Reprogramming of lipid metabolism in cancer-associated fibroblasts potentiates migration of colorectal cancer cells.
ABSTRACT: Metabolic interaction between cancer-associated fibroblasts (CAFs) and colorectal cancer (CRC) cells plays a major role in CRC progression. However, little is known about lipid alternations in CAFs and how these metabolic reprogramming affect CRC cells metastasis. Here, we uncover CAFs conditioned medium (CM) promote the migration of CRC cells compared with normal fibroblasts CM. CAFs undergo a lipidomic reprogramming, and accumulate more fatty acids and phospholipids. CAFs CM after protein deprivation still increase the CRC cells migration, which suggests small molecular metabolites in CAFs CM are responsible for CRC cells migration. Then, we confirm that CRC cells take up the lipids metabolites that are secreted from CAFs. Fatty acids synthase (FASN), a crucial enzyme in fatty acids synthesis, is significantly increased in CAFs. CAF-induced CRC cell migration is abolished by knockdown of FASN by siRNA or reducing the uptake of fatty acids by CRC cells by sulfo-N-succinimidyloleate sodium in vitro and CD36 monoclonal antibody in vivo. To conclude, our results provide a new insight into the mechanism of CRC metastasis and suggest FASN of CAFs or CD36 of CRC cells may be potential targets for anti-metastasis treatment in the future.
Project description:Fatty acid metabolism is closely associated with the occurrence and development of tumors. The aim of the present study was to investigate whether the key enzyme involved in fatty acid synthesis, fatty acid synthase (FASN), mediates fatty acid changes that affect the activity and migration of breast cancer cells, and whether specific fatty acids play a role in tumor metastasis. The difference in serum fatty acid profiles between patients with invasive ductal carcinoma (IDC) and healthy controls was evaluated by gas chromatography-mass spectrometry (GC-MS) fatty acid profile analysis, and it was revealed that five types of fatty acids may be potential tumor markers in IDC. Immunohistochemistry and GC-MS analysis revealed that FASN expression affected the serum fatty acid profiles of patients with IDC. Following FASN knockdown, the migration of SK-Br-3 breast cancer cells was inhibited, and the contents of various fatty acids both inside and outside the cell decreased, while the contents of various fatty acids inside and outside the cell increased following FASN overexpression. The results of the present study revealed that the expression level of FASN affected the content of fatty acids in IDC tissues and breast cancer cell lines, and that FASN-mediated changes in specific fatty acids promoted tumor cell migration.
Project description:Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.
Project description:Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC-EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in 'normalization' of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF(189), upregulation of antiangiogenic isoform VEGF(165b) and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC.
Project description:Metastasis is the major cause of death in colorectal cancer (CRC) patients. Inhibition of metastasis will prolong the survival of patients with CRC. Cancer cells bring their own soil, cancer-associated fibroblasts (CAFs), to metastasize together, promoting the survival and colonization of circulating cancer cells. However, the mechanism by which CAFs metastasize remains unclear. In this study, CAFs were derived from adipose mesenchymal stem cells (MSCs) after co-culture with CRC cell lines. Transwell assays showed that CAFs have stronger migration and invasion abilities than MSCs. In a nude mouse subcutaneous xenograft model, CAFs metastasized from the primary tumour to the lung and promoted the formation of CRC metastases. The expression of HIF-1α was upregulated when MSCs differentiated into CAFs. Inhibition of HIF-1α expression inhibited the migration and invasion of CAFs. Western blot and ChIP assays were used to identify the genes regulated by HIF-1α. HIF-1α regulated the migration and invasion of CAFs by upregulating miR-210 transcription. Bioinformatics analysis and luciferase reporter assays revealed that miR-210 specifically targeted the 3'UTR of VMP1 and regulated its expression. Downregulation of VMP1 enhanced the migration and invasion of CAFs. In vivo, inhibition of miR-210 expression in CAFs reduced the metastasis of CAFs and tumour cells. Therefore, the HIF-1α/miR-210/VMP1 pathway might regulate the migration and invasion of CAFs in CRC. Inhibition of CAF metastasis might reduce CRC metastasis.
