Sofía Torres

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.

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.

FGFR4 Role in Epithelial-Mesenchymal Transition and Its Therapeutic Value in Colorectal Cancer

Fibroblast growth factor receptor 4 (FGFR4) is vital in early development and tissue repair. FGFR4 expression levels are very restricted in adult tissues, except in several solid tumors including colorectal cancer, which showed overexpression of FGFR4. Here, FGFR4 mutation analysis discarded the presence of activating mutations, other than Arg388, in different colorectal cancer cell lines and tumoral samples. Stable shRNA FGFR4-silencing in SW480 and SW48 cell lines resulted in a significant decrease in cell proliferation, adhesion, cell migration and invasion. This decrease in the tumorigenic and invasive capabilities of colorectal cancer cells was accompanied by a decrease of Snail, Twist and TGFβ gene expression levels and an increase of E-cadherin, causing a reversion to a more epithelial phenotype, in three different cell lines. In addition, FGFR4-signaling activated the oncogenic SRC, ERK1/2 and AKT pathways in colon cancer cells and promoted an increase in cell survival. The relevance of FGFR4 in tumor growth was supported by two different strategies. Kinase inhibitors abrogated FGFR4-related cell growth and signaling pathways at the same extent than FGFR4-silenced cells. Specific FGFR4-targeting using antibodies provoked a similar reduction in cell growth. Moreover, FGFR4 knock-down cells displayed a reduced capacity for in vivo tumor formation and angiogenesis in nude mice. Collectively, our data support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer. In addition, FGFR4 targeting demonstrated its applicability for colorectal cancer therapy.

IL13 Receptor α2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis

IL13 signaling through its receptor IL13Rα2 plays a critical role in colon cancer invasion and liver metastasis, but the mechanistic features of this process are obscure. In this study, we identified a scaffold protein, FAM120A (C9ORF10), as a signaling partner in this process. FAM120A was overexpressed in human colon cancer cell lines and 55% of human colon cancer specimens. IL13Rα2-FAM120A coimmunoprecipitation experiments revealed further signaling network associations that could regulate the activity of IL13Rα2, including FAK, SRC, PI3K, G-protein-coupled receptors, and TRAIL receptors. In addition, FAM120A associated with kinesins and motor proteins involved in cargo movement along microtubules. IL13Rα2-triggered activation of the FAK and PI3K/AKT/mTOR pathways was mediated by FAM120A, which also recruited PI3K and functioned as a scaffold protein to enable phosphorylation and activation of PI3K by Src family kinases. FAM120A silencing abolished IL13-induced cell migration, invasion, and survival. Finally, antibody blockade of IL13Rα2 or FAM120A silencing precluded liver colonization in nude mice or metastasis. In conclusion, we identified FAM120A in the IL13/IL13Rα2 signaling pathway as a key mediator of invasion and liver metastasis in colon cancer.

An RGD Motif Present in Cadherin 17 Induces Integrin Activation and Tumor Growth

Background: The interaction between cadherin 17 and α2β1 integrin promotes cell adhesion and proliferation. Results: Cadherin 17 contains an RGD motif that constitutes the critical switch for integrin binding and activation. Conclusion: The cadherin RGD motif is a critical ligand for tumor growth and metastasis. Significance: Cadherin 17 is the first known integrin-ligand RGD-cadherin. Other RGD-cadherins might play important roles in cancer metastasis. Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in β1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2β1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins.

Sporadic colon cancer murine models demonstrate the value of autoantibody detection for preclinical cancer diagnosis

Although autoantibody detection has been proposed for diagnosis of colorectal cancer, little is known about their initial production and development correlation with cancer progression. Azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice developed colon adenocarcinoma in the distal colon similar to human sporadic colon cancer. We assessed this model together with AOM and DSS-only models for their applicability to early detection of cancer. All AOM/DSS-treated mice produced autoantibodies to tumor-associated antigens analogous to those observed in human colon cancer patients. Autoantibody response was related to tumor antigen overexpression. Cancer autoantibodies were detected 21 days after starting treatment, when no malignant histopathological features were detectable, and they increased according to tumor progression. When carcinogenesis was induced separately by AOM or DSS, only those mice that developed malignant lesions produced significant levels of autoantibodies. These findings demonstrate that autoantibody development is an early event in tumorigenesis and validates its use for preclinical colon cancer diagnosis.

LOXL2 Is Highly Expressed in Cancer-Associated Fibroblasts and Associates to Poor Colon Cancer 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, TGFβ 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 = 0.001 HR, 5.38 [95% confidence interval (CI), 1.70–17.01] and overall survival P = 0.001 HR, 8.52 (95% CI, 1.90–38.29). IHC analysis revealed a prognostic value for LOXL2 in stage II patients. Conclusions: We identified LOXL2 to be associated with the outcome of colon cancer patients. Furthermore, it can be used to stratify patients at stages II and III for further therapeutic decisions. Clin Cancer Res; 21(21); 4892–902. ©2015 AACR.

A Proteomic Analysis Reveals That Snail Regulates the Expression of the Nuclear Orphan Receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and Interleukin 17 (IL-17) to Inhibit Adipocyte Differentiation

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.

Combined miRNA profiling and proteomics demonstrates that different miRNAs target a common set of proteins to promote colorectal cancer metastasis

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