Rubén Álvaro Bartolomé

Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1α (SDF-1α). Using Matrigel as a model, we show that SDF-1α promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1α was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1α triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1α, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-β1, a Matrigel component, whereas anti-transforming growth factor-β antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1α. The identification of SDF-1α as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

Activation of Vav/Rho GTPase Signaling by CXCL12 Controls Membrane-Type Matrix Metalloproteinase–Dependent Melanoma Cell Invasion

Melanoma cells express the chemokine receptor CXCR4, which confers invasive signals on binding to its ligand CXCL12. We show here that knocking down membrane-type matrix metalloproteinase (MT1-MMP) expression translates into a blockade of invasion across reconstituted basement membranes and type I collagen gels in response to CXCL12, which is the result of lack of MMP-2 activation. Interference with MMP-2 expression further confirms its important role during this invasion. Vav proteins are guanine-nucleotide exchange factors for Rho GTPases that regulate actin dynamics and gene expression. We show that melanoma cells express Vav1 and Vav2, which are activated by CXCL12 involving Jak activity. Blocking Vav expression by RNA interference results in impaired activation of Rac and Rho by CXCL12 and in a remarkable inhibition of CXCL12-promoted invasion. Importantly, up-regulation of MT1-MMP expression by CXCL12, a mechanism contributing to melanoma cell invasion, is blocked by knocking down Vav expression or by inhibiting Jak. Together, these data indicate that activation of Jak/Vav/Rho GTPase pathway by CXCL12 is a key signaling event for MT1-MMP/MMP-2-dependent melanoma cell invasion.

MT1-MMP Is Required for Myeloid Cell Fusion via Regulation of Rac1 Signaling

Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse under various circumstances, but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant-cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas.

Moesin orchestrates cortical polarity of melanoma tumour cells to initiate 3D invasion

Tumour cell dissemination through corporal fluids (blood, lymph and body cavity fluids) is a distinctive feature of the metastatic process. Tumour cell transition from fluid to adhesive conditions involves an early polarization event and major rearrangements of the submembrane cytoskeleton that remain poorly understood. As regulation of cortical actin-membrane binding might be important in this process, we investigated the role of ezrin and moesin, which are key crosslinking proteins of the ERM (ezrin, radixin, moesin) family. We used short interfering RNA (siRNA) to show that moesin is crucial for invasion by melanoma cells in 3D matrices and in early lung colonization. Using live imaging, we show that following initial adhesion to the endothelium or 3D matrices, moesin is redistributed away from the region of adhesion, thereby generating a polarized cortex: a stable cortical actin dome enriched in moesin and an invasive membrane domain full of blebs. Using Lifeact-GFP, a 17-amino-acid peptide that binds F-actin, we show the initial symmetry breaking of cortical actin cytoskeleton during early attachment of round cells. We also demonstrated that ezrin and moesin are differentially distributed during initial invasion of 3D matrices, and, specifically, that moesin controls adhesion-dependent activation of Rho and subsequent myosin II contractility. Our results reveal that polarized moesin plays a role in orienting Rho activation, myosin II contractility, and cortical actin stability, which is crucial for driving directional vertical migration instead of superficial spreading on the fluid-to-solid tissue interface. We propose that this mechanism of cortical polarization could sustain extravasation of fluid-borne tumour cells during the process of metastasis.

Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Purpose: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer. Experimental Design: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further validations of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues. Results: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis. Conclusion: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer. Clin Cancer Res; 19(21); 6006–19. ©2013 AACR.

The Chemokine Receptor CXCR4 and the Metalloproteinase MT1-MMP Are Mutually Required during Melanoma Metastasis to Lungs

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.

High Expression of IL-13 Receptor α2 in Colorectal Cancer Is Associated with Invasion, Liver Metastasis, and Poor Prognosis

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion, Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.

Overexpression of E-cadherin on Melanoma Cells Inhibits Chemokine-promoted Invasion Involving p190RhoGAP/p120ctn-dependent Inactivation of RhoA

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.

Rap1-GTP-interacting Adaptor Molecule (RIAM) Protein Controls Invasion and Growth of Melanoma Cells

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces β1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.