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La reprogrammation métabolique est l’un des marqueurs de la carcinogenèse. Au cœur de cette reprogrammation se trouvent les mitochondries qui produisent l’énergie sous forme de molécules d’ATP. La régulation spatio-temporelle de la production d’ATP, indispensable pour fournir l’énergie au bon endroit et au bon moment, est assurée par le transport intracellulaire des mitochondries. Les complexes Miro/TRAK présents à la surface des mitochondries se lient aux protéines motrices de la cellule (dynéine, kinésine, myosine) pour transporter les mitochondries le long du cytosquelette. Ces acteurs du transport mitochondrial sont souvent dérégulés dans le cancer. Nous présentons dans cette revue les mécanismes par lesquels le transport mitochondrial contribue à la migration, à la division cellulaire et à la réponse au stress des cellules cancéreuses. Décrypter ces mécanismes pourrait ouvrir la voie à de nouvelles approches thérapeutiques en oncologie.

Movie 4 AVI file - 879K, Time-lapse videomicroscopy of wound healing assay using GFP-ATIP3-transiently transfected D3H2LN cells

Movie 3 AVI file - 997K, Time-lapse videomicroscopy of wound healing assay using GFP-transiently transfected D3H2LN cells

Supplementary Tables XLS file - 200K, Table SI : breast tumors and normal samples data; Table SII: MTUS1 expression level in invasive breast tumors collected at Institut Gustave Roussy; Table SIII: Time-course of metastasis formation

Movie 2 AVI file - 2424K, Time-lapse videomicroscopy of wound healing assay using GFP-ATIP3-transfected D3H2LN clone (Cl3)

Movie 5 AVI file - 6439K, Time-lapse videomicroscopy of EB1-GFP comets in siRNA-silenced HeLa cells

Supplementary Information PDF file - 115K, Supplemental materials and methods, legends to Movies and supplemental references

Movie 6 AVI file - 5618K, Time-lapse videomicroscopy of EB3-GFP comets in wild type and mCherry-ATIP3 transfected MRC5 cells

Supplementary Figures PDF file - 486K, Figure S1. Low levels of ATIP3 associate with decreased overall and relapse-free survival of the patients;Figure S2. Anti-metastatic effects of ATIP3 in vivo; Figure S3. ATIP3 inhibits breast cancer cell proliferation and migration; Figure S4. ATIP3 regulates MT dynamics; Figure S5. Characterization of ATIP3 domains

Movie 1 AVI file - 2575K, Time-lapse videomicroscopy of wound healing assay using GFP-transfected D3H2LN clone

Movie 7 AVI file - 629K, Time-lapse videomicroscopy of wound healing assay in GFP-D2-transiently transfected D3H2LN cells

Cancer cells are characterized by a deregulation of their metabolic activity, which allows them to meet a high energy demand. Mitochondria are key organelles that control several metabolic processes and represent the main source of energy in the form of ATP. Intracellular transport of mitochondria is essential for addressing these organelles to the right place at the right time according to energy requirement. Mitochondrial transport in cancer cells involves mitochondria-associated Miro/TRAK complexes that bind to motor proteins (kinesins, dyneins and myosins) to promote mitochondrial displacement along microtubules or actin filaments. This review focuses on the molecular players of intracellular mitochondrial transport along microtubules during cell migration and mitosis, and their deregulation in tissues from cancer patients. Intercellular mitochondrial transport upon cancer cell exposure to hypoxia or chemotherapy is also presented. This field of investigation opens new interesting perspectives in oncology, as targeting mitochondrial transport may represent an innovative strategy for treating cancer.Le transport mitochondrial - Quel impact dans le cancer ?La reprogrammation métabolique est l’un des marqueurs de la carcinogenèse. Au cœur de cette reprogrammation se trouvent les mitochondries qui produisent l’énergie sous forme de molécules d’ATP. La régulation spatio-temporelle de la production d’ATP, indispensable pour fournir l’énergie au bon endroit et au bon moment, est assurée par le transport intracellulaire des mitochondries. Les complexes Miro/TRAK présents à la surface des mitochondries se lient aux protéines motrices de la cellule (dynéine, kinésine, myosine) pour transporter les mitochondries le long du cytosquelette. Ces acteurs du transport mitochondrial sont souvent dérégulés dans le cancer. Nous présentons dans cette revue les mécanismes par lesquels le transport mitochondrial contribue à la migration, à la division cellulaire et à la réponse au stress des cellules cancéreuses. Décrypter ces mécanismes pourrait ouvrir la voie à de nouvelles approches thérapeutiques en oncologie.

