expression level of CTTN and EZR in vitro through LC-MS/MS analyses and western blot technology. Furthermore, we found that Fra-1 was correlated with dysregulation PI3K/Akt and p53 signaling pathway in gastric cancer tissues in vitro. Moreover, we found that Fra-1 overexpression affected the expression of PI3K, Akt, MDM2 and p53 in vivo. In summary, our results suggest that Fra-1 is upregulated in gastric cancer tissues and plays its function by affecting the PI3K/Akt and p53 signaling pathway in gastric cancer.

Expression of cortactin in human gliomas and its effect on migration and invasion of glioma cells.

The aim of the present study was to investigate the role of cortactin in the infiltrative behavior of glioma cells and the potential mechanism of cortactin in promoting the migration and invasion of glioma cells. The expression of cortactin was detected by immunohistochemistry in 40 human glioma specimens and 8 non-tumor brain specimens. U251, LN229 and SNB19 glioma cells were employed for the in vitro study and assigned into the siRNA-cortactin (transfected with siRNA specific to cortactin), siRNA-NC (transfected with negative control RNA sequence) and siRNA-N (transfected with empty vector) groups. The expression of cortactin in different treated glioma cell groups was detected using western blot analysis and RT-qPCR. The migration and invasion of glioma cells under different treatments were assessed using a wound-healing assay and Transwell-chamber invasion assay, respectively. The lamellipodia of glioma cells following treatment were observed by immunofluorescence (IF) and changes of lamellipodia over time were imaged using an inverted microscope. The distribution of cortactin and the actin-related protein 2/3 (Arp2/3) complex in glioma cells were observed after IF detection. The expression of cortactin in the glioma specimens was significantly higher than that in non-tumor brain tissue (P<0.05) and positively correlated with the malignancy of glioma specimens (r=0.912, P=0.00). The cortactin expression in glioma cells was markedly inhibited (P<0.05) and their migration and invasion ability was also impaired significantly following treatment with siRNA (P<0.05) compared with the other two groups. The size and persistence time of lamellipodia were reduced after cortactin expression was inhibited in glioma cells. Cortactin and the Arp2/3 complex were co-localized in the front of glioma cells, where actin was polymerized and lamellipodia formed. Thus, the results revealed that, cortactin is crucial in invasion and migration of glioma cells, which may promote the migration and invasion of glioma cells by regulating lamellipodia formation, a process requiring the combination of cortactin and the Arp2/3 complex.

Podocalyxin-like 1 promotes invadopodia formation and metastasis through activation of Rac1/Cdc42/cortactin signaling in breast cancer cells.

Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXL-knockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network.

Expression of LGR8 and related biomarkers in hepatocellular carcinoma: correlation with clinicopathological parameters.

This study aimed to evaluate the relationship between expression of LGR8, VEGF, MMP-2, MMP-9, fascin-1 and cortactin with clinicopathological parameters in hepatocellular carcinoma (HCC). The six biomarkers were investigated immunohistochemically using tissue microarrays of 93 HCC specimens. The tumor cells showed significant expression of LGR8, VEGF, MMP-9, fascin-1 and cortactin, but not of MMP-2. In addition, higher immunostaining scores for LGR8 in HCC showed negative correlation with T and AJCC clinical stages and upregulation of MMP-9, but no correlation with poorer survival rate; cortactin expression is correlated with poorer tumor differentiation in HCC. Thus, our data suggest that higher expression of LGR8 may facilitate tumor invasiveness in the early clinical stage of hepatocellular carcinoma, and synergic effects of cortactin also play a crucial role in the intrahepatic metastasis. Although tumor biological evidence implicates the relaxin-like hormone family as endocrine mediators of critical cellular action in cancer, characterization of target molecules and signaling pathways specific for LGR8 in defined tumor entities and crosstalk of the relaxin receptors with other receptor systems relevant to carcinogenesis will be of significant clinical relevance and may contribute to novel therapeutic strategies against hepatocellular carcinoma.

Expression of cortactin correlates with a poor prognosis in patients with stages II-III colorectal adenocarcinoma.

