A Network of 17 Microtubule-Related Genes Highlights Functional Deregulations in Breast Cancer.

A wide panel of microtubule-associated proteins and kinases is involved in coordinated regulation of the microtubule cytoskeleton and may thus represent valuable molecular markers contributing to major cellular pathways deregulated in cancer. We previously identified a panel of 17 microtubule-related (MT-Rel) genes that are differentially expressed in breast tumors showing resistance to taxane-based chemotherapy. In the present study, we evaluated the expression, prognostic value and functional impact of these genes in breast cancer. We show that 14 MT-Rel genes (KIF4A, ASPM, KIF20A, KIF14, TPX2, KIF18B, KIFC1, AURKB, KIF2C, GTSE1, KIF15, KIF11, RACGAP1, STMN1) are up-regulated in breast tumors compared with adjacent normal tissue. Six of them (KIF4A, ASPM, KIF20A, KIF14, TPX2, KIF18B) are overexpressed by more than 10-fold in tumor samples and four of them (KIF11, AURKB, TPX2 and KIFC1) are essential for cell survival. Overexpression of all 14 genes, and underexpression of 3 other MT-Rel genes (MAST4, MAPT and MTUS1) are associated with poor breast cancer patient survival. A Systems Biology approach highlighted three major functional networks connecting the 17 MT-Rel genes and their partners, which are centered on spindle assembly, chromosome segregation and cytokinesis. Our studies identified mitotic Aurora kinases and their substrates as major targets for therapeutic approaches against breast cancer.

Predictive biomarkers for personalized medicine in breast cancer.

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.

[Mitochondrial transport: How does it impact cancer?] / Le transport mitochondrial - Quel impact dans le cancer ?

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.

Title: Le transport mitochondrial - Quel impact dans le cancer ? Abstract: 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.

Mitochondrial Metabolism in Carcinogenesis and Cancer Therapy.

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.

Microtubule-Associated Protein ATIP3, an Emerging Target for Personalized Medicine in Breast Cancer.

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.

Predicting and Overcoming Taxane Chemoresistance.

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.

Reciprocal regulation of Aurora kinase A and ATIP3 in the control of metaphase spindle length.

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 identified 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 flux 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.

Organotypic Modeling of the Tumor Landscape.

Cancer is a complex disease and it is now clear that not only epithelial tumor cells play a role in carcinogenesis. The tumor microenvironment is composed of non-stromal cells, including endothelial cells, adipocytes, immune and nerve cells, and a stromal compartment composed of extracellular matrix, cancer-associated fibroblasts and mesenchymal cells. Tumorigenesis is a dynamic process with constant interactions occurring between the tumor cells and their surroundings. Even though all connections have not yet been discovered, it is now known that crosstalk between actors of the microenvironment drives cancer progression. Taking into account this complexity, it is important to develop relevant models to study carcinogenesis. Conventional 2D culture models fail to represent the entire tumor microenvironment properly and the use of animal models should be decreased with respect to the 3Rs rule. To this aim, in vitro organotypic models have been significantly developed these past few years. These models have different levels of complexity and allow the study of tumor cells alone or in interaction with the microenvironment actors during the multiple stages of carcinogenesis. This review depicts recent insights into organotypic modeling of the tumor and its microenvironment all throughout cancer progression. It offers an overview of the crosstalk between epithelial cancer cells and their microenvironment during the different phases of carcinogenesis, from the early cell autonomous events to the late metastatic stages. The advantages of 3D over classical 2D or in vivo models are presented as well as the most promising organotypic models. A particular focus is made on organotypic models used for studying cancer progression, from the less complex spheroids to the more sophisticated body-on-a-chip. Last but not least, we address the potential benefits of these models in personalized medicine which is undoubtedly a domain paving the path to new hopes in terms of cancer care and cure.

ATIP3 deficiency facilitates intracellular accumulation of paclitaxel to reduce cancer cell migration and lymph node metastasis in breast cancer patients.

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.

From tumorigenesis to cell death: the aneuploidy paradox.

Aneuploidy, an abnormal chromosome number, is a hallmark of cancer. We recently showed that depletion of microtubule-associated protein ATIP3 (AT2 receptor-interacting protein 3) induces aneuploidy and sensitizes breast cancer cells to taxanes. Combining taxane treatment with ATIP3 depletion cooperates to reach a detrimental level of aneuploidy.

Microtubule-associated tumor suppressors as prognostic biomarkers in breast cancer.

PURPOSE: 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 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 suppressor effects in breast cancer. RESULTS: 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. CONCLUSIONS: 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.

Improving breast cancer sensitivity to paclitaxel by increasing aneuploidy.

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.

Activation of the Kinin B1 Receptor by Its Agonist Reduces Melanoma Metastasis by Playing a Dual Effect on Tumor Cells and Host Immune Response.

Metastatic melanoma is an aggressive type of skin cancer leading half of the patients to death within 8-10 months after diagnosis. Kinins are peptides that interact with B1 and B2 receptors playing diverse biological roles. We investigated whether treatment with B1 receptor agonist, des-Arg9-bradykinin (DABK), has effects in lung metastasis establishment after melanoma induction in mice. We found a lower number of metastatic colonies in lungs of 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 animals that did not receive treatment. To understand whether the effects of DABK observed were due to the activation of the B1 receptor in the tumor cells or in the host, we treated wild-type (WT) and kinin B1 receptor knockout (B1-/-) mice with DABK. No significant differences in the number of melanoma colonies established in lungs were seen between WT and B1-/-mice; however, B1-/-mice presented higher VCAM-1 expression and lower CD8+T-cell infiltration. In conclusion, we believe that activation of kinin B1 receptor by its agonist in the host stimulates the immune response more efficiently, promoting CD8+T-cell recruitment to the metastatic lungs and interfering in VCAM-1 expression. Moreover, treatment with DABK reduced establishment of metastatic colonies by mainly acting on tumor cells; hence, this study brings insights to explore novel approaches to treat metastatic melanoma targeting the B1 receptor.

Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis.

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.

Regulation of end-binding protein EB1 in the control of microtubule dynamics.

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.

Host kinin B1 receptor plays a protective role against melanoma progression.

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.

Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

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.

G-protein coupled receptors of the renin-angiotensin system: new targets against breast 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 (AngII) 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, AngII being considered as harmful and Ang1-7 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.

Vemurafenib resistance selects for highly malignant brain and lung-metastasizing melanoma cells.

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.

Astrocytes facilitate melanoma brain metastasis via secretion of IL-23.

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.