Vimentin Promoter Research Articles

Vimentin promotes glioma progression and maintains glioma cell resistance to oxidative phosphorylation inhibition.

Glioma has been demonstrated as one of the most malignant intracranial tumors and currently there is no effective treatment. Based on our previous RNA-sequencing data for oxidative phosphorylation (OXPHOS)-inhibition resistant and OXPHOS-inhibition sensitive cancer cells, we found that vimentin (VIM) is highly expressed in the OXPHOS-inhibition resistant cancer cells, especially in glioma cancer cells. Further study of VIM in the literature indicates that it plays important roles in cancer progression, immunotherapy suppression, cancer stemness and drug resistance. However, its role in glioma remains elusive. This study aims to decipher the role of VIM in glioma, especially its role in OXPHOS-inhibition sensitivity, which may provide a promising therapeutic target for glioma treatment. The expression of VIM in glioma and the normal tissue has been obtained from The Cancer Genome Atlas (TCGA) database, and further validated in Human Protein Atlas (HPA) and Chinese Glioma Genome Atlas (CGGA). And the single-cell sequencing data was obtained from TISCH2. The immune infiltration was calculated via Tumor Immune Estimation Resource (TIMER), Estimation of Stromal and Immune Cells in Malignant Tumors using Expression Data (ESTIMATE) and ssGSEA, and the Immunophenoscore (IPS) was calculated via R package. The differentiated expressed genes were analyzed including GO/KEGG and Gene Set Enrichment Analysis (GSEA) between the VIM-high and -low groups. The methylation of VIM was checked at the EWAS and Methsurv. The correlation between VIM expression and cancer stemness was obtained from SangerBox. We also employed DepMap data and verified the role of VIM by knocking down it in VIM-high glioma cell and over-expressing it in VIM-low glioma cells to check the cell viability. Vim is highly expressed in the glioma patients compared to normal samples and its high expression negatively correlates with patients' survival. The DNA methylation in VIM promoters in glioma patients is lower than that in the normal samples. High VIM expression positively correlates with the immune infiltration and tumor progression. Furthermore, Vim is expressed high in the OXPHOS-inhibition glioma cancer cells and low in the OXPHOS-inhibition sensitive ones and its expression maintains the OXPHOS-inhibition resistance. In conclusion, we comprehensively deciphered the role of VIM in the progression of glioma and its clinical outcomes. Thus provide new insights into targeting VIM in glioma cancer immunotherapy in combination with the current treatment.

Circulating tumor DNA for monitoring colorectal cancer: A prospective observational study to assess the presence of methylated SEPT9 and VIM promoter genes and its role as a biomarker in colorectal cancer management.

Methylation status of Septin9 (SEPT9) and vimentin (VIM) genes in circulating tumor DNA of colorectal cancer (CRC) patients is a promising bio-marker for the early detection of CRC. The aim of the present study was to identify the methylation status in promoter regions of the SEPT9 and VIM genes in a cohort of Indian patients with biopsy proven colorectal cancer. Forty-five consecutive patients of colorectal cancer were recruited. 10 mL venous samples were collected from each patient and processed for isolation of cell-free DNA, bisulfite conversion of cell-free DNA, polymerase chain reaction (PCR) amplification and detection of SEPT9 and VIM genes. Partial methylation in vimentin was present in 42.22% of the patients and 57.78% showed no methylation and none of the tumors had complete methylation. Only three (6.66%) patients showed complete methylation patterns in SEPT9 and the remaining 42 (93.33%) tumors showed partial methylation. Considering the two genes together, only three (6.66%) out of 45 showed complete methylation. The association of methylation patterns in both genes (complete, partial, and no methylation) with sex, age, T stage, N stage, M stage, CEA, histology, and location (right or left colon) were explored and none of these parameters were statistically significant. In our study, only 6.66% CRC patients showed hypermethylation and there was no association of methylation patterns in the both genes (complete, partial, and no methylation) with any of the parameters like age, sex, TNM stage, CEA, and histology.

Vimentin epigenetic deregulation in Bladder Cancer associates with acquisition of invasive and metastatic phenotype through epithelial-to-mesenchymal transition.