Breast cancer is one of the most frequent malignancies among women worldwide. Based on clinical and molecular features of breast tumors, patients are treated with chemotherapy, hormonal therapy and/or radiotherapy and more recently with immunotherapy or targeted therapy. These different therapeutic options have markedly improved patient outcomes. However, further improvement is needed to fight against resistance to treatment. In the rapidly growing area of research for personalized medicine, predictive biomarkers - which predict patient response to therapy - are essential tools to select the patients who are most likely to benefit from the treatment, with the aim to give the right therapy to the right patient and avoid unnecessary overtreatment. The search for predictive biomarkers is an active field of research that includes genomic, proteomic and/or machine learning approaches. In this review, we describe current strategies and innovative tools to identify, evaluate and validate new biomarkers. We also summarize current predictive biomarkers in breast cancer and discuss companion biomarkers of targeted therapy in the context of precision medicine.

International audience; Breast cancer is the leading cause of death by malignancy among women worldwide. Clinical data and molecular characteristics of breast tumors are essential to guide clinician’s therapeutic decisions. In the new era of precision medicine, that aims at personalizing the treatment for each patient, there is urgent need to identify robust companion biomarkers for new targeted therapies. This review focuses on ATIP3, a potent anti-cancer protein encoded by candidate tumor suppressor gene MTUS1, whose expression levels are markedly down-regulated in breast cancer. ATIP3 is a microtubule-associated protein identified both as a prognostic biomarker of patient survival and a predictive biomarker of breast tumors response to taxane-based chemotherapy. We present here recent studies pointing out ATIP3 as an emerging anti-cancer protein and a potential companion biomarker to be combined with future personalized therapy against ATIP3-deficient breast cancer.

International audience; Maintaining the integrity of the mitotic spindle in metaphase is essential to ensure normal cell division. We show here that depletion of microtubule-associated protein ATIP3 reduces metaphase spindle length. Mass spectrometry analyses identi-ied the microtubule minus-end depolymerizing kinesin Kif2A as an ATIP3 binding protein. We show that ATIP3 controls metaphase spindle length by interacting with Kif2A and its partner Dda3 in an Aurora kinase A-dependent manner. In the absence of ATIP3, Kif2A and Dda3 accumulate at spindle poles, which is consistent with reduced poleward microtubule lux and shortening of the spindle. ATIP3 silencing also limits Aurora A localization to the poles. Transfection of GFP-Aurora A, but not kinase-dead mutant, rescues the phenotype, indicating that ATIP3 maintains Aurora A activity on the poles to control Kif2A targeting and spindle size. Collectively, these data emphasize the pivotal role of Aurora kinase A and its mutual regulation with ATIP3 in controlling spindle length.

International audience; Taxanes are microtubule-targeting drugs used as cytotoxic chemotherapy to treat most solid tumors. The development of resistance to taxanes is a major cause of therapeutic failure and overcoming chemoresistance remains an important challenge to improve patient's outcome. Extensive efforts have been made recently to identify predictive biomarkers to select populations of patients who will benefit from taxane-based chemotherapy and avoid inefficient treatment of patients with innate resistance. This, together with the discovery of new mechanisms of resistance that include metabolic reprogramming and dialogue between tumor and its microenvironment, pave the way to a new era of personalized medicine. In this review, we recapitulate recent insights into taxane resistance and present promising emerging strategies to overcome chemoresistance in the future.

