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Análisis genético y funcional de biomarcadores en cáncer colorrectal y estudio proteómico del papel de snail en fibroblastos tumorales

AutorPeláez-García, Alberto
DirectorCasal, J. Ignacio ; Barderas, Rodrigo
Fecha de publicación21-abr-2015
EditorCSIC - Centro de Investigaciones Biológicas (CIB)
Universidad Complutense de Madrid
ResumenColorectal cancer (CRC) is the third most prevalent cancer in the western world. The development of the disease takes decades and involves multiple sequential events. CRC is the most prevalent cancer in Spain, with an incidence of 32.240 new cases in 2012, and the second cancer in mortality rate because most of the patients are diagnosed at advanced stages due to the general reluctance to use invasive diagnostic tools like colonoscopy. Currently, CRC research focuses on i) the identification of biomarkers to detect early stages to improve the survival of CRC patients, and ii) the discovery of new therapeutic targets for a more effective chemotherapy. These objectives require a deep knowledge of CRC regarding the biology of the primary tumor, tumor microenvironment and the metastatic process. Biomarkers are specific markers of biological and/or pathological stages. Cancer biomarkers are used for diagnosis and prognosis, to stratify patients and to identify recurrences of the disease. Actually, only few proteins have been described as biomarkers in CRC, among others: carcinoembryonic antigen (CEA), CA19.9 and CA125, although none of them is recommended for clinical screening. Blood is the optimal source for the diagnostic screening of large human populations based on noninvasive markers. Moreover, blood circulation facilitates the contact with every body tissue, including representative tumour antigens. Then, the implementation of simpler and non-invasive methods for the early detection of CRC should be based on the identification of proteins or antibodies in serum or plasma. However, these tumour antigens are probably present at a very low range of concentration in plasma and they probably suffer from extensive proteolysis in a relative short period of time; making the search for tumour-specific antigens a complicated task. Accordingly, the humoral response produced against tumour-associated antigens should be an alternative to the detection of tumour antigens in blood for the development of diagnostic tools. Indeed, autoantibodies can be detected at early cancer stages and prior to cancer diagnosis, revealing their potential as biomarkers. Self-proteins (autoantigens) altered during tumour formation and progression by specific point mutations, misfolding, overexpression, aberrant modifications as truncation or degradation, or different combinations of these factors, can be the target of autoantibodies in cancer patients. In fact, several tumour associated autoantigens (TAAs): i.e. p53, HER2, NY-ESO1 or MUC1 have been previously identified in different studies involving autoantibody screening in CRC and other tumours. The discovery of autoantibodies and their target tumor-associated antigens has been revitalized in the last years by the use of high-throughput proteomic approaches, including protein microarrays.
Protein microarrays provide a platform for the identification of both autoantibodies and their respective TAAs for diagnosis purposes by the screening of serum from cancer patients. Previously, we have used commercially available highdensity protein microarrays (Protoarrays) and home-made T7-phage microarrays for the identification of autoantibody signatures and tumour-associated antigens in colorectal cancer. Our results confirmed the presence of a specific autoantibody signature for CRC with the potential to diagnose the disease with a higher specificity and sensitivity than previously reported CRC serum markers. Furthermore, since most of the identified TAAs were kinases (FGFR4, STK4 or PIM1), we hypothesized that they could be implicated in CRC progression or being new therapeutic targets of the disease. In addition, one intriguing question not yet solved is how the autoimmune response to tumour-associated antigens occurs. Little is known about the mechanism of immune recognition of altered gene or protein products, despite their potential relevance to tumour immunity, autoimmunity and for diagnosis. On the other hand, proteomics is actively used for the identification of cancer associated biomarkers and for cancer characterization involving to primary tumour, tumour stroma or metastasis. In depth genetic studies followed by cell-biology analyses demonstrate that tumour growth is not just determined by malignant cancer cells themselves, but also by the tumour stroma. The main components of stroma are fibroblasts, which form the structural scaffold and synthesize extracellular matrix (ECM) components, such as collagens and fibronectin. Fibroblasts are the most abundant cell type in connective tissues, and some subpopulations are able to differentiate to other cell types as adipocytes or osteoblasts. In normal conditions, fibroblasts are in an inactive quiescent state; however fibroblasts become activated in wound healing and fibrosis. Fibroblast activation is induced by several stimuli, including growth factors such as TGF-β. Furthermore, an increased expression of TGFβ in colorectal cancer correlates with the accumulation of fibrotic desmoplastic tissue. Snail1 transcription is up-regulated by this cytokine in epithelial cells. In fibroblasts, Snail1 levels are modulated by serum and TGFβ. In addition, Snail1 is not only up-regulated by TGF-β, but also induces the transcription of TGFβ, creating a self-activation loop for the production of TGFβ. It is becoming increasingly clear that fibroblasts are modifiers of cancer progression. Moreover, further knowledge of the role of resting and activated fibroblasts in cancer is needed, since recent evidences indicate that subpopulations of fibroblasts, the so-called cancer-associated fibroblasts (CAFs), are important promoters of tumour growth and progression.
