Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion.

The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression. To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and three-dimensional cultures of MCF-10░A cells. We show that upon Dbl expression MCF-10░A cells undergo EMT. In addition, we found that Dbl overexpression sustain

p130Cas is an essential transducer element in ErbB2 transformation

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.-Cabodi, S., Tinnirello, A., Bisaro, B., Tornillo, G., Camacho-Leal, M. P., Forni, G., Cojoca, R., Iezzi, M., Amici, A., Montani, M., Eva, A., Di Stefano, P., Muthuswamy, S. K., Tarone, G., Turco, E., Defilippi, P. p130Cas is an essential transducer element in ErbB2 transformation

Partitioning Interpolant-Based Verificationfor effective Unbounded Model Checking

Interpolant-based model checking has been shown to be effective on large verification instances, as it efficiently combines automated abstraction and reachability fixed-point checks. On the other hand, methods based on variable quantification have proved their ability to remove free inputs, thus projecting the search space over state variables. In this paper we propose an integrated approach which combines the abstraction power of interpolation with techniques that rely on AIG and/or BDD representations of states, directly supporting variable quantification and fixed-point checks. The underlying idea of this combination is to adopt AIG- or BDD-based quantifications to limit and restrict the search space and the complexity of the interpolant-based approach. The exploited strategies, most of which are individually well-known, are integrated with a new flavor, specifically designed to improve their effectiveness on difficult verification instances. Experimental results, specifically oriented to hard-to-solve verification problems, show the robustness of our approach

Focal adhesion kinase contributes to proliferative potential of ErbB2 mammary tumour cells but is dispensable for ErbB2 mammary tumour induction in vivo

INTRODUCTION: Activation of focal adhesion kinase (FAK) is hypothesized to play an important role in the pathogenesis of human breast cancer. METHODS: To directly evaluate the role of FAK in mammary tumour progression, we have used a conditional FAK mouse model and mouse mammary tumour virus (MMTV)-driven Cre recombinase strain to inactivate FAK in the mammary epithelium of a transgenic mouse model of ErbB2 breast cancer. RESULTS: Although mammary epithelial disruption of FAK in this model resulted in both a delay in onset and a decrease in the number of neoplastic lesions, mammary tumours occurred in 100% of virgin female mice. All of the tumours and derived metastases that developed were proficient for FAK due to the absence of Cre recombinase expression. The hyperplastic epithelia where Cre-mediated recombination of FAK could be detected exhibited a profound proliferative defect. Consistent with these observations, disruption of FAK in established tumour cells resulted in reduced tumour growth that was associated with impaired proliferation. To avoid the selection for FAK-proficient ErbB2 tumour epithelia through escape of Cre-mediated recombination, we next intercrossed the FAK conditional mice with a separate MMTV-driven ErbB2 strain that co-expressed ErbB2 and Cre recombinase on the same transcriptional unit. CONCLUSIONS: While a delay in tumour induction was noted, FAK-deficient tumours arose in 100% of female animals indicating that FAK is dispensable for ErbB2 tumour initiation. In addition, the FAK-null ErbB2 tumours retained their metastatic potential. We further demonstrated that the FAK-related Pyk2 kinase is still expressed in these tumours and is associated with its downstream regulator p130Cas. These observations indicate that Pyk2 can functionally substitute for FAK in ErbB2 mammary tumour progression

SNIP/p140Cap mRNA expression is an unfavourable prognostic factor in breast cancer and is not expressed in normal breast tissue

The prevalence and clinical relevance of SNIP/p140Cap has not been extensively investigated. Here SNIP/p140Cap mRNA expression was studied in 103 breast tumour biopsies, where it was detected in ∼37% of tumour specimens, but not in any normal breast specimens. Expression correlated significantly with unfavourable overall survival. This suggests that SNIP/p140Cap may be a useful diagnostic and prognostic marker for breast cancer and its expression in breast cancer, but not in normal breast tissue, suggests that it may have potential as a therapeutic target

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

Background: Anoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. in our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.Methods: the beta 1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and beta 1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.Results: Differential association among Timp1, CD63 and beta 1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. in human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.Conclusions: Our results show that Timp1 is assembled in a supramolecular complex containing CD63 and beta 1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. in addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilLudwig Inst Canc Res, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Microbiol Immunol & Parasitol Dept, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/12306-1FAPESP: 2010/18715-8CAPES: 2867/10Web of Scienc