Taxane-based chemotherapy is frequently used in neoadjuvant treatment of breast cancer patients to reduce tumor growth and lymph node metastasis. However, few patients benefit from chemotherapy and predictive biomarkers of chemoresistance are needed. The microtubule-associated protein ATIP3 has recently been identified as a predictive biomarker whose low levels in breast tumors are associated with increased sensitivity to chemotherapy. In this study, we investigated whether ATIP3 deficiency may impact the effects of paclitaxel on cancer cell migration and lymph node metastasis. Expression levels of ATIP3 were analyzed in a cohort of 133 breast cancer patients and classified according to lymph node positivity following neoadjuvant chemotherapy. Results showed that low ATIP3 levels are associated with reduced axillary lymph node metastasis. At the functional level, ATIP3 depletion increases cell migration, front-rear polarity and microtubule dynamics at the plus ends, but paradoxically sensitizes cancer cells to the inhibitory effects of paclitaxel on these processes. ATIP3 silencing concomitantly increases the incorporation of fluorescent derivative of Taxol along the microtubule lattice. Together our results support a model in which alterations of microtubule plus ends dynamics in ATIP3-deficient cells may favor intracellular accumulation of paclitaxel, thereby accounting for increased breast tumor sensitivity to chemotherapy.

International audience; Predictive biomarkers for tumor response to neoadjuvant chemotherapy are needed in breast cancer. This study investigates the predictive value of 280 genes encoding proteins that regulate microtubule assembly and function. By analyzing 3 independent multicenter randomized cohorts of breast cancer patients, we identified 17 genes that are differentially regulated in tumors achieving pathological complete response (pCR) to neoadjuvant chemotherapy. We focused on the MTUS1 gene, whose major product, ATIP3, is a microtubule-associated protein down-regulated in aggressive breast tumors. We show here that low levels of ATIP3 are associated with an increased pCR rate, pointing to ATIP3 as a predictive biomarker of breast tumor chemosensitivity. Using preclinical models of patient-derived xenografts and 3-dimensional models of breast cancer cell lines, we show that low ATIP3 levels sensitize tumors to the effects of taxanes but not DNA-damaging agents. ATIP3 silencing improves the proapoptotic effects of paclitaxel and induces mitotic abnormalities, including centrosome amplification and multipolar spindle formation, which results in chromosome missegregation leading to aneuploidy. As shown by time-lapse video microscopy, ATIP3 depletion exacerbates cytokinesis failure and mitotic death induced by low doses of paclitaxel. Our results favor a mechanism by which the combination of ATIP3 deficiency and paclitaxel treatment induces excessive aneuploidy, which in turn results in elevated cell death. Together, these studies highlight ATIP3 as an important regulator of mitotic integrity and a useful predictive biomarker for a population of chemoresistant breast cancer patients.

International audience; Metastatic melanoma is an aggressive type of skin cancer leading half of the patients todeath within 8–10 months after diagnosis. Kinins are peptides that interact with B1 andB2 receptors playing diverse biological roles. We investigated whether treatment with B1receptor agonist, des-Arg9-bradykinin (DABK), has effects in lung metastasis establishmentafter melanoma induction in mice. We found a lower number of metastatic colonies in lungsof DABK-treated mice, reduced expression of vascular cell adhesion molecule 1 (VCAM-1), and increased CD8+T-cell recruitment to the metastatic area compared to animalsthat did not receive treatment. To understand whether the effects of DABK observedwere due to the activation of the B1 receptor in the tumor cells or in the host, we treatedwild-type (WT) and kinin B1 receptor knockout (B1−/−) mice with DABK. No significantdifferences in the number of melanoma colonies established in lungs were seen betweenWT and B1−/−mice; however, B1−/−mice presented higher VCAM-1 expression and lowerCD8+T-cell infiltration. In conclusion, we believe that activation of kinin B1 receptor by itsagonist in the host stimulates the immune response more efficiently, promoting CD8+Tcellrecruitment to the metastatic lungs and interfering in VCAM-1 expression. Moreover,treatment with DABK reduced establishment of metastatic colonies by mainly actingon tumor cells; hence, this study brings insights to explore novel approaches to treatmetastatic melanoma targeting the B1 receptor.