The transcription factor Snail1 is a major inducer of the epithelial-mesenchymal transition (EMT) during embryonic development and cancer progression. Recent reports indicate that Snail ectopic expression in mesenchymal cells abrogated their differentiation to osteoblasts or adipocytes. In addition, Snail1 knock-down caused a large decrease in the number of bone marrow murine mesenchymal stem cells (MSCs). This depletion is accompanied of an acceleration of their differentiation to osteoblasts or adipocytes. In this way, Snail would exert its effect on maintaining stemness and pluripotency in MSCs. Snail effects on adipogenesis have been proposed to be mediated, among others, by a lack of response to TGFβ or by an apparent inhibition of PPARγ and C/EBPα expression. Similar results were obtained in preventing the differentiation of bone marrow-derived murine mesenchymal stem cells (mMSC) to osteoblasts or adipocytes. Still, the molecular mechanisms underlying the effect of Snail on MSCs differentiation and the blocking of adipogenesis are far from being established. Confluent 3T3-L1 preadipocytes and mMSCs differentiate to adipocytes upon exposure to a cocktail of adipogenic inducers. During adipogenic differentiation Snail expression is almost negligible in 3T3-L1 cells. As a consequence, Snail1 expression plays an inhibitory effect on adipocyte differentiation program, suggesting a functional role for Snail in obesity and caquexia. Obesity is associated with incidence of CRC and cancer associated caquexia is responsible for approximately 20% of total deaths in cancer patients. Then, the role of Snail in these processes needs to be further studied. In this context, the work that makes up this Doctoral Thesis is divided in two welldefined blocks with two main objectives: 1. The first block focused on the study of the basis of the autoimmunity in CRC patients. We aimed to decipher some of the molecular mechanisms that control the induction of an immune response against FGFR4 and PIM1, with the following partial objectives to clarify the alterations in FGFR4 and PIM1 susceptible of inducing the humoral response in CRC: a. To analyse FGFR4 and PIM1 expression levels in CRC cell lines and paired normal/tumoral tissue by immunoblotting, immunohistochemistry and meta-analysis using publicly available databases. b. To analyse for the presence of mutations in the TAAs and/or identify new splice isoforms. We used a collection of tissue and plasma samples from patients with elevated autoantibody responses for mRNA isolation and cDNA sequencing. c. To define the role of wild-type and mutated proteins in tumour progression. We studied the effect of FGFR4 and PIM1 in CRC cell lines through gain-of-function and loss-of-function experiments with stably and transiently transfected with shRNA, siRNA and expression vectors cells. We evaluated cell growth, adhesion, invasion and survival in vitro in transfected cells, and tumour growth in vivo in Swiss nude mice subcutaneously injected with cells to analyse the relevance of FGFR4 and PIM1.