Bladder cancer (BlCa) is the ninth most common cancer worldwide, associated with significant morbidity and mortality. Thus, understand the biological mechanisms underlying tumour progression is of great clinical significance. Vimentin (VIM) is (over)expressed in several carcinomas, putatively in association with EMT. We have previously found that VIM promoter methylation accurately identified BlCa and VIM expression associated with unfavourable prognosis. Herein, we sought to investigate VIM expression regulation and its role in malignant transformation of BlCa. Analysis of tissue samples disclosed higher VIM transcript, protein, and methylation levels in BlCa compared with normal urothelium. VIM protein and transcript levels significantly increased from non-muscle invasive (NMIBC) to muscle-invasive (MIBC) cases and to BlCa metastases. Inverse correlation between epithelial CDH1 and VIM, and a positive correlation between mesenchymal CDH2 and VIM were also observed. In BlCa cell lines, exposure to demethylating agent increased VIM protein, with concomitant decrease in VIM methylation. Moreover, exposure to histone deacetylases pan-inhibitor increased the deposit of active post-translational marks (PTMs) across VIM promoter. In primary normal urothelium cells, lower levels of active PTMs with concomitant higher levels of repressive marks deposit were observed. Finally, VIM knockdown in UMUC3 cell line increased epithelial-like features and decreased migration and invasion in vitro, decreasing tumour size and angiogenesis in vivo. We demonstrated that VIM promoter is epigenetically regulated in normal and neoplastic urothelium, which determine a VIM switch associated with EMT and acquisition of invasive and metastatic properties. These findings might allow for development of new, epigenetic-based, therapeutic strategies for BlCa.

EBF3 reactivation by inhibiting the EGR1/EZH2/HDAC9 complex promotes metastasis via transcriptionally enhancing vimentin in nasopharyngeal carcinoma.

Metastasis is the major reason for treatment failure and accounts for cancer-related death in patients with nasopharyngeal carcinoma. However, the genetic alterations and molecular mechanisms that cause nasopharyngeal carcinoma metastasis are elusive. Herein, we performed RNA sequencing in patients with or without metastasis, and found that the early B-cell factor 3 (EBF3) was significantly elevated in the samples with metastasis. Mechanistically, EBF3 promoted metastasis by directly combining with the promoter of Vimentin and transcriptionally upregulating it. In addition, EBF3 was epigenetically silenced by EGR1/EZH2/HDAC9 complexes via sustaining the high level of H3K27-Me3 at its promoter. Clinically, there was a positive correlation between EBF3 and Vimentin in nasopharyngeal carcinoma tissues. Moreover, high expression of EBF3 or Vimentin was correlated with poor overall survival, while the combination of high EBF3 and Vimentin expression was associated with more significant poor prognosis. Therefore, specific agents targeting EBF3 or stabilizing the EGR1/EZH2/HDAC9 complex could be novel therapeutic strategies for cancer metastasis.

Abstract PO-090: TGF-β induced EMT gene expression is associated with promoter demethylation in pancreatic cancer

Abstract INTRODUCTION: Transforming growth factor-β (TGF-β), a ubiquitous molecule in pancreatic ductal adenocarcinoma (PDA) tumors, acts as a potent inducer of tumor invasion by regulating proteins involved in metastasis and driving epithelial-mesenchymal transition (EMT). We hypothesized that TGF-β signaling-induced EMT is regulated by DNA methylation. To evaluate this, we investigated the association between EMT gene promoter methylation status and gene expression in the TCGA PDA dataset. We also investigated vimentin gene (a mesenchymal marker)-specific changes in DNA methylation due to TGF-β treatment in PDA cells. METHODS: The methylation status of EMT-related genes (ZEB2, CDH2, Vimentin) was interrogated in the TCGA PDA data set. β-values (proportion of methylated gene probes in a location) were compared to gene expression levels. Next, a PDA cell line (Panc-1) was treated with TGF-β (10ng/ml), vehicle control, DNA methyltransferase inhibitor (5-azacytidine, AZA, 10mM), or TGF-β plus AZA to evaluate the effects on the DNA methylation status of the vimentin gene with methylation-specific PCR (MSP) against the promoter region of vimentin. Unmethylation and methylation levels at the vimentin promoter region were then compared. RESULTS: We found that β values of promotor regions of ZEB2, CDH2, and Vimentin were moderately negatively correlated with gene expression (Pearson r varies from 0.45 to 0.63) in the PDA TCGA data set. In Panc-1 cells, vimentin gene expression was increased following treatment with AZA compared to controls suggesting that DNA demethylation increases vimentin gene expression. MSP demonstrated that TGF-β treatment increased unmethylated vimentin gene levels compared to vehicle control treatment. TGF-β treatment increased unmethylated vimentin gene levels more so than methylated gene levels. CONCLUSIONS: These results demonstrate that gene expression of EMT-related genes may be at least partially regulated by DNA methylation. We demonstrate that TGF-β treatment leads to increased Vimentin promotor demethylation (lower β value) and increased gene expression in Panc-1 cells. This observation is consistent with our findings in the TCGA data set that lower β-values are associated with increased expression of mesenchymal genes, indicative of EMT. Further studies are underway to investigate the relationship between DNA methylation and TGF-β-induced EMT in general. Citation Format: Manjul Rana, Abul Elahi, Abidemi O. Ajidahun, Rita G. Kansal, Anders E. Berglund, David Shibata, Evan S. Glazer. TGF-β induced EMT gene expression is associated with promoter demethylation in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-090.

