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Introduction: Tissue-based proteomic studies of colorectal cancer (CRC) metastasis have delivered fragmented results, with very few therapeutic targets and prognostic biomarkers moving beyond the discovery phase. This situation is likely due to the difficulties in obtaining and analyzing large numbers of patient-derived metastatic samples, the own heterogeneity of CRC, and technical limitations in proteomics discovery. As an alternative, metastatic CRC cell lines provide a flexible framework to investigate the underlying mechanisms and network biology of metastasis for target discovery., Areas Covered: In this perspective, we comment on different in-depth proteomic studies of metastatic versus non-metastatic CRC cell lines. Identified metastasis-related proteins are introduced and discussed according to the spatial location in different cellular fractions, with special emphasis on membrane/adhesion proteins, secreted proteins, and nuclear factors, including miRNAs associated with liver metastasis. Moreover, we analyze the biological significance and potential therapeutic applications of the identified liver metastasis-related proteins., Expert Opinion: The combination of protein discovery and functional analysis is the only way to accelerate the progress to clinical translation of the proteomic-derived findings in a relatively fast pace. Patient-derived organoids represent a promising alternative to patient tissues and cell lines, but further optimizations are still required for achieving solid and reproducible results.

The necessity to accurately predict recurrence and clinical outcome in early stage colorectal cancer (CRC) is critical to identify those patients who may benefit from adjuvant chemotherapy. Here, we developed and validated a gene-based risk-score algorithm for patient stratification and personalised treatment in early stage disease based on alterations in the secretion of metastasis-related proteins. A quantitative label-free proteomic analysis of the secretome of highly and poorly metastatic CRC cell lines with different genetic backgrounds revealed 153 differentially secreted proteins (fold-change >5). These changes in the secretome were validated at the transcriptomic level. Starting from 119 up-regulated proteins, a six-gene/protein-based prognostic signature composed of IGFBP3, CD109, LTBP1, PSAP, BMP1, and NPC2 was identified after sequential discovery, training,and validation in four different cohorts. This signature was used to develop a risk-score algorithm, named SEC6,for patient stratification. SEC6 risk-score components showed higher expression in the poor prognosis CRC sub types: consensus molecular subtype 4 (CMS4), CRIS-B, and stem-like. High expression of the signature was also associated with patients showing dMMR, CIMP+ status, and BRAF mutations. In addition, the SEC6 signature was associated with lower overall survival, progression-free interval, and disease-specific survival in stage II and III patients. SEC6-based risk stratification indicated that 5-FU treatment was beneficial for low-risk patients,whereas only aggressive treatments (FOLFOX and FOLFIRI) provided benefits to high-risk patients in stages II and III. In summary, this novel risk-score demonstrates the value of the secretome compartment as a reliable source for the retrieval of biomarkers with high prognostic and chemotherapy-predictive capacity, providing a potential new tool for tailoring decision-making in patient care.

Background: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells., Methods: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments., Results: A significant association of high AIP expression with poor CRC patients’ survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver., Conclusions: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7’s role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

La descripción se refiere a agentes que se unen específicamente a la cadherina 17 humana (CDH17) y/o a la cadherina 5 humana (CDH5) y/o a la cadherina 6 humana (CDH6) y/o a la cadherina 20 humana (CDH20). La divulgación también se relaciona con el uso de estos agentes en terapia, métodos para diagnóstico y/o pronóstico y/o estratificación de un cáncer en un sujeto, y composiciones farmacéuticas que comprenden dichos agentes. La divulgación también se refiere a marcadores de cáncer y marcadores de metástasis.

[EN] The present invention relates to a monoclonal antibody that specifically binds to an epitope comprising the amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and capable of blocking the binding of IL-13 to IL13Ra2 or inhibiting the IL13/IL13R2 signaling, as well as the antiserum, the kit and the pharmaceutical composition comprising said monoclonal antibody. The invention also relates to the use of the antibody, the antiserum and the pharmaceutical composition in the treatment of cancer, specifically, cancer metastasis., [FR] La présente invention concerne un anticorps monoclonal qui se lie spécifiquement à un épitope comprenant la séquence d'acides aminés SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4 ou SEQ ID NO : 5 et capable de bloquer la liaison de IL-13 à IL13Ra2 ou d'inhiber la signalisation de IL13/IL13R2, ainsi que l'antisérum, le kit et la composition pharmaceutique comprenant ledit anticorps monoclonal. L'invention concerne également l'utilisation de l'anticorps, de l'antisérum et de la composition pharmaceutique dans le traitement du cancer, en particulier des métastases cancéreuses.