BACKGROUND: The present study was designed to specifically investigate the clinicopathological role of expression of cortactin, as well as the correlation with clinical outcomes in stages II-III colorectal cancer (CRC). METHODS: Two hundred and five stages II-III CRC patients were included in this study. Formalin-fixed paraffin-embedded specimens were stained for cortactin and the correlation between the staining, its clinicopathological parameters, and its prognostic power were analyzed statistically. RESULTS: Of the 205 patients studied, 113 cases (55.1%) were strongly positive for cortactin. Cortactin expression correlated with tumor invasion (P = 0.018), histological grade (P = 0.004), and preoperative CEA level (P < 0.001). In univariate analysis, tumor invasion, American Joint Committee on Cancer (AJCC) stage, lymphovascular invasion, preoperative CEA level, and cortactin expression were significant prognostic factors for disease-free survival (P = 0.034, 0.009, 0.043, 0.004, and 0.004, respectively), while for overall survival, tumor invasion, AJCC stage, pathologic grade, preoperative CEA level, and cortactin expression were significant prognostic factors (P = 0.003, 0.008, 0.038, 0.017, and <0.001, respectively). In multivariate analysis, tumor invasion, preoperative CEA level, and cortactin expression maintained their independent prognostic influence on disease-free survival (P = <0.001, 0.003, and 0.008, respectively). However, tumor invasion, AJCC stage, and cortactin expression influenced overall survival (P = 0.036, <0.001, and 0.004, respectively). CONCLUSIONS: Cortactin may be a good biomarker to be applied in the clinical setting to predict the prognosis of patients with completely resected pathologic stages II-III CRC.

Fascin, cortactin and survivin expression of melanocytic neoplasms and association with clinicopathological parameters and anatomic locations in Chinese people.

Distinct genetic aberrations between melanomas in different anatomical locations have been confirmed in recent years. However, the associations between immunohistochemical expression, tumor sites, and clinical parameters are not clear. We examined the correlation of protein expression of fascin, cortactin and survivin with clinicopathological parameters and lesion locations in patients with cutaneous melanoma. We collected 170 melanocytic lesions, including 106 cutaneous malignant melanoma (MM) from acral (AM) and non-acral (NAM) sites, 24 dysplastic nevi (DN), and 40 common melanocytic nevi (CMN). Tissue micro arrays were constructed and immunohistochemical expression for cortactin, fascin, and survivin was assessed with correlation with clinical parameters. Cytoplasmic immunostaining for fascin was found to be significantly higher in CMN than DN, and survivin staining was significantly higher in DN than MM. Positive nuclear immunoreactivity for survivin was seen in a large subset of melanomas but much less frequently in CMN and DN. In addition, nuclear survivin immunoreactivity was significantly more common in NAM than in AM. Nuclear survivin staining in patients with NAM was significantly correlated with poor survival. The significantly different expression of immunoreactivities (nuclear survivin) between AM and NAM and the different mortality risks of melanomas on acral versus non-acral sites might change site-specific therapeutic concepts in melanoma.

Cytogenetic characterization of head and neck squamous cell carcinoma cell lines as model systems for the functional analyses of tumor-associated genes.

Head and neck squamous cell carcinoma (HNSCC) is a solid malignant neoplasm exhibiting aggressive phenotypes and high recurrence rates. To improve its clinical management, understanding the molecular basis of HNSCC development is of critical importance. For the investigation of tumor-associated genes, functional analyses in well-characterized tumor cell systems are required. To establish an experimental platform, a set of 20 HNSCC cell lines was screened for genetic imbalances by chromosomal comparative genomic hybridization (cCGH). Frequent DNA copy number gains were detected on 3q26.3-qter, 5p, 7p11-p13, 8q23-qter, 9p11-p13, 9q31-qter, 11q13 and 20q13.1, whereas copy number losses were found on 3p, 4p, 4q32.1-qter, 8p11-p12 and 18q22 in agreement with previous observations on genetic aberrations detected in primary HNSCC specimens. Subsequent mRNA expression analysis of 11q13 candidate genes CCND1 and CTTN revealed that HNSCC cell lines exhibiting a DNA copy number gain on 11q13 had a higher transcript level of CCND1 and CTTN compared with HNSCC cell lines without 11q13 copy number gain (P = 0.014 and P = 0.009, respectively). Furthermore, CCND1 and CTTN amplification as detected by fluorescence in situ hybridization correlated with protein expression as assessed by immunocytochemistry. In summary, the cytogenetic characterization illustrates that this set of HNSCC cell lines is representative for the HNSCC genome and provides tumor model systems for detailed analysis of genes with a possible role in the pathomechanism of head and neck tumors.

Overexpression of cortactin increases invasion potential in oral squamous cell carcinoma.

Cortactin, an F-actin binding protein, stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin has been reported in several human cancers. Cortactin stimulates cell migration, invasion, and experimental metastasis. However, the underlying mechanism is not still understood. In the present study, we therefore evaluated the possibility that cortactin could be appropriate as a molecular target for cancer gene therapy. In 70 primary oral squamous cell carcinomas and 10 normal oral mucosal specimens, cortactin expression was evaluated by immunological analyses, and the correlations of the overexpression of cortactin with clinicopathologic factors were evaluated. Overexpression of cortactin was detected in 32 of 70 oral squamous cell carcinomas; significantly more frequently than in normal oral mucosa. Cortactin overexpression was more frequent in higher grade cancers according to T classification, N classifications, and invasive pattern. Moreover, RNAi-mediated decrease in cortactin expression reduced invasion. Downregulation of cortactin expression increased the expression levels of E-cadherin, ß-catenin, and EpCAM. The siRNA of cortactin also reduced PTHrP expression via EGF signaling. These results consistently indicate that the overexpression of cortactin is strongly associated with an aggressive phenotype of oral squamous cell carcinoma. In conclusion, we propose that cortactin could be a potential molecular target of gene therapy by RNAi targeting in oral squamous cell carcinoma.