International audience; Breast cancer is the most common malignancy in women worldwide. Although important therapeutic progress was achieved over the past decade, this disease remains a public health problem. In the light of precision medicine, the identification of new prognostic biomarkers in breast cancer is urgently needed to stratify populations of patients with poor clinical outcome who may benefit from new personalized therapies. The microtubule cytoskeleton plays a pivotal role in essential cellular functions and is an interesting target for cancer therapy. Microtubule assembly and dynamics are regulated by a wide range of microtubule-associated proteins (MAPs), some of which have oncogenic or tumor suppressors effects in breast cancer. This review covers current knowledge on microtubule-associated tumor suppressors (MATS) in breast cancer and their potential value as prognostic biomarkers. We present recent studies showing that combinatorial expression of ATIP3 and EB1, two microtubule-associated biomarkers with tumor suppressor and oncogenic effects, respectively, improves breast cancer prognosis compared to each biomarker alone. These findings are discussed regarding the increasing complexity of protein networks composed of MAPs that coordinate microtubule dynamics and functions. Further studies are warranted to evaluate the prognostic value of combined expression of different MATS and their interacting partners in breast cancer.

Simple Summary Reprogramming metabolism is a hallmark of cancer. Warburg’s effect, defined as increased aerobic glycolysis at the expense of mitochondrial respiration in cancer cells, opened new avenues of research in the field of cancer. Later findings, however, have revealed that mitochondria remain functional and that they actively contribute to metabolic plasticity of cancer cells. Understanding the mechanisms by which mitochondrial metabolism controls tumor initiation and progression is necessary to better characterize the onset of carcinogenesis. These studies may ultimately lead to the design of novel anti-cancer strategies targeting mitochondrial functions. Abstract Carcinogenesis is a multi-step process that refers to transformation of a normal cell into a tumoral neoplastic cell. The mechanisms that promote tumor initiation, promotion and progression are varied, complex and remain to be understood. Studies have highlighted the involvement of oncogenic mutations, genomic instability and epigenetic alterations as well as metabolic reprogramming, in different processes of oncogenesis. However, the underlying mechanisms still have to be clarified. Mitochondria are central organelles at the crossroad of various energetic metabolisms. In addition to their pivotal roles in bioenergetic metabolism, they control redox homeostasis, biosynthesis of macromolecules and apoptotic signals, all of which are linked to carcinogenesis. In the present review, we discuss how mitochondria contribute to the initiation of carcinogenesis through gene mutations and production of oncometabolites, and how they promote tumor progression through the control of metabolic reprogramming and mitochondrial dynamics. Finally, we present mitochondrial metabolism as a promising target for the development of novel therapeutic strategies.

International audience; Purpose: The identification of molecular biomarkers for classification of breast cancer is needed to better stratify the patients and guide therapeutic decisions. The aim of this study was to investigate the value of MAPRE1 gene encoding microtubule-end binding proteins EB1 as a biomarker in breast cancer and evaluate whether combinatorial expression of MAPRE1 and MTUS1 gene encoding EB1-negative regulator ATIP3 may improve breast cancer diagnosis and prognosis.Methods: Probeset intensities for MAPRE1 and MTUS1 genes were retrieved from Exonhit splice array analyses of 45 benign and 120 malignant breast tumors for diagnostic purposes. Transcriptomic analyses (U133 Affymetrix array) of one exploratory cohort of 150 invasive breast cancer patients and two independent series of 130 and 155 samples were compared with clinical data of the patients for prognostic studies. A tissue microarray from an independent cohort of 212 invasive breast tumors was immunostained with anti-EB1 and anti-ATIP3 antibodies.Results: We show that MAPRE1 gene is a diagnostic and prognostic biomarker in breast cancer. High MAPRE1 levels correlate with tumor malignancy, high histological grade and poor clinical outcome. Combination of high-MAPRE1 and low-MTUS1 levels in tumors is significantly associated with tumor aggressiveness and reduced patient survival. IHC studies of combined EB1/ATIP3 protein expression confirmed these results.Conclusions: These studies emphasize the importance of studying combinatorial expression of EB1 and ATIP3 genes and proteins rather than each biomarker alone. A population of highly aggressive breast tumors expressing high-EB1/low-ATIP3 may be considered for the development of new molecular therapies.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

International audience; The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of "endogenous EB1 antagonists" and emphasize the importance of finely regulating EB1 function at growing microtubule ends.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.