Vimentin affects colorectal cancer proliferation, invasion, and migration via regulatedby activator protein 1.

Uncontrolled recurrence and metastasis are important reasons for the high mortality rate of malignant tumors. Vimentin is positively correlated with the degree of malignancy of cancer cells. Vimentin is also highly expressed in colorectal cancer (CRC) cells andplays a critical rolein the metastasis and prognosis of CRC. However, the molecular mechanism of vimentin in the progression of CRC is incompletely understood. Therefore, the most active regions (nucleotides: 785-1085 nt) of the vimentin promoter in CRC were identified using luciferase experiments. By transcription factor sequence search and mutation analysis, the activator protein 1 (AP-1) binding site in the region of 785-1085 nt was confirmed. The vimentin promoter activity was enhanced by overexpression of AP-1. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that the binding site was recognized by AP-1. By cell proliferation assay, colony-forming assay, scratch-wound assay, cell migration assay, and cell invasion assay, we demonstrated that the AP-1 overexpression increased CRC cell proliferation, migration, and invasion. However, when vimentin was knocked down by vimentin small hairpin RNA in the CRC cell of AP-1 overexpression, this trend disappeared. Animal experiments and immunohistochemistry showed that AP-1 promoted tumor growth by regulating the vimentin gene. In summary, AP-1 affected metastasis, invasion of CRC cells in vitro, and tumor growth in vivo by activating the vimentin promoter. This study might provide new insights into the molecular mechanisms of the development of CRC and provide potential therapeutic targets for CRC.

Hsa_circ_001659 serves as a novel diagnostic and prognostic biomarker for colorectal cancer

Colorectal cancer (CRC) is prevalent worldwide and novel diagnostic and prognostic biomarkers are needed to improve precision medicine. Circular RNAs (circRNAs) are currently being considered as emerging tumor biomarkers. Herein, we aimed to explore the possible clinical application of circRNAs in the early diagnosis and prognostic prediction of CRC. First, candidate circRNA was selected by integrating analysis of Gene Expression Omnibus (GEO) database using GEO2R program. ROC curve analysis demonstrated the predictive values and likelihood ratios of circ_001659 were satisfactory for the diagnosis of CRC, including patients in early-stage disease or patients with carcinoembryonic antigen (CEA)-negative status. Moreover, serum circ_001659 may be a novel biomarker in the assessment of successful treatment and remission of cancer tracking. We further investigated the oncogenic role of circ_001659. In vivo and in vitro experiments indicated that circ_001659 could promote CRC cell invasion and migration. Mechanistically, circ_001659 was localized in the nucleus, recruited the RBBP5 to Vimentin promoter and increased H3K4 trimethylation level on the Vimentin promoter region, which epigenetically activated Vimentin transcription. Our findings demonstrate that circ_001659 could be a useful serum biomarker for CRC diagnosis and prognosis. Targeting circ_001659 and its pathway may be meaningful for treating patients with CRC.