Cytoskeleton alterations in melanoma: aberrant expression of cortactin, an actin-binding adapter protein, correlates with melanocytic tumor progression.

Cortactin is a multidomain actin-binding protein important for the functions of cytoskeleton by regulating cortical actin dynamics. It is involved in a diverse array of basic cellular functions. Tumorigenesis and tumor progression involves alterations in actin cytoskeleton proteins. We sought to study the role of cortactin in melanocytic tumor progression using immunohistochemistry on human tissues. The results reveal quantitative differences between benign and malignant lesions. Significantly higher cortactin expression is found in melanomas than in nevi (P<0.0001), with levels greater in metastatic than in invasive melanomas (P<0.05). Qualitatively, tumor tissues often show aberrant cortactin localization at the cell periphery, corresponding to its colocalization with filamentous actin in cell cortex of cultured melanoma cells. This suggests an additional level of protein dysregulation. Furthermore, in patients with metastatic disease, high-level cortactin expression correlates with poor disease-specific survival. Our data, in conjunction with outcome data on several other types of human cancers and experimental data from melanoma cell lines, supports a potential role of aberrant cortactin expression in melanoma tumor progression and a rational for targeting key elements of actin-signaling pathway for developmental therapeutics in melanomas.

Testicular gene expression profiling following 2-methoxyethanol and 2-ethoxyethanol exposure in male rats reveals abnormal expression of the actin binding protein cortactin in degenerating spermatocytes.

The glycol ether solvents 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) produce testicular toxicity characterized by spermatocyte degeneration, while a similar glycol ether, 2-butoxyethanol (2-BE), has no testicular effects. The goal of the current study was to better understand the mechanism of glycol ether testicular toxicity through gene expression profiling and functional classification of differentially expressed genes. Male rats were administered 2-ME (150 and 50mg/kg/day), 2-EE (500 mg/kg/day), 2-BE (125 mg/kg/day), or vehicle for 3 days, and testes were collected for histopathological and gene expression analysis. Histopathological changes in the testes were observed only in animals given 150 mg/kg/day 2-ME, consisting of degeneration and necrosis of spermatocytes and reductions in spermatocyte numbers. Microarray analysis of testicular samples from these animals revealed a large number of differentially expressed genes from animals exposed to 2-EE or to 50mg/kg or 150 mg/kg 2-ME (>900 each at >1.5-fold changed), compared to 28 genes from 2-BE treated animals. Expression Analysis Systematic Explorer (EASE) analysis of these genes demonstrated statistical enrichment in genes in categories including protein transport, endocytosis, protein kinase activity, cell cycle, and meiosis. Quantitative PCR confirmation of select genes confirmed increased expression of the actin binding protein cortactin and the transcription factor Wilm's tumor 1 (Wt1) following 2-ME exposure. Increased localization of cortactin in abnormal spermatocytes was also observed by immunohistochemistry, consistent with a possible role for this protein in the mechanism of toxicity.

Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer.

INTRODUCTION: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, namely CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Over-expression of cyclin D1 protein, however, confers tamoxifen resistance but not a tamoxifen-induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein over-expression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we examined a selected marker for this event. METHOD: Array comparative genomic hybridization analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients who were randomly assigned to either tamoxifen or no adjuvant treatment. The protein expression was also compared with gene expression data in a subset of 56 breast cancer samples. RESULTS: Cortactin and FADD (Fas-associated death domain) over-expression was linked to CCND1 amplification, determined by fluorescence in situ hybridization, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1 (checkpoint kinase 1), was associated with an impaired tamoxifen response, and interestingly with low proliferative breast cancer of low grade. For Pak1 (p21-activated kinase 1) and cyclin D1 the protein expression corresponded to the gene expression data. CONCLUSIONS: The results indicate that many 11q13 associated gene products are over-expressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response.

Association of cortactin, fascin-1 and epidermal growth factor receptor (EGFR) expression in ovarian carcinomas: correlation with clinicopathological parameters.

