Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling.

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The Ras-like protein R-Ras2/TC21 is important for proper mammary gland development

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.R-Ras2/TC21 is a GTPase with high sequence and signaling similarity with Ras subfamily members. Although it has been extensively studied using overexpression studies in cell lines, its physiological role remains poorly characterized. Here we used RRas2-knockout mice expressing â-galactosidase under the regulation of the endogenous RRas2 promoter to investigate the function of this GTPase in vivo. Despite its expression in tissues critical for organismal viability, RRas2 -/- mice show no major alterations in viability, growth rates, cardiovascular parameters, or fertility. By contrast, they display a marked and specific defect in the development of the mammary gland during puberty. In the absence of R-Ras2/TC21, this gland forms reduced numbers of terminal end buds (TEBs) and ductal branches, leading to a temporal delay in the extension and arborization of the gland tree in mammary fat pads. This phenotype is linked to cell-autonomous proliferative defects of epithelial cells present in TEBs. These cells also show reduced Erk activation but wild type - like levels of phosphorylated Akt. Using compound RRas2-, HRas-, and NRas-knockout mice, we demonstrate that these GTPases act in a nonsynergistic and nonadditive manner during this morphogenic process. © 2012 Larive et al.XRB work was supported by grants from the Spanish Association Against Cancer, the Spanish Ministry of Economy and Competitiveness (SAF2009-07172 and RD06/0020/0001) and the Castilla-León Autonomous Government (GR97). ES and BA work is supported by grants from the Spanish Ministry of Economy and Competitiveness (RD06/0020/0000 and RD06/0020/1002, respectively). RML, AA and CMC are supported by funding from the RD06/0020/0001 grant. Most Spanish funding is co-sponsored by the European FEDER program.Peer Reviewe

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness

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The rho exchange factors vav2 and vav3 control a lung metastasis-specific transcriptional program in breast cancer cells

The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, andmany of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs. Microarray analyses revealed that Vav2 and Vav3 controlled a vast transcriptional program in breast cancer cells through mechanisms that were shared between the two proteins, isoform-specific or synergistic. Furthermore, the abundance of Vavregulated transcripts wasmodulated by Rac1-dependent and Rac1-independent pathways. This transcriptome encoded therapeutically targetable proteins that played nonredundant roles in primary tumorigenesis and lung-specific metastasis, such as integrin-linked kinase (Ilk), the transforming growth factor-β family ligand inhibin βA, cyclooxygenase-2, and the epithelial cell adhesionmolecule Tacstd2. It also contained gene signatures that predicted disease outcome in breast cancer patients. These results identify possible targets for treating breast cancer and lung metastases and provide a potential diagnostic tool for clinical use.X.R.B.’s work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2009-07172 and RD06/0020/0001), the Castilla-León Autonomous Government (GR97), the 7th Framework European Union Program (FP7-HEALTH-2007-A-201862), and the Spanish Association Against Cancer. A portion of X.R.B.’s funding comes from the European Regional Development Fund. Work in J.M.P.’s laboratory is partially supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2006-00121 and RD06/0020/0029) and the Madrid Autonomous Government (S2006/BIO-0232). C.C.’s and M.M.-M.’s salaries were partially supported by the JAE-Doc program, the Spanish National Research Council, the RD06/0020/0001 grant, and the European Regional Development Fund.Peer Reviewe

Estimating spatial distribution of oxygen and hypoxia in tumor microenvironment: a mechanistic approach

International audienceBeing a hallmark of several solid tumors, hypoxia - a state of reduced level of tissue oxygen tension and a result of aberrant vasculature - leads to several alterations in the tumor microenvironment. Hypoxic regions of neoplasm are prone to be more resistant towards radiation therapy than compared to well oxygenated ones (A. L. Harris 2002). Furthermore, hypoxia and its mediators influence multiple signaling pathways and gene regulation to promote neovascularization, invasion, migration, adhesion, metastasis, and phenotypic switches (D. S. Widmer et al. 2013, A. Tameemi et al., 2019). Hence hypoxia is one of the leading factors which contributes towards intratumor heterogeneity and resistance against treatments, these two features being particularly important and common in many invasive tumors including melanoma (B. Bedogni et al. 2009, D'Aguanno et al. 2021). Estimation of accurate hypoxia profile would be key for better prognosis and design of more efficient treatment approaches. Mathematical modeling has been proven a useful tool to understand and predict such complex dynamics. Several computational and mathematical models have been proposed to describe tissue oxygenation, however the majority of them are restricted to synthetic data and qualitative results, lacking application to and connection with real tumor tissues and experimental results.We propose mechanistic modeling frameworks, which are driven by experimental data, to explain and mimic oxygen-hypoxia dynamics. The data is in the form of tissue scans of Patient Derived Xenograft (PDX) of breast, ovarian and pancreatic as well as human melanoma tumors. These scans of tumor tissue slices are immunohistochemical stained with CD31 -cluster of differentiation 31, marking the presence of endothelial cells- and CAIX- carbonic anhydrase IX, regulated by the hypoxia-inducible factor (HIF) 1, is an intrinsic marker of tumor hypoxia - markers. Keeping the data availability in mind, the distribution of oxygen is described by a reaction-diffusion partial differential equation with the source term incorporating the contribution from blood vessel density (obtained from CD31 staining) for the 2D model and from the vasculature architecture and the geometry of each blood vessel (reconstructed from several 2D tissue slices) for the 3D model. Next, hypoxia is modeled from the obtained oxygen distribution using an algebraic equation. The further steps include estimation of parameters and validation. The obtained parameters demonstrate biological relevance. 3D reconstruction, which is underway, is required for obtaining 3D profiles of oxygen and hypoxia. This requirement leads to another aspect of this work consisting in quantification of the error made when 2D models are used instead of more realistic 3D models. This is important since the 3D reconstruction is not always feasible, especially for patient tissue samples. A framework to quantify this approximation error would be essential for evaluating the hypoxia profile for clinical applications. Future work involves development of a general framework, applicable to most of the solid tumors, to estimate oxygen and hypoxia distribution based on the 3D reconstruction of blood vessels as well as for the 2D case with an error bound due to the approximation

Data driven mechanistic modeling of oxygen distribution and hypoxia profile in tumor microenvironment

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Data driven mechanistic modeling of oxygen distribution and hypoxia profile in tumor microenvironment

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PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness

International audienceClinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization

3D Reconstruction of H&E Whole Slide Images in Melanoma

International audienceCutaneous melanoma is an invasive cancer with a worldwide annual death toll of 57,000 (Arnold et al., JAMA Dermatol 2022). Changes in the tumor microenvironment, including the evolution of the metabolic states, have been suggested to be associated with poor chemotherapeutic response. To elucidate differing metabolic states and their correlation with treatment response, we have developed a vascular-based 3D pipeline for whole slide images (WSI). In this presentation, we share details of the pipeline, provide quantitative results to validate its performance, and share the challenges experienced and the solutions created to manage and process WSIs