2. In the second block, we investigated those molecules mediating Snail1 transcriptional control for blocking 3T3-L1 and MSC differentiation to adipocytes. As partial objectives, we proposed the following objectives: a. We carried out in-depth quantitative proteomic analysis of Snailtransfected cells to identify the cellular and molecular mechanisms controlled by Snail. We used either stable isotopic metabolic labeling (SILAC) for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT) for mMSCs. Since Snail mediates its effects mainly through regulation of other TFs, we focused the proteomic analysis on the nuclear fraction. b. Validation of Snail-deregulated proteins in 3T3-L1 and mMSC cells was performed by PCR and WB. In silico and experimental analysis revealed putative Snail1 E-box consensus motifs in different promoters of quantified deregulated proteins. We used wild-type 3T3-L1 cells to analyse the effect of siRNAs against various TFs on differentiation. c. Characterization of the role of Nr2f6 and IL-17 in adipogenesis and differentiation. No new SNPs or activating mutations were found in FGFR4. Our results suggest that FGFR4 overexpression is the major determinant to induce a FGFR4 humoral response in CRC patients. In addition, loss-of-function experiments revealed a major role of FGFR4 in the tumorigenic properties of colorectal cancer cells, since its depletion abrogated proliferation, adhesion, migration and invasion. We identified a new role for FGFR4 as regulator of the epithelial-to-mesenchymal transition and invasion in colorectal cancer. Silencing of FGFR4 in colorectal cancer cells produced a reversion to a more epithelial phenotype and the reduction of tumorigenic properties of colorectal cancer cells. FGFR4 regulated the expression and the stability of TGFβ, SNAIL and TWIST genes, as well as the MAP kinase (proliferation) and AKT (survival) pathways. Finally, the relevance of FGFR4 as therapeutic target in CRC was demonstrated with specific antibodies and multikinase inhibitors (PD173074 and TKI-258). Regarding PIM1, we found multiple somatic mutations and the presence of the His48Asp SNP in CRC cell lines and colorectal tumour tissues. PIM1 expression levels analyses on paired tumoral/normal tissue showed an intense staining in tumoral tissues with a more abundant expression in late stages. Our results suggest that overexpression is not the reason for the induction of an immune response. In PIM1, the presence of mutations seems to play a more relevant role in early humoral response.
In addition, we demonstrated the proliferative, invasive and antiapoptotic effects of PIM1 overexpression. We examined the effect of identified PIM1 mutations in CRC cell lines respect to PIM1 wild-type and control. We observed an increased in proliferation, invasion and survival in PIM1 A315T mutant and His48Asp SNP transfected cells. These mutations caused a constitutive activation of the kinase PIM1. Additionally, we studied the frequency of His48Asp SNP in colorectal cancer patients, which was found to not be associated with the clinical outcome of CRC patients. In conclusion, the combination of different proteomic strategies for the identification of the autoimmune response against TAAs showed not only a great potential for the discovery of new biomarkers for diagnosis and prognosis in cancer, the identification of key players in CRC progression and new therapeutic targets for intervention. In the second block of this thesis, we used a sensitive and quantitative proteomic analysis to study Snail effects on adipocyte differentiation in mesenchymal cells through the inhibition of the nuclear orphan receptor Nr2f6, which antagonizes the expression of the pro-inflammatory cytokine IL-17. Snail also inhibited a number of different transcription factors, cytokines, growth factors and immunomodulators. This capacity of Snail to bind and regulate these transcription factors was confirmed by WB, PCR, luciferase and ChIP assays. In addition to the observed repression of TFs, Snail expression increased the abundance of different components of the NurD complex, and other chromatin associated proteins. NuRD belongs to the chromatin remodelling complexes and plays important roles in transcription, chromatin assembly, cell cycle progression and genomic stability. Collectively, our results suggest an early effect of Snail on adipogenesis, upstream of C/EBPα and PPARγ proteins, which would be mediated through Nr2f6 and IL-17. We demonstrated the capacity of Snail to induce IL-17 expression in fibroblasts or mesenchymal cells. These results suggest that the action of IL-17 is more farreaching than an expression restricted to T lymphocytes and might have an impact on cancer microenvironment. Our results support a link between Snail1 expression, inflammation and adipogenesis. Snail, as a master regulator, plays a central role at different levels to favour the expression and/or repression of a cascade of multiple transcription factors that control adipogenic gene expression at different levels. Further work is required to define the fine specificity of other identified transcription factors and the cascade of signalling events. These results provide a functional role for Snail in obesity that goes beyond the control of the EMT process and epithelial plasticity.
Descripción191 p.-57 fig.-13 tab.
URIhttp://hdl.handle.net/10261/122614
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