Cortactin, fascin-1 and EGFR are recognized as important factors in tumor progression. We tested the hypothesis that cortactin, fascin-1 and EGFR expression correlates with clinicopathological parameters of the four most common ovarian surface epithelial carcinomas--serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid adenocarcinoma, and clear cell carcinoma. Immunohistochemical analysis of cortactin, fascin-1 and EGFR was performed using tissue microarrays of 172 specimens comprising 69 serous cystadenocarcinomas, 44 mucinous cystadenocarcinomas, 45 endometrioid adenocarcinomas and 14 clear cell carcinomas. All ovarian carcinomas showed significant expression of cortactin, fascin-1 and EGFR in staining intensity, tumor percentages and immunostaining scores. In addition, higher immunostaining scores of fascin-1 correlated with more advanced cancer stages (TNM), poorer histological differentiation and poorer survival rate of mucinous cystadenocarcinoma. Similarly, higher immunostaining scores of cortactin correlated with T stages and histological differentiation of serous cystadenocarcinoma. The immunostaining scores of EGFR did not correlate with TNM stages, tumor differentiation or prognosis in the four ovarian surface epithelial carcinomas. Our findings suggest that cortactin and fascin-1 may serve as good biomarkers in evaluating aggressiveness of ovarian serous and mucinous cystadenocarcinoma. And the pharmacological inhibitors of fascin-1 activity may slow down tumor progression and prolong survival time in patients with mucinous cystadenocarcinoma.

Aberrant expression of cortactin in head and neck squamous cell carcinoma cells is associated with enhanced cell proliferation and resistance to the epidermal growth factor receptor inhibitor gefitinib.

The CTTN gene (formerly designated EMS1), encodes cortactin, a key regulator of dynamic actin networks. Both CTTN and CCND1, the latter encoding the cell cycle regulator cyclin D1, reside at chromosomal locus 11q13, a region commonly amplified in breast cancers and head and neck squamous cell carcinoma (HNSCC). Previously, we identified a novel role for cortactin in cancer cells, whereby cortactin overexpression attenuated ligand-induced down-regulation of the epidermal growth factor (EGF) receptor (EGFR), leading to sustained signaling. However, how this affected growth factor-induced cellular responses was unclear. Here, by modulation of cortactin expression in a panel of HNSCC cell lines, we show that cortactin overexpression enhances serum- and EGF-stimulated proliferation under both anchorage-dependent and anchorage-independent conditions and also increases resistance to anoikis (detachment-induced apoptosis). These effects are associated with increased activation of extracellular signal-regulated kinase and/or AKT. Furthermore, we report that cortactin stabilizes the c-MET receptor tyrosine kinase and enhances hepatocyte growth factor-induced mitogenesis and cell scattering. Therefore, cortactin may modulate signaling by a broader range of receptors than originally proposed and thereby affect a variety of responses. Finally, we have determined that cortactin overexpression, either alone or in combination with cyclin D1 up-regulation, promotes resistance to the EGFR kinase inhibitor gefitinib. These findings indicate that cortactin may play multiple roles in progression of HNSCC and should be evaluated as a marker of prognosis, disease progression, and therapeutic responsiveness, particularly to EGFR-directed agents.

Association of cortactin and fascin-1 expression in gastric adenocarcinoma: correlation with clinicopathological parameters.

Cortactin and fascin-1 are important factors in tumor progression. We tested the hypothesis that cortactin and fascin-1 expression correlates with clinicopathological parameters of gastric adenocarcinoma. Immunohistochemical analysis of cortactin and fascin-1 was done using tissue microarrays of 100 surgical specimens, including 20 well-differentiated, 20 moderately differentiated, and 60 poorly differentiated gastric adenocarcinomas. Among the 20 well-differentiated gastric adenocarcinomas, 15 cases (75%) showed negative or weak staining (1+); 5 cases (25%) had moderate (2+) or strong (3+) cortactin expression. Among the 60 poorly differentiated gastric adenocarcinomas, more than three-quarters of the cases (76.7%) had moderate or strong cortactin expression; 14 cases (23.3%) had weak staining. Of 20 well-differentiated gastric adenocarcinoma cases, 14 (70%) showed negative or weak staining of fascin-1, whereas nearly one-third (30%) had moderate or strong expression. Among the 60 poorly differentiated gastric adenocarcinomas, 32 (53.3%) exhibited moderate or strong fascin-1 expression; fewer than half of the cases showed negative or weak staining. Higher intensity of cortactin and fascin-1 staining correlated directly with more-advanced cancer stages (TNM) and inversely with survival rates. Our findings suggest the possibility that pharmacological inhibitors of cortactin and fascin-1 activity may slow down tumor progression and prolong survival time in patients with gastric adenocarcinomas.

Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia.

Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in cortactin by RNA interference knockdown methods. In these cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3-positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with cortactin expression levels. MMP inhibitor treatment of control cells mimicked the cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients.