[Resistance to BRAF inhibitors: A lesson from clinical observations]. / La résistance aux inhibiteurs de BRAF - Les leçons de la clinique.

The MAPK/ERK pathway is an essential intracellular signaling pathway. Its deregulation is involved in tumor transformation and progression. The discovery of activating mutations of BRAF in various cancers has opened new therapeutic avenues with BRAF protein kinase inhibitors. Depending on the type of cancers, these inhibitors have shown either insufficient efficacy due to primary resistance of tumor cells or transient efficacy due to the development of acquired resistance. In this review, we revisit the discoveries that led to the development of BRAF inhibitors and detail the molecular and cellular mechanisms of resistance in cancers treated with these inhibitors. Understanding these mechanisms is crucial for developing more efficient therapeutic strategies.

Title: La résistance aux inhibiteurs de BRAF - Les leçons de la clinique. Abstract: La voie de signalisation MAPK/ERK est une voie centrale de la signalisation intracellulaire. Sa dérégulation participe à la transformation et la progression tumorales. Dans plusieurs cancers, la découverte de mutations activatrices de BRAF, à l'origine de l'activation de cette voie, a ouvert de nouvelles perspectives thérapeutiques avec le développement d'inhibiteurs spécifiques de la protéine. Selon les cancers, ces inhibiteurs ont cependant montré soit une efficacité insuffisante, due à la résistance primaire des cellules tumorales, soit une efficacité transitoire, due à l'apparition d'une résistance acquise. Dans cette revue, nous revenons sur les découvertes qui ont conduit au développement de ces inhibiteurs de BRAF. Nous détaillons également les mécanismes moléculaires et cellulaires de la résistance à ces inhibiteurs observée dans différents types de cancers. Comprendre ces mécanismes est en effet primordial pour développer des stratégies thérapeutiques qui soient plus efficaces.

Comparison of SYK Signaling Networks Reveals the Potential Molecular Determinants of Its Tumor-Promoting and Suppressing Functions.

Spleen tyrosine kinase (SYK) can behave as an oncogene or a tumor suppressor, depending on the cell and tissue type. As pharmacological SYK inhibitors are currently evaluated in clinical trials, it is important to gain more information on the molecular mechanisms underpinning these opposite roles. To this aim, we reconstructed and compared its signaling networks using phosphoproteomic data from breast cancer and Burkitt lymphoma cell lines where SYK behaves as a tumor suppressor and promoter. Bioinformatic analyses allowed for unveiling the main differences in signaling pathways, network topology and signal propagation from SYK to its potential effectors. In breast cancer cells, the SYK target-enriched signaling pathways included intercellular adhesion and Hippo signaling components that are often linked to tumor suppression. In Burkitt lymphoma cells, the SYK target-enriched signaling pathways included molecules that could play a role in SYK pro-oncogenic function in B-cell lymphomas. Several protein interactions were profoundly rewired in the breast cancer network compared with the Burkitt lymphoma network. These data demonstrate that proteomic profiling combined with mathematical network modeling allows untangling complex pathway interplays and revealing difficult to discern interactions among the SYK pathways that positively and negatively affect tumor formation and progression.

Imidazo[1,2-a]quinoxalines for melanoma treatment with original mechanism of action.

The malignant transformation of melanocytes causes several thousand deaths each year, making melanoma an important public health concern. Melanoma is the most aggressive skin cancer, which incidence has regularly increased over the past decades. We described here the preparation of new compounds based on the 1-(3,4-dihydroxyphenyl)imidazo[1,2-a]quinoxaline structure. Different positions of the quinoxaline moiety were screened to introduce novel substituents in order to study their influence on the biological activity. Several alkylamino or alkyloxy groups were also considered to replace the methylamine of our first generation of Imiqualines. Imidazo[1,2-a]pyrazine derivatives were also designed as potential minimal structure. The investigation on A375 melanoma cells displayed interesting in vitro low nanomolar cytotoxic activity. Among them, 9d (EAPB02303) is particularly remarkable since it is 20 times more potent than vemurafenib, the reference clinical therapy used on BRAF mutant melanoma. Contrary to the first generation, EAPB02303 does not inhibit tubulin polymerization, as confirmed by an in vitro assay and a molecular modelisation study. The mechanism of action for EAPB02303 highlighted by a transcriptomic analysis is clearly different from a panel of 12 well-known anticancer drugs. In vivoEAPB02303 treatment reduced tumor size and weight of the A375 human melanoma xenografts in a dose-dependent manner, correlated with a low mitotic index but not with necrosis.

