Epithelial-to-mesenchymal Transition In Colorectal Cancer Cells Research Articles

DJ-1 promotes epithelial-to-mesenchymal transition via enhancing FGF9 expression in colorectal cancer.

ABSTRACTTumor metastasis is the main contributor to high recurrence and mortality in colorectal cancer (CRC). In a previous study, we found that DJ-1 plays an important role in CRC metastasis, and is the main target in Ciclopirox olamine (CPX)-treated CRC. However, the mechanism underlying DJ-1-induced CRC metastasis remains elusive. In the present study, our results showed that DJ-1 could activate Wnt signaling resulting in enhanced invasive potential and epithelial-to-mesenchymal transition (EMT) in CRC cells. RNA-seq and bioinformatics analysis reveals that the DJ-1/Wnt signaling pathway may promote CRC cells’ EMT by regulating fibroblast growth factor 9 (FGF9) expression. Molecular validation showed that expression of FGF9 was upregulated by the DJ-1/Wnt signaling pathway and decreasing FGF9-expression impeded DJ-1-induced CRC invasive ability and EMT, suggesting that FGF9 is involved in DJ-1-enhanced CRC metastasis. In addition, we show that FGF9 was overexpressed in CRC human specimens and was significantly associated with tumor differentiation. High FGF9 expression was correlated with worse overall survival, and a correlation exhibited between FGF9 and EMT markers (E-cadherin and Vimentin) in CRC samples. Together, our results determined that FGF9 was involved in DJ-1-induced invasion and EMT in CRC cells, and may represent a promising therapeutic candidate for CRC anti-metastatic strategies.

Sodium Butyrate Selectively Kills Cancer Cells and Inhibits Migration in Colorectal Cancer by Targeting Thioredoxin-1.

BackgroundSodium butyrate (NaB) is a short-chain fatty acid which is produced by bacterial fermentation of nondigestible dietary fiber and has been reported to exert anti-tumor effects in many tumors including colorectal cancer (CRC). However, the role of thioredoxin-1 (Trx-1) in NaB-induced anti-tumor effect has not been completely clarified.Materials and MethodsEffects of NaB on the growth of CRC cell lines HT29 and SW480 were detected by the Cell Counting Kit-8 (CCK-8) and colony formation assays. The apoptotic cells were determined by flow cytometry, and cell migration was assessed by a Transwell assay. Western blot analysis was used to test the Trx-1 and epithelial-to-mesenchymal transition (EMT)-related proteins level. Reactive oxygen species (ROS) level was determined and N-acetylcysteine (NAC) recovery experiment was performed in CRC cells. In addition, mice xenograft model was established to test the effect of NaB on CRC growth in vivo. Further, the effects of NaB on CRC cells with overexpression or knockdown were tested by the CCK-8 and Transwell assays.ResultsNaB treatment significantly inhibited cell growth and decreased Trx-1 protein expression in CRC cells but not in normal colon epithelial cells. NaB also induced apoptosis, inhibited colony formation, migration and EMT in CRC cells. Besides, NaB increased ROS level in CRC cells and NAC reversed NaB-induced inhibition of cell proliferation. Moreover, downregulation of Trx-1 significantly enhanced NaB-induced inhibitory effects on cell growth and migration, whereas overexpression of Trx-1 attenuated NaB-induced inhibitory effects on growth and migration in CRC cells.ConclusionThese findings indicate that the NaB-mediated anti-tumor effects on CRC cells are related to downregulation of Trx-1.

Twist1 induces chromosomal instability (CIN) in colorectal cancer cells.

Twist1 is a basic helix-loop-helix transcription factor, essential during early development in mammals. While Twist1 induces epithelial-to-mesenchymal transition (EMT), here we show that Twist1 overexpression enhances nuclear and mitotic aberrations. This is accompanied by an increase in whole chromosomal copy number gains and losses, underscoring the role of Twist1 in inducing chromosomal instability (CIN) in colorectal cancer cells. Array comparative genomic hybridization (array CGH) analysis further shows sub-chromosomal deletions, consistent with an increased frequency of DNA double strand breaks (DSBs). Remarkably, Twist1 overexpression downmodulates key cell cycle checkpoint factors—Bub1, BubR1, Mad1 and Mad2—that regulate CIN. Mathematical simulations using the RACIPE tool show a negative correlation of Twist1 with E-cadherin and BubR1. Data analyses of gene expression profiles of patient samples from The Cancer Genome Atlas (TCGA) reveal a positive correlation between Twist1 and mesenchymal genes across cancers, whereas the correlation of TWIST1 with CIN and DSB genes is cancer subtype-specific. Taken together, these studies highlight the mechanistic involvement of Twist1 in the deregulation of factors that maintain genome stability during EMT in colorectal cancer cells. Twist1 overexpression enhances genome instability in the context of EMT that further contributes to cellular heterogeneity. In addition, these studies imply that Twist1 downmodulates nuclear lamins that further alter spatiotemporal organization of the cancer genome and epigenome. Notwithstanding their genetic background, colorectal cancer cells nevertheless maintain their overall ploidy, while the downstream effects of Twist1 enhance CIN and DNA damage enriching for sub-populations of aggressive cancer cells.

