Vimentin Research Articles

Overexpression of ALDH1 and EMT marker profile are linked with unfavorable outcome in head and neck cancer.

Background The aim of this research was to assess the expression of aldehyde dehydrogenase 1 (ALDH1) and epithelial-mesenchymal transition (EMT) markers in head and neck squamous cell carcinoma (HNSCC), and to correlate them with the clinical and histopathological parameters of a patient cohort with follow-up over an 8-year period. Material and Methods For this, seventeen HNSCC and non-neoplastic adjacent epithelium (AE) samples were subjected to laser microdissection and real-time PCR to evaluate the mRNA expression of ALDH1, E-cadherin (E-CAD), N-cadherin (N-CAD), and vimentin (VIM). Also, immunohistochemistry was performed for ALDH1, E-CAD, N-CAD, and VIM in the tumor center (TC), invasion front (IF), and AE of the seventeen samples. Mann-Whitney, Kruskal-Wallis and Chi-square tests were used to correlate the mRNA and immunohistochemical expression with different variables, considering p<0.05. Kaplan-Meier curves were produced for local recurrence, regional metastasis and treatment. Results A mRNA overexpression of ALDH1 in primary tumors was associated with regional metastasis and a high ALDH1 immunostaining was related to metastasis and a worse patient outcome. Additionally, a favorable outcome was associated with the transition phase and an unfavorable outcome was associated with EMT event. An overall 26.9 months was observed with longer survival associated with surgery and radiotherapy. Conclusions However, due to the intense variability inherent to the indicator proteins in the EMT process, the complete profile markers related to this biological process should be continuous investigated. Key words:Aldehyde dehydrogenase 1, epithelial mesenchymal transition, head and neck cancer, squamous cell carcinoma, follow-up study.

Sonic Hedgehog/Gli1 Signaling Pathway Regulates Cell Migration and Invasion via Induction of Epithelial-to-mesenchymal Transition in Gastric Cancer.

Background: The aberrant activation of the Sonic hedgehog (Shh) signaling pathway is involved in progression of several types of cancer, including gastric cancer (GC). However, it remains uncertain whether it also plays a critical role in promoting cancer initiation and progression by inducing epithelial-to-mesenchymal transition (EMT) in GC. Thus, the aim of the present study was to determine whether the Shh pathway is involved in GC, and to investigate the function of the Shh pathway in the induction of EMT in GC.Materials and methods: The expression levels of Shh pathway members and EMT markers were examined in GC tissues by immunohistochemistry. The association between these factors and patient clinicopathological characteristics was analyzed. In addition, Gli-antagonist 61 (GANT61) was used to block Shh/Gli1 pathway activity, and recombinant Shh proteins (N-Shh) were used to activate the Shh pathway in GC cells. Wound healing and Transwell invasion and migration assays were performed to assess the effects of the Shh pathway on the migration and invasion of GC cells in vitro. Furthermore, western blot analysis was used to examine the changes in protein expression.Results: The results demonstrated that these Shh/Gli1 pathway members were upregulated in GC tissues, and that Gli1 upregulation was associated with tumor progression and a poor prognosis. Gli1 expression was negatively associated with E-cadherin (E-Cad) expression, and positively with Vimentin (VIM) expression in GC specimens. Further analysis revealed that when the Shh/Gli1 pathway was activated, the migratory and invasive abilities of GC cells were enhanced, and the expression levels of Gli1 and VIM were increased, while E-Cad expression was decreased. Opposite results were observed when the Shh/Gli1 pathway was blocked by GANT61.Conclusions: The present study indicated that the Shh/Gli1 pathway exhibits an abnormal activation pattern in GC with possible predictive and prognostic significance. The Shh/Gli1 pathway may promote the migratory and invasive potential of GC cells by inducing EMT. The Shh/Gli1 pathway can thus be considered as a potential therapeutic target for GC.

Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10.