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness.

Clinical 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.

The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex.

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, ß-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.

Network Reconstruction and Significant Pathway Extraction Using Phosphoproteomic Data from Cancer Cells.

Protein phosphorylation acts as an efficient switch controlling deregulated key signaling pathway in cancer. Computational biology aims to address the complexity of reconstructed networks but overrepresents well-known proteins and lacks information on less-studied proteins. A bioinformatic tool to reconstruct and select relatively small networks that connect signaling proteins to their targets in specific contexts is developed. It enables to propose and validate new signaling axes of the Syk kinase. To validate the potency of the tool, it is applied to two phosphoproteomic studies on oncogenic mutants of the well-known phosphatidyl-inositol 3-kinase (PIK3CA) and the unfamiliar Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites (SRMS) kinase. By combining network reconstruction and signal propagation, comprehensive signaling networks from large-scale experimental data are built and multiple molecular paths from these kinases to their targets are extracted. Specific paths from two distinct PIK3CA mutants are retrieved, and their differential impact on the HER3 receptor kinase is explained. In addition, to address the missing connectivities of the SRMS kinase to its targets in interaction pathway databases, phospho-tyrosine and phospho-serine/threonine proteomic data are integrated. The resulting SRMS-signaling network comprises casein kinase 2, thereby validating its currently suggested role downstream of SRMS. The computational pipeline is publicly available, and contains a user-friendly graphical interface (http://doi.org/10.5281/zenodo.3333687).

Vav proteins maintain epithelial traits in breast cancer cells using miR-200c-dependent and independent mechanisms.

The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.

Reconstruction and signal propagation analysis of the Syk signaling network in breast cancer cells.

The ability to build in-depth cell signaling networks from vast experimental data is a key objective of computational biology. The spleen tyrosine kinase (Syk) protein, a well-characterized key player in immune cell signaling, was surprisingly first shown by our group to exhibit an onco-suppressive function in mammary epithelial cells and corroborated by many other studies, but the molecular mechanisms of this function remain largely unsolved. Based on existing proteomic data, we report here the generation of an interaction-based network of signaling pathways controlled by Syk in breast cancer cells. Pathway enrichment of the Syk targets previously identified by quantitative phospho-proteomics indicated that Syk is engaged in cell adhesion, motility, growth and death. Using the components and interactions of these pathways, we bootstrapped the reconstruction of a comprehensive network covering Syk signaling in breast cancer cells. To generate in silico hypotheses on Syk signaling propagation, we developed a method allowing to rank paths between Syk and its targets. We first annotated the network according to experimental datasets. We then combined shortest path computation with random walk processes to estimate the importance of individual interactions and selected biologically relevant pathways in the network. Molecular and cell biology experiments allowed to distinguish candidate mechanisms that underlie the impact of Syk on the regulation of cortactin and ezrin, both involved in actin-mediated cell adhesion and motility. The Syk network was further completed with the results of our biological validation experiments. The resulting Syk signaling sub-networks can be explored via an online visualization platform.

Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease.

R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2.

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

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, and many 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 Vav-regulated transcripts was modulated 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-ß family ligand inhibin ßA, cyclooxygenase-2, and the epithelial cell adhesion molecule 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.

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

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 ß-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.

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

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.