Introduction Neo-adjuvant chemotherapy combining taxanes and anthracyclines represents an interesting option for a number of breast cancer patients. However, the proportion of patients who achieve a pathological complete response (pCR) after treatment remains low, reaching 15%–20% in the whole population. Thus, identifying predictive biomarkers of drug resistance is necessary to select the patients who are most likely to benefit from chemotherapy. Taxanes (paclitaxel, docetaxel) are potent stabilisers of the microtubule cytoskeleton, which functions are tightly regulated by a panel of MT-associated proteins and mitotic kinases. In the present study, we evaluated whether taxane resistance in breast cancer may be associated with alterations of genes encoding MT-regulatory proteins. Material and methods Expression levels of 411 MT-related genes were analysed from Affymetrix U133 microarray of breast tumours from three independent multicentered clinical trials, namely REMAGUS-02 (115 patients), REMAGUS-04 (142 patients) and MD Anderson (133 patients). Only patients treated with neoadjuvant taxane-based chemotherapy were included in this study. Expression level of MT-related genes in tumours was compared with the ability to achieve pCR. Results and discussions By comparing expression levels of 411 MT-related genes in three independent cohorts of breast cancer patients treated with taxane-based neo-adjuvant chemotherapy, we have identified a total of 94 MT-related genes that are differentially expressed in tumours with pCR. Among those, 18 MT-related genes are common to the three cohorts. Interestingly, all these genes appear as potential prognostic biomarkers of overall survival. Ongoing studies aim at functionally validating these candidates using siRNA screening in two different breast cancer cell lines. Conclusion This study should lead to the identification of novel predictive biomarkers to select breast cancer patients who are likely to benefit from taxane-based chemotherapy. This should also pave the way for future targeted treatments of chemoresistant breast tumours.

AT2, the second subtype of angiotensin II receptors, is a major component of the renin-angiotensin system involved in cardiovascular and neuronal functions. AT2 belongs to the superfamily of G protein-coupled receptors, but its intracellular signaling pathways have long remained elusive. Over the past few years, efforts to characterize this atypical receptor have led to the identification of novel molecular scaffolds that directly bind to its intracellular tail. The present review focuses on a family of AT2 receptor-interacting proteins (ATIPs) involved in neuronal differentiation, vascular remodeling and tumor suppression. Recent findings that ATIPs and ATIP-related proteins associate with microtubules suggest that they might constitute a novel family of multifunctional proteins regulating a wide range of physiopathological functions.

Angiotensin II (Ang II) AT2 receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10-7 M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT2 antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT2 receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT2 or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT2 or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT2 receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT2 receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT2 subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes. Fil: Alvarez, Sergio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Seguin, Leonardo Roque. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Villarreal, Rodrigo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina Fil: Nahmias, Clara. Universite de Paris V. Institut Cochin.; Francia Fil: Ciuffo, Gladys Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia; Argentina

Sao Paulo State Research Foundation (FAPESP) FAPESP Melanoma is a very aggressive tumor that arises from melanocytes. Late stage and widely spread diseases do not respond to standard therapeutic approaches. The kallikrein-kinin system (KKS) participates in biological processes such as vasodilatation, pain and inflammatory response. However, the role of KKS in tumor formation and progression is not completely understood. The role of the host kinin B1 receptor in melanoma development was evaluated using a syngeneic melanoma model. Primary tumors and metastasis were respectively induced by injecting B16F10 melanoma cells, which are derived from C57BL/6 mice, subcutaneously or in the tail vein in wild type C57BL/6 and B1 receptor knockout mice (B1(-/-)). Tumors developed in B1(-/-) mice presented unfavorable prognostic factors such as increased incidence of ulceration, higher levels of IL-10, higher activation of proliferative pathways such as ERK1/2 and Akt, and increased mitotic index. Furthermore, in the metastasis model, B1(-/-) mice developed larger metastatic colonies in the lung and lower CD8(+) immune effector cells when compared with WT animals. Altogether, our results provide evidences that B1(-/-) animals developed primary tumors with multiple features associated with poor prognosis and unfavorable metastatic onset, indicating that the B1 receptor may contribute to improve the host response against melanoma progression. Univ Sao Paulo, Dep Biochem & Immunol, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Pathol, BR-14049900 Ribeirao Preto, Brazil Univ Fed Sao Paulo, Dept Biophys, BR-04039032 Sao Paulo, Brazil Inst Gustave Roussy, INSERM, U981, F-94800 Villejuif, France Univ Fed Sao Paulo, Dept Biophys, BR-04039032 Sao Paulo, Brazil FAPESP: 2010/13346-4 FAPESP: 2012/20148-0 FAPESP: 2011/02144-4 Web of Science

Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development, but also in pathological conditions to protect the brain against stroke; however, the detailed mechanisms are unclear. Here, we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2, one of the ubiquitin-conjugating enzyme variants. The AT2 receptor, MMS2, Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1), and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner, reaching a peak after 4 h of stimulation, and this effect was enhanced with AT1 receptor blocker, valsartan, but inhibited by AT2 receptor blocker PD123319. Moreover, we observed that an AT2 receptor agonist, CGP42112A, alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover, the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor, sodium orthovanadate. After AT2 receptor stimulation, ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore, increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1.

// Lauriane Velot 1,2,3,* , Angie Molina 1,2,3,* , Sylvie Rodrigues-Ferreira 1,2,3 , Anne Nehlig 1,2 , Benjamin Pierre Bouchet 4 , Marina Morel 3 , Ludovic Leconte 5 , Laurence Serre 6 , Isabelle Arnal 6 , Diane Braguer 7,8 , Ariel Savina 9 , Stephane Honore 7,8 and Clara Nahmias 1,2,3 1 Inserm U981, Institut Gustave Roussy Department of Molecular Medicine, Villejuif, France 2 Universite Paris-Saclay, Villejuif, France 3 CNRS UMR8104, Institut Cochin, Paris, France 4 Cell Biology, Faculty of Science, Utrecht University, Padualaan, CH Utrecht, The Netherlands 5 Cell and Tissue Imaging Core Facilty, PICT-IBiSA, CNRS UMR144 Institut Curie, Centre de Recherche, Paris, France 6 Inserm U836, Grenoble Institut des Neurosciences, Grenoble, France 7 Aix Marseille Universite, Inserm, CRO2 UMR_S 911, Marseille, France 8 APHM, Hopital Timone, Marseille, France 9 Scientific Partnerships Roche SAS, Boulogne Billancourt, France * These authors have contributed equally to this work Correspondence to: Clara Nahmias, email: // Keywords : EB1, MTUS1, protein interaction, +TIP, microtubule dynamics Received : May 07, 2015 Accepted : October 14, 2015 Published : October 20, 2015 Abstract The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor.

V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer.

G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules and vasoactive peptides. Among those, angiotensins (angiotensin II and angiotensin 1-7) are the major biologically active products of the classical and alternative Renin-Angiotensin System (RAS). These peptides bind and activate three different subtypes of GPCRs, namely AT1, AT2 and Mas receptors, to regulate cardiovascular functions. Over the past decade, the contribution of several RAS components in tumorigenesis has emerged as a novel important concept, Angiotensin II being considered as harmful and Angiotensin 1-7 as protective against cancer. Development of selective ligands targeting each RAS receptor may provide novel and efficient targeted therapeutic strategies against cancer. In this review, we focus on breast cancer to summarize current knowledge on angiotensin receptors (AT1, AT2, and Mas), and discuss the potential use of angiotensin receptor agonists and antagonists in clinics.