Evaluating the role of hsa-miR-200c in reversing the epithelial to mesenchymal transition in prostate cancer

Deregulated epithelial-to-mesenchymal transition constitutes one of the major aspects of cancer progression. In this study, to identify key molecular principles of EMT pathway in prostate carcinogenesis, an elaborate gene expression profiling was conducted by qRT-PCR and Western blot analyses. A preponderance of mesenchymal trait was observed in the pathological samples of prostate cancer. To simulate an appropriate in vitro model, PC3 cell line was subjected to hypoxic stress, which resulted in elevated expression of vimentin along with EMT-mediating transcription factors Zeb1 and Slug. To conciliate this mesenchymal behavior of PC3 cells, hsa-miR-200c was deliberately overexpressed which led to a marked reduction of cell motility and expression of vimentin, N-cadherin, Zeb1 and Slug with concurrent increase in level of β-catenin. hsa-miR-200c was demonstrated to appease hypoxia-aggravated changes in cellular morphology by coordinated repression of vimentin, Zeb1 and Slug. Mode of action for hsa-miR-200c was mediated through transcriptional repression of Zeb1 and Slug interacting with E-box sequences in the vimentin promoter as documented by promoter assay. This ability of hsa-miR-200c to reclaim epithelial traits leads to the anticipation that molecular reprogramming of Zeb1-Slug/vimentin axis may relieve aggressiveness of prostate cancer.

Visualization of epithelial-mesenchymal transition in an inflammatory microenvironment\u2013colorectal cancer network

Epithelial-mesenchymal transition (EMT) is a biological process by which epithelial cells acquire mesenchymal characteristics. In malignant tumors, EMT is crucial for acquisition of a mesenchymal phenotype with invasive and metastatic properties, leading to tumor progression. An inflammatory microenvironment is thought to be responsible for the development and progression of colorectal cancer (CRC); however, the precise role of inflammatory microenvironments in EMT-related CRC progression remains unclear. Here, we show the spatiotemporal visualization of CRC cells undergoing EMT using a fluorescence-guided EMT imaging system in which the mesenchymal vimentin promoter drives red fluorescent protein (RFP) expression. An inflammatory microenvironment including TNF-α, IL-1β, and cytokine-secreting inflammatory macrophages induced RFP expression in association with the EMT phenotype in CRC cells. In vivo experiments further demonstrated the distribution of RFP-positive CRC cells in rectal and metastatic tumors. Our data suggest that the EMT imaging system described here is a powerful tool for monitoring EMT in inflammatory microenvironment–CRC networks.

Aberrant histone deacetylase 1 expression upregulates vimentin expression via an NF‑κB‑dependent pathway in hepatocellular carcinoma

Aberrantly elevated expression levels of histone deacetylase 1 (HDAC1) and vimentin are closely associated with disease progression in hepatocellular carcinoma (HCC). It was previously demonstrated that knocking down expression of HDAC1 resulted in a concurrent decrease in the expression levels of vimentin. However, a causal link between these two proteins has not yet been demonstrated, to the best of our knowledge. In the present study, the association between HDAC1 and vimentin was investigated using an HDAC1 overexpression platform. HDAC1 and vimentin were significantly increased in HCC cells, and HDAC1 overexpression enhanced vimentin mRNA and protein expression levels in an HDAC1 dose-dependent manner. Subsequently, truncation and mutation of a vimentin promoter demonstrated that HDAC1-induced vimentin expression was dependent on a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding site in the vimentin promoter sequence. Furthermore, HDAC1 induced vimentin expression by promoting NF-κB translocation between the cytoplasm and the nucleus, as opposed to modulating the total expression level of vimentin directly. The data in the present study demonstrated that HDAC1 is overexpressed in HCC and that HDAC1 may upregulate vimentin expression through the NF-κB signaling pathway, thus demonstrating a causal link between HDAC1 and vimentin in HCC, and may provide valuable information in understanding the pathogenesis of HCC.

ZEB1 promotes tumorigenesis and metastasis in hepatocellular carcinoma by regulating the expression of vimentin.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and its prognosis remains poor. Epithelial‑to‑mesenchymal transition (EMT)‑induced markers have emerged as key regulators of tumor development and progression in HCC. The aim of the present study was to investigate the role of zinc finger E‑box‑binding homeobox1 (ZEB1) in the tumorigenesis of HCC and to elucidate the mechanism underlying the correlation between ZEB1 and vimentin (VIM). The expression levels of ZEB1 and VIM were assessed by immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction analysis in HCC tissues and cell lines. The biological significance of ZEB1 was examined by downregulating the expression of ZEB1 in Huh‑7 cells. A luciferase reporter assay was used to investigate the association between ZEB1 and VIM. The expression levels of ZEB1 and VIM were higher in tumor tissues compared with those in adjacent normal tissues, and they were significantly associated with a poor prognosis in patients with HCC, whereas ZEB1 silencing led to the attenuation of HCC cell proliferation, invasion and migration. Furthermore, it was observed that ZEB1 was able to bind to a certain site in the VIM promoter and regulate the transcriptional activity of VIM. Therefore, the present study demonstrated that ZEB1 is a potential biomarker of the tumorigenesis and progression of HCC, and it may regulate transcription of the VIM gene.

Abstract 1101: Visualization of epithelial-mesenchymal transition in inflammatory microenvironment-colorectal cancer network in vitro and in vivo

Abstract Background: Epithelial-mesenchymal transition (EMT) is a biologic process by which epithelial cancer cells acquire mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancer (CRC). However, the precise role of inflammatory microenvironment in the EMT-related tumor progression remains unclear because EMT network is regulated by complex crosstalk between tumor and normal cells. In this study, we developed a fluorescence-guided live-cell imaging system for the spatiotemporal visualization of EMT state, and assessed the in vitro and in vivo visualization of EMT state in human CRC cells under the inflammatory microenvironment. Methods: We used two human CRC cell lines, HCT116 and RKO. The promoter of mesenchymal marker vimentin was used for inducing the EMT-dependent red fluorescence protein (RFP) expression in human CRC cells. The RFP expression was quantitatively analyzed to evaluate its association with EMT-related markers and malignant phenotype. Inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or co-culture with inflammatory mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide (LPS) (200 ng/mL) were used as an inflammatory microenvironment. Biodistribution of EMT cells was analyzed using in vivo experiments with primary and metastatic CRC xenograft tumors. Results: Inflammatory cytokines reversely induced the RFP expression in association with EMT-related malignant phenotype, such as morphologic change, EMT marker modulation, and invasion ability, in HCT116 and RKO cells. Co-culture with LPS-stimulated inflammatory macrophages also induced the RFP expression by secreting inflammatory cytokines. In vivo experiments revealed that EMT cells were detected within tumor tissues at colon, liver and peritoneum. Conclusions: Our results suggest that inflammatory microenvironment has a great potential to induce EMT program in CRC cells. This fluorescence-guided EMT imaging system is a useful tool for monitoring the EMT state in the inflammation microenvironment-CRC network in vitro and in vivo. Citation Format: Hiroshi Tazawa, Takeshi Ieda, Shuya Yano, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Masahiko Nishizaki, Shunsuke Kagawa, Takashi Saitou, Takeshi Imamura, Toshiyoshi Fujiwara. Visualization of epithelial-mesenchymal transition in inflammatory microenvironment-colorectal cancer network in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1101.

Polyphyllin I inhibits invasion and epithelial-mesenchymal transition via CIP2A/PP2A/ERK signaling in prostate cancer.

PolyphyllinI (PPI) is a natural compound extracted from the rhizomes of Paris polyphylla and has been used to treat fevers and headaches in China. In the present study, the antitumor activity of PPI in prostate cancer(PC) cells was evaluated. At low doses, PPI decreased proliferation, invasion and epithelial-mesenchymal transition(EMT) in PC cells. PPI decreased the expression of matrix metalloproteinase7(MMP7), an enzyme that is critical for tumor metastasis. PPI also decreased the expression of Snail and vimentin, which are EMT-associated factors. Additionally, PPI suppressed AP-1 transcriptional activity and AP-1 binding to the MMP7 and vimentin promoters. The results demonstrated that PPI downregulated the phosphorylation of extracellular signaling‑related kinase(ERK), which is upstream modulator of AP-1. The results of the present study demonstrated that PPI may inhibit the cancerous inhibitor of protein phosphatase2A(CIP2A)/protein phosphatase2A(PP2A)/ERK axis, downregulate the expression of MMP7, vimentin, and Snail, and suppress tumor invasion and EMT. A PC xenograft mouse model was employed and the results revealed that PPI may decrease tumor growth and weight. Additionally, PPI may inhibit proliferating cell nuclear antigen expression and CIP2A/PP2A/ERK signaling pathway in PPI-treated tumors. Therefore, the results of the present study suggest that PPI may suppress the growth, invasion and EMT of PC cells via inhibition of CIP2A/PP2A/ERK signaling axis. As a result, PPI may be a novel target for the treatment of PC.

Damage-induced reactive oxygen species regulate vimentin and dynamic collagen-based projections to mediate wound repair.

Tissue injury leads to early wound-associated reactive oxygen species (ROS) production that mediate tissue regeneration. To identify mechanisms that function downstream of redox signals that modulate regeneration, a vimentin reporter of mesenchymal cells was generated by driving GFP from the vimentin promoter in zebrafish. Early redox signaling mediated vimentin reporter activity at the wound margin. Moreover, both ROS and vimentin were necessary for collagen production and reorganization into projections at the leading edge of the wound. Second harmonic generation time-lapse imaging revealed that the collagen projections were associated with dynamic epithelial extensions at the wound edge during wound repair. Perturbing collagen organization by burn wound disrupted epithelial projections and subsequent wound healing. Taken together our findings suggest that ROS and vimentin integrate early wound signals to orchestrate the formation of collagen-based projections that guide regenerative growth during efficient wound repair.

NF-κB-vimentin is involved in steroidogenesis stimulated by mono-butyl phthalate in primary cultured ovarian granulosa cells

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP) are the most common endocrine disrupting chemicals. Many studies indicated the effects of MBP on male steroidogenesis, however, little attention have been paid on the effects of low levels of MBP on female steroidogenesis. This study was aimed to assess steroidogenesis stimulated by low-dose MBP on primary cultured ovarian granulosa cells (mGCs). Ovarian granulosa cells were isolated from ICR female mice. Hormone levels in medium were detected by ELISA, mRNA and protein expressions of vimentin, NF-κB p65 and phosphorylation of NF-κB p65 (p-p65) were assayed by qRT-PCR, western blot and immunohistochemistry, respectively. Besides, confocal immunofluorescence and electrophoretic mobility shift assay (EMSA) were used for detecting vimentin expression and activity of NF-κB p65 binding to the promoter of vimentin, respectively. Progesterone levels, mRNA and protein levels of vimentin and p-p65 in cells were increased significantly in mGCs treated by MBP at 10−10M. Additionally, MBP-induced steroidogenesis was blocked when vimentin protein was knocked down or activity of NF-κB was inhibited. EMSA assay showed that binding activity of NF-κB to the promoter regions of vimentin was boosted after MBP exposure. Accordingly, the results suggested that MBP could up-regulated steroidogenesis through NF-κB-vimentin signal in mGCs.

MiR-375 Regulates Invasion-Related Proteins Vimentin and L-Plastin

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our invitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion invitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

Cyclic AMP responsive element-binding protein induces metastatic renal cell carcinoma by mediating the expression of matrix metallopeptidase-2/9 and proteins associated with epithelial-mesenchymal transition.

Renal cell carcinoma (RCC) is the most frequently occurring malignancy of the kidney worldwide. Anti-angiogenic targeted therapies inhibit the progression of RCC, however, limited effects on the invasion or metastasis of tumor cells have been observed. Cyclic AMP responsive element‑binding protein (CREB) is a serine/threonine kinase that has been implicated in the regulation of cell proliferation, apoptosis, cycle progression and metastasis, amongst others. Our previous research demonstrated that phosphorylated CREB (pCREB) was upregulated in human renal cancer cell lines and tissues, and decreased pCREB at the Ser133 site inhibited the growth and metastatic activity of OS‑RC‑2 cells. However, the role of CREB in RCC metastasis requires further investigation. Thus, the present study further investigated the role of CREB in RCC metastasis. The present study demonstrated that knockdown of CREB using small interfering RNA (siRNA) that targeted CREB (siCREB) significantly inhibited the migration and invasion of 786‑O and OS‑RC‑2 cells, however, the opposite effect was observed in ACHN cells. In addition, knockdown of CREB suppressed the expression of matrix metallopeptidase (MMP)‑2/9 and proteins associated with epithelial‑mesenchymal transition (EMT) in 786‑O and OS‑RC‑2 cells, and promoted expression in ACHN cells. Furthermore, the chromatin immunoprecipitation assay indicated that pCREB (Ser133) had a direct interaction with the fibronectin promoter, however, pCREB (Ser133) did not target the vimentin promoter in RCC. Therefore, the results of the present study indicate that CREB regulated metastatic RCC by mediating the expression of MMP‑2/9 and EMT‑associated proteins, however, CREB‑mediated MMP‑2/9 and EMT‑associated protein expression may be induced by different pathways in different RCC cells.

TET1 promotes cisplatin-resistance via demethylating the vimentin promoter in ovarian cancer.

The development of chemo-resistance impairs the outcome of the first line platinum-based chemotherapies for ovarian cancer. Deregulation of DNA methylation/demethylation provides a critical mechanism for the occurrence of chemo-resistance. The ten-eleven translocation (TET) family of dioxygenases including TET1/2/3 plays an important part in DNA demethylation, but their roles in cisplatin resistance have not been elucidated. Using cisplatin-sensitive and cisplatin-resistant ovarian cancer cell models, we found that TET1 was significantly upregulated in cisplatin-resistant CP70 cells compared with that in cisplatin-sensitive A2780 cells. Ectopic expression of TET1 in A2780 cells promoted cisplatin resistance and decreased cytotoxicity induced by cisplatin, while inhibition of TET1 by siRNA transfection in CP70 cells attenuated cisplatin resistance and enhanced cytotoxicity of cisplatin. Increased TET1 induced re-expression of vimentin through active DNA demethylation, and cause partial epithelial-to-mesenchymal (EMT) in A2780 cells. Contrarily, knocking down of TET1 in CP70 cells reduced vimentin expression and reversed EMT process. Immunohistochemical analysis of TET1 in human ovarian cancer tissues revealed that TET1 existed in nucleus and cytoplasm in ovarian cancer tissues. And the expression of nuclear TET1 was positively correlated with residual tumor and chemotherapeutic response. Thus, TET1 expression causes resistance to cisplatin and one of the targets of TET1 action is vimentin in ovarian cancer.

Methylation of SOX1 and VIM promoters in serum as potential biomarkers for hepatocellular carcinoma.

Aberrant methylation of tumor-related genes has been identified as a promising biomarker for hepatocellular carcinoma (HCC). This study aimed to investigate the diagnostic value of SRY (sex determining region Y)-box 1 (SOX1) and Vimentin (VIM) promoter methylation for HCC. The study included 360 subjects, 240 patients with HCC, 29 with liver cirrhosis (LC), 66 with chronic hepatitis B (CHB) and 25 healthy controls (HCs). The methylation status of SOX1 and VIM promoters in the serum was detected by methylation-specific polymerase chain reaction (MSP). The methylation frequencies of SOX1 and VIM promoters in HCC patients were significantly higher than those in LC (p<0.001 and p<0.001), CHB (p<0.001 and p<0.001) and HC (p<0.001 and p<0.001) subjects. Furthermore, hypermethylation of SOX1 and VIM promoters were found in patients with advanced TNM stage (III-IV) and larger tumor size (≥5 cm) compared with early stage (I-II) (p<0.001 and p=0.004) patients with smaller tumor size (<3 cm) (p=0.018 and p=0.001). Moreover, the VIM promoter methylation frequency was higher in patients with portal vein tumor thrombosis (PVTT) (p=0.006) and vascular invasion (p=0.003). In addition, the combination of α-fetoprotein (≥20 ng/ml) with SOX1 and VIM promoter methylation significantly improved their diagnostic value. In conclusions, aberrant methylation of SOX1 and VIM promoters may be potential biomarkers for noninvasive detection of HCC and HCC metastasis.

LSD1 binds to HPV16 E7 and promotes the epithelial-mesenchymal transition in cervical cancer by demethylating histones at the Vimentin promoter.

Lysine-specific demethylase 1 (LSD1), which specifically demethylates histone H3 lysine 4 (H3K4) and lysine 9 (H3K9), is dysregulated in several cancers. We found that ectopic expression of LSD1 in cervical cancer cells promoted invasion and metastasis in vitro and in vivo, reduced the expression of the epithelial marker E-cadherin, and induced the expression of the mesenchymal marker, Vimentin. By contrast, LSD1 knockdown had the opposite effect and attenuated the HPV16 E7-induced epithelial-mesenchymal transition (EMT). We proposed a novel mechanism, whereby LSD1 is recruited to the Vimentin promoter and demethylates H3K4me1 and H3K4me2. Notably, HPV16 E7 enhanced the expression of LSD1, formed a complex with LSD1, and suppressed LSD1 demethylase activity by hindering the recruitment of LSD1 to the Vimentin promoter. Thus, LSD1 is a primary and positive regulator of the HPV16 E7-induced EMT and an attractive therapeutic target for alleviating HPV16 E7-induced EMT and tumor metastasis.