ZEB2-AS1 Accelerates Epithelial/Mesenchymal Transition Through miR-1205/CRKL Pathway in Colorectal Cancer.

Background: Accumulating reports have demonstrated that long-noncoding RNAs (lncRNAs) play critical roles in the pathological progression of colorectal cancer (CRC). However, the role of lncRNA zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1) in CRC remains largely unknown. Methods: The authors detected the ZEB2-AS1 expression in CRC tissue sample and CRC cell lines. The effects of ZEB2-AS1 on CRC were identified through in vitro assays (i.e., transwell assay, wound-healing assay, immunofluorescence assay, and Western blot) in a ZEB2-AS1 knockdown system. The molecular mechanism of ZEB2-AS1 was explored via bioinformatic tools, quantitative real-time polymerase chain reaction (qRT-PCR), dual-luciferase reporter assay, RNA immunoprecipitation assay, and so on. Moreover, a series of gain-of-function experiments were performed to identify the effect of ZEB2-AS1 and miR-1205 on epithelial-to-mesenchymal transition (EMT) in CRC cells. Results: This analysis clarified that ZEB2-AS1 was upregulated in both CRC tissue sample and cells lines; meanwhile, the high expression of ZEB2-AS1 was correlated with poor overall survival rate. ZEB2-AS1 knockdown significantly suppresses the EMT in CRC cells. Furthermore, the authors identified that the expression of ZEB2-AS1 was negatively correlated with expression of miR-1205, and CRKL could be a direct target of miR-1205. Through the gain-of-function experiments, they found that ZEB2-AS1 accelerates EMT in CRC cells via modulating the expression of miR-1205 and CRKL. Conclusion: Taken together, this study revealed that ZEB2-AS1 accelerates EMT in CRC through the miR-1205/CRKL pathway, suggesting that ZEB2-AS1 may potentially serve as a target of CRC.

Cigarette smoke extract modulates E-Cadherin, Claudin-1 and miR-21 and promotes cancer invasiveness in human colorectal adenocarcinoma cells

BackgroundCigarette smoke is considered a risk factor for lung and colorectal cancer. A convincing link between epithelial-to-mesenchymal transition (EMT) with colorectal cancer progression and therapeutic resistance has emerged. Deregulated expression of E-Cadherin and Claudin-1 and increased miR-21 expression and invasiveness represent hallmarks of EMT. The effects of cigarette smoke exposure on EMT in colorectal adenocarcinoma cells are largely unknown. AimThe aim of the study is to evaluate the effect of cigarette smoke extract (CSE) on miR-21, Claudin-1 and E-Cadherin, molecules associated to EMT in colorectal cancer cells. MethodsA human colorectal adenocarcinoma cell line (Caco-2) was treated with CSE at different concentration (5% and 10%) and for different time points (3 h and 24 h). Metabolic activity (by MTS assay), cell necrosis/cell apoptosis (evaluating Propidium Iodide/Annexin V expression by flow cytometry), miR-21, Claudin-1 and E-Cadherin gene expression were evaluated by Real time PCR. Cell permeability, actin polymerization and cancer cell migration was assessed by Trans-Epitelial Electrical Resistance (TEER), Phalloidin expression and matrigel system, respectively. ResultsCSE at all the tested concentrations and at all time points reduced cell necrosis. CSE at 10% increased miR-21 and reduced the metabolic activity, cell necrosis, Claudin-1 and E-cadherin mRNA at 3 h. Cell permeability, actin polymerization and cancer cell migration were all increased upon CSE exposure. ConclusionThese results showed that CSE increasing miR-21, Claudin-1 and E-Cadherin and enhancing the aggressiveness of cancer cells, may concur to colorectal cancer progression.

Long noncoding RNA VIM-AS1 promotes colorectal cancer progression and metastasis by inducing EMT

Emerging evidence indicates that lncRNAs play crucial roles in the initiation and progression of various malignant tumors. VIM-AS1 RNA is an lncRNA that transcribes from a shared bidirectional promoter with vimentin mRNA and its function in cancer cells is largely unknown. This study assessed the clinical significance of VIM-AS1 expression in colorectal cancer (CRC). We found that the VIM-AS1 transcript was significantly upregulated in high-grade, lymph node metastasis and vascular invasion tumors. Loss-of-function experiments revealed that the downregulation of VIM-AS1 could inhibit tumor cell proliferation by inducing apoptosis, cellular senescence and arresting the cell cycle. Moreover, the obtained data demonstrated that VIM-AS1 might play a crucial role in cell migration as well as the epithelial to mesenchymal transition (EMT) of CRC cells. Collectively, for the first time, our data provide novel evidence for the biological and clinical significance of VIM-AS1 expression in CRC. Further, the findings of this study suggest that VIM-AS1 promotes tumor growth and metastasis by inducing EMT in CRC cells and could be considered as a novel tumor marker with probable value in diagnosis and CRC treatment.