L’endothéline-1 (ET-1) et l’angiotensine II (AngII) sont des peptides vasoactifs, mais aussi mitogènes et pro-angiogéniques. Tous deux exercent leurs actions par l’intermédiaire de récepteurs couplés aux protéines G : ETA-R et ETB-R pour ET-1 ; AT1R et AT2R pour AngII. L’expression des composants des systèmes ET-1 et AngII dans diverses tumeurs présente généralement une ou plusieurs des caractéristiques suivantes : surexpression du peptide et/ou du récepteur, modification du sous-type de récepteur exprimé et localisation nucléaire du récepteur. ET-1 et AngII agissent sur les différentes étapes de la progression tumorale, et l’utilisation d’antagonistes spécifiques de leurs récepteurs, ou d’inhibiteurs de leur synthèse, est efficace pour ralentir la croissance tumorale in vitro et in vivo dans différents modèles animaux. Des essais cliniques utilisant des antagonistes d’ETA-R donnent des résultats encourageants pour le traitement antitumoral, et une approche similaire ayant pour objectif de bloquer ETB-R ou AT1R est envisageable. De plus, une thérapie combinée ciblant les deux systèmes, ET-1 et AngII, pourrait se révéler bénéfique pour le traitement de tumeurs fortement angiogéniques., Endothelin-1 (ET-1) and angiotensin II (AngII), two potent vasoactive peptides involved in the regulation of cardiovascular homeostasis, also induce mitogenic and pro-angiogenic responses in vitro and in vivo. Both peptides are produced by cleavage of inactive precursors by metalloproteases (endothelin-converting enzyme and angiotensin-converting enzyme, respectively) and activate two subtypes of membrane receptors (ETA-R and ETB-R for ET-1, AT1R and AT2R for AngII) that all belong to the superfamily of G-protein coupled receptors. There is increasing evidence that ETA-R, ETB-R and AT1R are expressed in a variety of cancer cells and tissues, and may play a role on tumor growth, angiogenesis and invasion in vivo. This review summarizes the similarities and differences between the ET-1 and AngII systems with regard to their reported effects on various aspects of cancer. In addition to being expressed on vascular endothelium, ET-1 and AngII receptors participate in tumor angiogenesis through the production of the angiogenic factor VEGF. Furthermore, recent clinical studies indicate that a selective ETA-R antagonist has beneficial effects in prostate cancer, suggesting that a similar approach using ETB-R and AT1R blockers might be envisioned. Experimental data presented here suggest that a combined therapy targeting both ET-1 and AngII systems may prove valuable for future treatments of highly angiogenic tumors.

There is increasing evidence that Angiotensin II (AngII), a major regulator of blood pressure and cardiovascular homeostasis, is involved in the regulation of cell proliferation, angiogenesis, inflammation and tissue remodeling, which suggests that this peptide might also play a role in cancer. This review focuses on the expression and function of Angiotensin I-converting enzyme (ACE) and AngII receptors in various aspects of cancer. Recent experimental data suggests that ACE inhibitors and AngII type 1 receptor (AT1R) antagonists have beneficial effects on tumor progression, vascularization and metastasis, and that the AngII type 2 receptor (AT2R) subtype has a potential role in cancer. An overview of the major intracellular signaling pathways associated with AT1R and AT2R activation in cancer cells, as well as in endothelial and inflammatory cells, is presented here.

In the present study we examined the effects of angiotensin II (Ang II) type 2 (AT(2)) receptor stimulation on AT(1) receptor-mediated monocyte chemoattractant protein-1 (MCP-1) expression and the possible mechanisms of AT(2) receptor-mediated signaling in cultured rat fetal vascular smooth muscle cells, which express both AT(1) and AT(2) receptors. Ang II stimulation induced MCP-1 mRNA expression as well as an increase in nuclear factor-kappaB (NF-kappaB) binding to the corresponding cis DNA element of the MCP-1 promoter region and a decrease in the cytosolic inhibitory protein-kappaB (IkappaB) protein level via AT(1) receptor stimulation, whereas stimulation of the AT(2) receptor decreased Ang II-induced MCP-1 expression, NF-kappaB DNA binding, and IkappaB degradation, suggesting that activation of the AT(2) receptor attenuated AT(1) receptor-mediated MCP-1 expression via a decrease in NF-kappaB DNA binding and an increase in IkappaB stability. Moreover, we demonstrated that AT(2) receptor stimulation attenuated TNFalpha-mediated NF-kappaB activation and MCP-1 expression. A tyrosine phosphatase inhibitor, orthovanadate, attenuated the AT(2) receptor-mediated increase in IkappaB protein. Moreover, we observed that two IkappaB subunits (IkappaBalpha and IkappaBbeta) were tyrosine-phosphorylated after Ang II stimulation. Transfection of a dominant-negative Src homology protein tyrosine phosphatase-1 mutant into vascular smooth muscle cells inhibited the AT(2) receptor-mediated increase in IkappaB, leading to a significant increase in AT(1) receptor-induced NF-kappaB activation and MCP-1 expression. Taken together, our results demonstrated that AT(2) receptor stimulation attenuated MCP-1 expression via IkappaB stabilization, and Src homology protein tyrosine phosphatase-1 might play a critical role in the transcriptional regulation of MCP-1 expression through the control of IkappaB protein stability.