Project description:Altered fatty acid metabolism continues to be an attractive target for therapeutic intervention in cancer. We previously found that colorectal cancer (CRC) cells with a higher metastatic potential express a higher level of fatty acid translocase (CD36). However, the role of CD36 in CRC metastasis has not been studied. Here, we demonstrate that high expression of CD36 promotes invasion of CRC cells. Consistently, CD36 promoted lung metastasis in the tail vein model and GI metastasis in the cecum injection model. RNA-Seq analysis of CRC cells with altered expression of CD36 revealed an association between high expression of CD36 and upregulation of MMP28, a novel member of the metallopeptidase family of proteins. Using shRNA-mediated knockdown and overexpression of CD36, we confirmed that CD36 regulates MMP28 expression in CRC cells. siRNA-mediated knockdown of MMP28 decreases invasion of CRC cells, suggesting that MMP28 regulates the metastatic properties of cells downstream of CD36. Importantly, high expression of MMP28 leads to a significant decrease in active E-cadherin and an increase in the products of E-cadherin cleavage, CTF1 and CTF2. In summary, upregulation of CD36 expression promotes the metastatic properties of CRC via upregulation of MMP28 and an increase in E-cadherin cleavage, suggesting that targeting the CD36-MMP28 axis may be an effective therapeutic strategy for CRC metastasis.
Project description:Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.
Project description:Recent studies have shown that the tumor microenvironment plays a significant role in the progression of solid tumors. As an abundant component of the tumor microenvironment, cancer-associated fibroblasts (CAFs) have been shown to promote tumorigenesis and cancer aggressiveness, but their molecular characteristics remain poorly understood. In the present study, paired CAFs and normal fibroblasts (NFs) were isolated from five colorectal cancer (CRC) tissues from patients who underwent surgical resection. The gene expression profiles of CAFs and NFs identified by RNA sequencing were compared to understand the complex role of CAFs in cancer progression. Gene Set Enrichment Analysis revealed that the gene sets related to the Wnt signaling pathway were highly enriched in CAFs, as well as TGF? signaling, which is considered to be a regulator of CAFs. Among the components of this pathway, Wnt2 was specifically expressed. The observations led us to speculate that Wnt2 is extremely involved in regulating CRC progression by CAFs. Thus, we performed immunohistochemical analysis on Wnt2 in 171 patients who underwent surgery for colorectal adenocarcinoma. Positive staining for Wnt2 was mainly observed in cancer stroma, although the immunoreactivity was weak in cancer cells. Wnt2 expression in CAFs was significantly correlated with depth of tumor (P < .001), lymph node metastasis (P = .044), TNM stage (P = .010), venous invasion (P < .001), and recurrence (P = .013). Subsequent in vitro analyses were conducted using conditioned medium (CM) from immortalized CAFs transfected with siRNA targeting Wnt2. As a result, cancer cell invasion and migration were significantly decreased in the CM from immortalized CAFs transfected with siRNA targeting Wnt2. Our findings indicated that Wnt2 protein released from CAFs enhances CRC cell invasion and migration. In conclusion, Wnt2 secreted by CAFs plays a key role in cancer progression and is a potential therapeutic target for CRC.
Project description:Colorectal cancer (CRC) is a highly malignant tumor associated with poor prognosis, yet the molecular mechanisms are not fully understood. In this study, we showed that LYAR, a nucleolar protein, is expressed at a higher level in CRC tissue than in adjacent normal tissue and that LYAR expression is closely associated with distant CRC metastasis. LYAR not only significantly promotes the migration and invasion of CRC cells <i>in vitro</i>, but knockdown (KD) of LYAR in CRC cells also inhibits xenograft tumor metastasis <i>in vivo</i>. Microarray analysis of <i>LYAR</i> KD cells combined with a chromatin immunoprecipitation (ChIP) assay, gene reporter assay, and rescue experiment indicated that <i>FSCN1</i> (encoding fascin actin-bundling protein 1 (Fascin-1)) serves as a novel key regulator of LYAR-promoted migration and invasion of CRC cells. Knockdown of <i>FSCN1</i> significantly inhibits subcutaneous tumorigenesis of CRC cells and leads to the downregulation of <i>FASN</i> and <i>SCD</i>, genes encoding key enzymes in fatty acid synthesis. In summary, this study reveals a novel mechanism by which LYAR promotes tumor cell migration and invasion by upregulating <i>FSCN1</i> expression and affecting fatty acid metabolism in CRC.