Withaferin-A suppress AKT induced tumor growth in colorectal cancer cells.

The oncogenic activation of AKT gene has emerged as a key determinant of the aggressiveness of colorectal cancer (CRC); hence, research has focused on targeting AKT signaling for the treatment of advanced stages of CRC. In this study, we explored the anti-tumorigenic effects of withaferin A (WA) on CRC cells overexpressing AKT in preclinical (in vitro and in vivo) models. Our results indicated that WA, a natural compound, resulted in significant inhibition of AKT activity and led to the inhibition of cell proliferation, migration and invasion by downregulating the epithelial to mesenchymal transition (EMT) markers in CRC cells overexpressing AKT. The oral administration of WA significantly suppressed AKT-induced aggressive tumor growth in a xenograft model. Molecular analysis revealed that the decreased expression of AKT and its downstream pro-survival signaling molecules may be responsible for tumor inhibition. Further, significant inhibition of some important EMT markers, i.e., Snail, Slug, β-catenin and vimentin, was observed in WA-treated human CRC cells overexpressing AKT. Significant inhibition of micro-vessel formation and the length of vessels were evident in WA-treated tumors, which correlated with a low expression of the angiogenic marker RETIC. In conclusion, the present study emphasizes the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in CRC cells and suggests that WA may overcome AKT-induced cell proliferation and tumor growth in CRC.

An hTERT/ZEB1 complex directly regulates E-cadherin to promote epithelial-to-mesenchymal transition (EMT) in colorectal cancer

In human cancer, high telomerase expression is correlated with tumor aggressiveness and metastatic potential. Telomerase activation occurs through telomerase reverse transcriptase (hTERT) induction, which contributes to malignant transformation by stabilizing telomeres. Previous studies have shown that hTERT can promote tumor invasion and metastasis of gastric cancer, liver cancer and esophageal cancer. Epithelial-to-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis, plays a key role in cancer progression. Although hTERT promotes EMT through Wnt signaling in several cancers, it is unknown if other signaling pathways are involved. In the present study, we found that hTERT and ZEB1 form a complex, which directly binds to the E-cadherin promoter, and then inhibits E-cadherin expression and promots EMT in colorectal cancer cells. hTERT overexpression in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even during hTERT overexpression. Taken together, our findings suggest that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting them may prevent cancer progression.

Abstract 2370: Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition

Abstract During tumor formation, normal tissue microenvironment is transformed in an “altered” niche, composed of non-malignant supporting cells, which influence the homeostasis of cancer cells via paracrine regulators. Tumor-associated stromal cells (TASC) are the prominent stromal elements in most types of human carcinomas including colorectal cancer (CRC). The differentiation of TASC from other cell types, such as resident stromal cells or bone marrow-derived mesenchymal stem cells (BM-MSC) is mainly mediated by factors produced during the crosstalk with malignant cells. TASC produce various extracellular matrix proteins, chemokines, and other tumor-promoting factors which affect vascularization, proliferation, tumor cell invasiveness and survival. Emerging data suggests that they also play a critical role in determining response to therapy. For instance TASC-derived factors may contribute to the development of a protective milieu by influencing cell-cell/cell-matrix interactions, cancer cell survival, and suppression of anti-tumor immune responses. Moreover, physical contact between TASC and malignant cells supports tumor cell survival via activation of anti-apoptotic pathways or inducing epithelial-to-mesenchymal transition (EMT). We previously showed that BM-MSC are capable of triggering EMT in CRC cells, leading to increased tumor cell aggressiveness in vitro and in vivo. However, the role played by the stromal component and the processes induced on CRC cells remain to be fully elucidated. For this purpose we have addressed phenotypic and functional characterization of TASC in vitro and we have analyzed TASC-mediated effects on CRC development and progression in vivo. TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity was assessed upon injection in NOD/SCID mice and developing tumors were analyzed. Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells they express membrane-bound TGF-β, through which they are capable to initiate EMT in tumor cells. Moreover CRC cells acquire a more invasive phenotype after co-culture with TASC. Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured with TASC show a significantly faster growth kinetic and develop significantly larger tumor masses as compared to tumor cells alone. Thus our data show that the stromal component of CRC increases the tumor cells malignancy triggering EMT induction. Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Raoul Droeser, Daniel Oertli, Markus Zuber, Raffaele Rosso, Ivan Martin, Michael Heberer, Giulio C. Spagnoli, Giandomenica Iezzi. Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2015-2370