The mechanistic basis of liver metastasis in colorectal cancer remains poorly understood. We previously reported that the sclerostin domain containing-1 (SOSTDC1) protein is overexpressed in the secretome of metastatic colorectal cancer cells and can inhibit liver homing. Here, we investigated the mechanisms of SOSTDC1 for promoting invasiveness and progression of colorectal cancer liver metastasis. SOSTDC1 inhibition of BMP4 maintains the expression of cancer stem cell traits, including SOX2 and NANOG. Immunoprecipitation and mass spectrometry analyses reveal the association of SOSTDC1 with ALCAM/CD166, which was confirmed by confocal microscopy and competition ELISA. Interaction with ALCAM is mediated by the N-terminal region of SOSTDC1, which contains a sequence similar to the ALCAM-binding motif used by CD6. Knocking down either SOSTDC1 or ALCAM expression, or using blocking antibodies, reduces the invasive activity by inhibiting Src and PI3K/AKT signaling pathways. In addition, ALCAM interacts with the alpha2SS1 and alpha1SS1 integrins, providing a possible link to Src activation. Finally, inoculation of SOSTDC1-silenced metastatic cells increases mouse survival by inhibiting liver metastasis. In conclusion, SOSTDC1 promotes invasion and liver metastasis in colorectal cancer, by overcoming BMP4-specific antimetastatic signals and inducing ALCAM-mediated Src and PI3K/AKT activation. These experiments underscore the potential of SOSTDC1 as a therapeutic target in metastatic colorectal cancer.

The disclosure relates to agents binding specifically to human cadherin 17 (CDH17), and/or to human cadherin 5 (CDH5), and/or to human cadherin 6 (CDH6), and/or to human cadherin 20 (CDH20). The disclosure also relates to the use of these agents in therapy, methods for diagnosis and/or prognosis and/or stratification of a cancer in a subject, and pharmaceutical compositions comprising said agents. The disclosure also relates to cancer markers and markers of metastasis.

Glioblastoma (GBM) is the most common and aggressive brain tumor and is associated with poor prognosis. GBM cells are frequently resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and finding new combinatorial therapies to sensitize glioma cells to TRAIL remains an important challenge. PIM kinases are serine/threonine kinases that promote cell survival and proliferation and are highly expressed in different tumors. In this work, we studied the role of PIM kinases as regulators of TRAIL sensitivity in GBM cells. Remarkably, PIM inhibition or knockdown facilitated activation by TRAIL of a TRAIL-R2/DR5-mediated and mitochondria-operated apoptotic pathway in TRAIL-resistant GBM cells. The sensitizing effect of PIM knockdown on TRAIL-induced apoptosis was mediated by enhanced caspase-8 recruitment to and activation at the death-inducing signaling complex (DISC). Interestingly, TRAIL-induced internalization of TRAIL-R2/DR5 was significantly reduced in PIM knockdown cells. Phospho-proteome profiling revealed a decreased phosphorylation of p62/SQSTM1 after PIM knockdown. Our results also showed an interaction between p62/SQSTM1 and the DISC that was reverted after PIM knockdown. In line with this, p62/SQSTM1 ablation increased TRAIL-R2/DR5 levels and facilitated TRAIL-induced caspase-8 activation, revealing an inhibitory role of p62/SQSTM1 in TRAIL-mediated apoptosis in GBM. Conversely, upregulation of TRAIL-R2/DR5 upon PIM inhibition and apoptosis induced by the combination of PIM inhibitor and TRAIL were abrogated by a constitutively phosphorylated p62/SQSTM1(S332E) mutant. Globally, our data represent the first evidence that PIM kinases regulate TRAIL-induced apoptosis in GBM and identify a specific role of p62/SQSTM1( Ser332) phosphorylation in the regulation of the extrinsic apoptosis pathway activated by TRAIL.

The disclosure relates to agents binding specifically to human cadherin 17 (CDH17), and/or to human cadherin 5 (CDH5), and/or to human cadherin 6 (CDH6), and/or to human cadherin 20 (CDH20). The disclosure also relates to the use of these agents in therapy, methods for diagnosis and/or prognosis and/or stratification of a cancer in a subject, and pharmaceutical compositions comprising said agents. The disclosure also relates to cancer markers and markers of metastasis.

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.