Project description:Nontumour cells in the tumour microenvironment, especially fibroblasts, contribute to tumour progression and metastasis. The occurrence and evolution of colorectal cancer (CRC) is closely related to cancer-associated fibroblasts (CAFs). The aim of this work was to evaluate the effects of the growth factors and cytokines secreted by CAFs on CRC progression. The secreted cytokines were examined in CAFs by Human Cytokine Antibody array. We screened 37 differentially secreted cytokines in the culture supernatants of CAFs and NFs. CLEC3B, attractin, kallikrein 5 and legumain were selected for further verification. CLEC3B was more highly expressed in the stroma of CRC tissues than the other 3 cytokines. Immunohistochemistry revealed that CLEC3B expression was associated with serosal invasion by CRC. Patients with co-expression of CLEC3B and ?-SMA had worse survival outcomes than those with only CLEC3B or ?-SMA expression. CLEC3B secreted from CAFs may promote tumour migration. Knockdown of endogenous CLEC3B in CAFs markedly decreased CRC cell migration, while recombinant human CLEC3B clearly promoted CRC cell migration and actin remodelling. In conclusion, our findings suggest that CAFs promote the CRC cell migration and skeletal reorganization by secreting CLEC3B. CLEC3B might be a potential therapeutic molecule for CRC treatment.
Project description:<h4>Background</h4>Cancer associated fibroblasts (CAFs) are key stroma cells that play dominant roles in tumor progression. However, the CAFs-derived molecular determinants that regulate colorectal cancer (CRC) metastasis and chemoresistance have not been fully characterized.<h4>Methods</h4>CAFs and NFs were obtained from fresh CRC and adjacent normal tissues. Exosomes were isolated from conditioned medium and serum of CRC patients using ultracentrifugation method and ExoQuick Exosome Precipitation Solution kit, and characterized by transmission electronic microscopy, nanosight and western blot. MicroRNA microarray was employed to identify differentially expressed miRNAs in exosomes secreted by CAFs or NFs. The internalization of exosomes, transfer of miR-92a-3p was observed by immunofluorescence. Boyden chamber migration and invasion, cell counting kit-8, flow cytometry, plate colony formation, sphere formation assays, tail vein injection and primary colon cancer liver metastasis assays were employed to explore the effect of NFs, CAFs and exosomes secreted by them on epithelial-mesenchymal transition, stemness, metastasis and chemotherapy resistance of CRC. Luciferase report assay, real-time qPCR, western blot, immunofluorescence, and immunohistochemistry staining were employed to explore the regulation of CRC metastasis and chemotherapy resistance by miR-92a-3p, FBXW7 and MOAP1.<h4>Results</h4>CAFs promote the stemness, epithelial-mesenchymal transition (EMT), metastasis and chemotherapy resistance of CRC cells. Importantly, CAFs exert their roles by directly transferring exosomes to CRC cells, leading to a significant increase of miR-92a-3p level in CRC cells. Mechanically, increased expression of miR-92a-3p activates Wnt/?-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1, contributing to cell stemness, EMT, metastasis and 5-FU/L-OHP resistance in CRC. Clinically, miR-92a-3p expression is significantly increased in CRC tissues and negatively correlated with the levels of FBXW7 and MOAP1 in CRC specimens, and high expression of exosomal miR-92a-3p in serum was highly linked with metastasis and chemotherapy resistance in CRC patients.<h4>Conclusions</h4>CAFs secreted exosomes promote metastasis and chemotherapy resistance of CRC. Inhibiting exosomal miR-92a-3p provides an alternative modality for the prediction and treatment of metastasis and chemotherapy resistance in CRC.