The contribution of various components of the renin–angiotensin system in tumorigenesis is an important emerging concept that may lead to the development of novel targeted therapeutic strategies against cancer. There is increasing evidence that AT 1 receptors contribute to tumor progression, whereas Mas receptors have beneficial effects against cancer. Studies examining the expression and effects of the angiotensin II type 2 (AT 2 ) receptor in cancer have led to controversial results, suggesting that AT 2 functions may be context-dependent.

In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85alpha subunit of PI3K, but led to a significant reduction of insulin-induced p85alpha phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.

Accumulating evidence suggests that estrogen exerts cardioprotective effects and protects against neointima formation in response to vascular injury in vivo, whereas angiotensin (Ang) II stimulation via the Ang II type 1 (AT 1 ) receptor exaggerates vascular injury. We postulate that estrogen treatment antagonizes the AT 1 receptor-mediated growth-promoting effects in vascular smooth muscle cells (VSMCs). The present in vitro study was designed to explore this possibility and to establish the cellular mechanism whereby estrogen attenuates the growth of VSMCs. Primary cultures of VSMCs derived from male adult Sprague-Dawley rats express exclusively AT 1 receptors. Treatment with Ang II enhanced proliferation of VSMC and c- fos expression, whereas 17β-estradiol (E2) attenuated these vasotrophic effects of Ang II. We also demonstrated that E2 attenuated AT 1 receptor-mediated extracellular signal-regulated kinase activation and that this effect of E2 was restored by pretreatment with vanadate or okadaic acid. Moreover, we demonstrated that E2 enhanced SHP-1 activity, rapidly reaching a peak after 3 minutes of E2 stimulation, whereas E2 transactivated mitogen-activated protein kinase phosphatase-1 expression, showing a peak after 60 minutes of E2 treatment. SHP-1 activation was not influenced by actinomycin D treatment, whereas E2-mediated mitogen-activated protein kinase phosphatase-1 expression was attenuated. Taken together, our results suggest a novel mechanism of vasoprotection by which estrogen antagonizes the effect of the AT 1 receptor via the activation and induction of phosphatases through nongenomic as well as genomic signaling.

> Les microtubules, formes de l’assemblage dynamique de dimeres d’alpha et de beta-tubuline, jouent un role essentiel dans l’homeostasie cellulaire. Dans les cellules en interphase, le cytosquelette de microtubules intervient dans le maintien de l’architecture cellulaire, la migration et le trafic intracellulaire de proteines et d’organites ; en mitose, il se reorganise pour former le fuseau mitotique qui permet une repartition correcte des chromosomes [1]. La dynamique de polymerisation des microtubules, essentielle a leur fonction, est sous le controle d’un ensemble de proteines agissant de concert : les MAP (microtubule-associated proteins) dont l’activite est finement regulee dans l’espace et le temps [1, 2]. Les alterations de la structure ou de la regulation des MAP peuvent avoir des repercussions considerables dans de nombreuses situations physiopathologiques, comme c’est le cas par exemple pour la proteine APC (adenomatous polyposis coli) dans le cancer du colon, ou la proteine Tau dans la maladie d’Alzheimer.