The disclosure relates to agents binding specifically to human cadherin 17 (CDH17), and/or to human cadherin 5 (CDH5), and/or to human cadherin 6 (CDH6), and/or to human cadherin 20 (CDH20). The disclosure also relates to the use of these agents in therapy, methods for diagnosis and/or prognosis and/or stratification of a cancer in a subject, and pharmaceutical compositions comprising said agents. The disclosure also relates to cancer markers and markers of metastasis.

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.

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.

Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC-labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin-bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular fractions. A total of 1318 proteins with at least 1.5-fold change were deregulated in highly metastatic KM12SM cells respect to KM12C cells. The protein network organization, protein complexes and functional pathways associated to CRC metastasis was revealed with spatial resolution. Although 92% of the differentially expressed proteins showed the same deregulation in all subcellular compartments, a subset of 117 proteins (8%) showed opposite changes in different subcellular localizations. The chaperonin CCT, the Eif2 and Eif3 initiation of translation and the oxidative phosphorylation complexes together with an important number of guanine nucleotide-binding proteins, were deregulated in abundance and localization within the metastatic cells. Particularly relevant was the relationship of deregulated protein complexes with exosome secretion. The knowledge of the spatial proteome alterations at subcellular level contributes to clarify the molecular mechanisms underlying colorectal cancer metastasis and to identify potential targets of therapeutic intervention.

The process of liver colonization in colorectal cancer remains poorly characterized. Here, we addressed the role of microRNA (miRNA) dysregulation in metastasis. We first compared miRNA expression profiles between colorectal cancer cell lines with different metastatic properties and then identified target proteins of the dysregulated miRNAs to establish their functions and prognostic value. We found that 38 miRNAs were differentially expressed between highly metastatic (KM12SM/SW620) and poorly metastatic (KM12C/SW480) cancer cell lines. After initial validation, we determined that three miRNAs (miR-424-3p, −503, and −1292) were overexpressed in metastatic colorectal cancer cell lines and human samples. Stable transduction of non-metastatic cells with each of the three miRNAs promoted metastatic properties in culture and increased liver colonization in vivo. Moreover, miR-424-3p and miR-1292 were associated with poor prognosis in human patients. A quantitative proteomic analysis of colorectal cancer cells transfected with miR-424-3p, miR-503, or miR-1292 identified alterations in 149, 129, or 121 proteins, respectively, with an extensive overlap of the target proteins of the three miRNAs. Importantly, down-regulation of two of these shared target proteins, CKB and UBA2, increased cell adhesion and proliferation in colorectal cancer cells. The capacity of distinct miRNAs to regulate the same mRNAs boosts the capacity of miRNAs to regulate cancer metastasis and underscores the necessity of targeting multiple miRNAs for effective cancer therapy. Copyright © 2017 Pathological Society of Great Britain and Ireland.

The disclosure relates to agents binding specifically to human cadherin 17 (CDH17), and/or to human cadherin 5 (CDH5), and/or to human cadherin 6 (CDH6), and/or to human cadherin 20 (CDH20). The disclosure also relates to the use of these agents in therapy, methods for diagnosis and/or prognosis and/or stratification of a cancer in a subject, and pharmaceutical compositions comprising said agents. The disclosure also relates to cancer markers and markers of metastasis.

The disclosure relates to agents binding specifically to human cadherin 17 (CDH17), and/or to human cadherin 5 (CDH5), and/or to human cadherin 6 (CDH6), and/or to human cadherin 20 (CDH20). The disclosure also relates to the use of these agents in therapy, methods for diagnosis and/or prognosis and/or stratification of a cancer in a subject, and pharmaceutical compositions comprising said agents. The disclosure also relates to cancer markers and markers of metastasis.

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.

Purpose: Cancer-associated fibroblasts (CAF) are major mediators in tumor microenvironment. We investigated the changes in protein expression in colon cancer–associated fibroblasts compared with normal fibroblasts (NF) in the context of searching for prognostic biomarkers, particularly for stage II patients., Experimental Design: CAFs and NFs isolated from colon cancer patients were used to identify differentially expressed proteins using quantitative proteomics. Stromal expression of deregulated proteins was analyzed by IHC. Prognostic impact was studied using external gene-expression datasets for training,then quantitative PCR and IHC for validation in different cohorts of patients. Combined datasets were used for prediction of risk assessment at stages II and III., Results: A desmoplastic signature composed of 32 proteins,highly specific for stromal components in colon cancer, was identified. These proteins were enriched for extracellular matrix organization components, TGFb signaling pathway, fibrosis, and wound-healing proteins. The expression in CAFs of 11 upregulated proteins and four downregulated proteins, selected for biomarker validation, was verified by orthogonal techniques.LOXL2 displayed a high prognostic impact by using external independent datasets and further validation in two different cohorts of patients. High expression of LOXL2 was associated with higher recurrence P ¼ 0.001 HR, 5.38 [95% confidence interval (CI), 1.70–17.01] and overall survival P ¼ 0.001 HR,8.52 (95% CI, 1.90–38.29). IHC analysis revealed a prognostic value for LOXL2 in stage II patients.

Cell migration and invasion require regulated turnover of integrin-dependent adhesion complexes. Rap1-GTP-interacting adaptor molecule (RIAM) is an adaptor protein that mediates talin recruitment to the cell membrane, and whose depletion leads to defective melanoma cell migration and invasion. In this study, we investigated the potential involvement of RIAM in focal adhesion (FA) dynamics. RIAM-depleted melanoma and breast carcinoma cells displayed an increased number, size and stability of FAs, which accumulated centrally at the ventral cell surface, a phenotype caused by defective FA disassembly. Impairment in FA disassembly resulting from RIAM knockdown correlated with deficient integrin-dependent mitogen-activated protein kinase kinase (MEK)-Erk1/2 activation and, importantly, overexpression of constitutively active MEK resulted in rescue of FA disassembly and recovery of cell invasion. Furthermore, RIAM-promoted Ras homologue gene family, member A (RhoA) activation following integrin engagement was needed for subsequent Erk1/2 activation. In addition, RhoA overexpression partially rescued the FA phenotype in RIAM-depleted cells, also suggesting a functional role for RhoA downstream of RIAM, but upstream of Erk1/2. RIAM knockdown also led to enhanced phosphorylation of paxillin Tyr118 and Tyr31. However, expression of phosphomimetic and nonphosphorylatable mutants at these paxillin residues indicated that paxillin hyperphosphorylation is a subsequent consequence of the blockade of FA disassembly, but does not cause the FA phenotype. RIAM depletion also weakened the association between FA proteins, suggesting that it has important adaptor roles in the correct assembly of adhesion complexes. Our data suggest that integrin-triggered, RIAM-dependent MEK activation represents a key feedback event required for efficient FA disassembly, which could help explain the role of RIAM in cell migration and invasion

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes. © 2010 The Author(s).

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

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 p190RhoGAPdependent 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-cadherinbound 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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. Copyright © American Society for Investigative Pathology.

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

The adhesion molecule CD44 and the membrane-type matrix metalloproteinase MT1-MMP act coordinately in tumor cells to promote cell invasion through a yet unclear mechanism. We are interested in studying the interplay between CD44 and MT1-MMP in carcinoma cells embedded in HA containing three-dimensional collagen I matrices (3D HA-Col I) by time-lapse confocal microscopy imaging. Here we report the in vivo interaction between CD44 and MT1-MMP, revealed by fluorescence resonance energy transfer (FRET) microscopy. MT1-MMP interacts with CD44 preferentially at the trailing edge of the invading tumor cells during rear retraction and on membrane fragments released during the invasion process. A fluorescent biosensor designed to monitor the proteolytic processing of CD44 by live cell imaging demonstrates that cleavage of the CD44 extracellular domain is enriched in the retracting rear ends of invasive tumor cells. Invasion assays showed that MT1-MMP mediates CD44-dependent tumor-cell invasion, whereas CD44 is not essential for MT1-MMP-mediated invasion of 3D HA-Col I matrices. Together, our results support a role for MT1-MMP in cell retraction during CD44-mediated cell invasion

The GTPase RhoA is a downstream target of heterotrimeric G(13) proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Galpha(13) form (G(alpha)(13)QL) or lysophosphatidylcholine (LPC)-promoted signaling through G(alpha)(13)-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing G(alpha)(13)QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream G(alpha)(13) target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of G(alpha)(13)QL melanoma cells. Importantly, G(alpha)(13)QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that G(alpha)(13)-dependent downstream effects on RhoA activation and invasion tightly depend on cell type-specific GAP activities and that G(alpha)(13)-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis

The 4 integrins ( 4 1 and 4 7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of 4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor- 1 (TGF- 1) rapidly (0.5–5 min) and transiently up-regulated 4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF- 1 enhanced the 4 integrin-mediated adhesion of PBLs to tumor necrosis factor- –treated human umbilical vein endothelial cells, indicating the stimulation of 4 1/VCAM-1 interaction. Although TGF- 1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF- 1 was necessary for 4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF- 1 further increased cell adhesion mediated by 4 integrins in response to the chemokine stromal cellderived factor-1 . These data suggest that TGF- 1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by 4 integrins