Background: Epithelial ovarian cancer (EOC) is one of the most lethal malignancies in women worldwide. Many studies showed the transcription factor SNAI2-induced Epithelial-Mesenchymal Transition (EMT) through inhibiting E-cadherin (E-cad) expression. Our previous study reported that miR-222-3p was an important tumor-suppressive miRNA for EOC development and dissemination. The present study aimed to acquire a deeper mechanistic understanding of the role of miR-222-3p regulation that might contribute to improving current anti-metastasis strategies in EOC.Methods: A variety of techniques were used to measure mRNA and protein expression levels, including quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, immunohistochemical (IHC) staining, and immunofluorescence (IF). Four different microRNA (miRNA) target prediction databases were used to predict the target genes of miR-222. Luciferase assay was performed to determine the direct binding of miR-222-3p to the untranslated region (3'-UTR) of PDCD10. The biological effects of PDCD10 and miR-222-3p were also investigated in vitro by Transwell and wound healing assays, as well as in vivo by a xenograft mice model. Combining UCSC and JASPAR, as well as ENCODE public databases, we predicted that the transcription factor SNAI2 could affect miR-222-3p expression. Luciferase assay was utilized to examine the validity of putative SNAI2 binding sites for miR-222-3p regulation. Chromatin immunoprecipitation (ChIP) was used to explore the SNAI2's occupancy on the miR-222-3p promoter.Results: We observed the inhibitory effect of SNAI2 on miR-222-3p transcription and confirmed the tumor-suppressive function of miR-222-3p both in EOC cells and tissues. PDCD10 was upregulated and inversely correlated with miR-222-3p, both in vitro and in vivo, which was consistent with the information in bioinformatics databases. Furthermore, We observed direct binding of miR-222-3p to the 3'-UTR of PDCD10 and inhibition of PDCD10 translation, which, in turn, inhibited EOC cell migration in vitro and repressed EOC xenografted tumor metastasis in vivo. We found that genetic overexpression of PDCD10 (OE-PDCD10) increased cancer metastasis by down-regulating E-cad and enhancing Vimentin (VIM) thereby inducing EMT and promoting β-catenin/Wnt-mediated cell migration.

Long non‑coding RNA LINC00312 regulates breast cancer progression through the miR‑9/CDH1 axis.

Long non‑coding RNAs (lncRNAs) are important mediators of the initiation and progression of tumors, including breast cancer (BC). The exact role of long intergenic non‑coding RNA 00312 (LINC00312) in BC and its mechanism of action have not been reported to date. In the present study, LINC00312 was found to be downregulated in human BC tissues and cell lines by RT‑qPCR. The findings of a functional study indicated that overexpression of LINC00312 suppressed the proliferation, colony forming ability, migration and invasiveness of BC cell lines. Mechanistically, LINC00312 was found to induce suppression of cell migration and invasion by directly binding to miR‑9. Overexpression of LINC00312 increased the expression of cadherin1 (CDH1), a direct target of miR‑9, and decreased the expression of vimentin (VIM), a major cytoskeletal component of mesenchymal cells as determined by western blot analysis. miR‑9 partly abrogated the upregulation of CDH1 and downregulation of VIM induced by LINC00312. Taken together, the results of the present study indicate a role for the LINC00312/miR‑9/CDH1 axis in the progression of BC, and suggest a novel lncRNA‑based diagnostic biomarker or therapeutic target for BC.

Genetically Engineered Lung Cancer Cells for Analyzing Epithelial-Mesenchymal Transition.

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial–mesenchymal transition (EMT) and its inverse process, mesenchymal–epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future.

Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 expression and its contribution to the regulation of biological function of human clear cell renal cell carcinoma cell line

Objective To investigate the clinical significance and biological function of human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) in human clear cell renal cell carcinoma (ccRCC). Methods Down-regulation of HHLA2 was performed by using RNA interference (RNAi) method to investigate the role of HHLA2 in regulation of biological behaviors in human clear cell renal cell carcinoma cell lines 786-O and ACHN. The cell counting kit-8 (CCK-8) assay, wound healing assay, and transwell invasion assay were used to examine the cellular function after HHLA2 knockdown of these cells. We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis. Results To investigate the functions of HHLA2 in human ccRCC, we successfully constructed HHLA2 knockdown expression in human ccRCC cell lines by using the RNAi method. CCK-8 assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell proliferation. The wound healing assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell migration (LV-HHLA2-sh1: 786-O: F=99.340, P<0.01, ACHN: F=113.500, P<0.01; LV-HHLA2-sh2: 786-O: F=35.320, P<0.01, ACHN: F=26.470, P<0.01). Transwell invasion assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell invasion. The cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, according to the microarray data, the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly changed after knockdown of HHLA2 expression. After knockdown of HHLA2 in 786-O and ACHN, the expression of E-cadherin was significantly increased, meanwhile the expression of N-cadherin and Vimentin were significantly decreased. Conclusion Our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy. Key words: Human endogenous retrovirus subfamily H long terminal repeat associating protein 2; Human clear cell renal cell carcinoma; RNA interference

Abstract B018: Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes

Abstract Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018

Abstract A032: Clinical significance of vimentin-positive AXL-expressing circulating tumor cells in advanced non-small-cell lung cancer patients

Abstract Purpose: Circulating tumor cells (CTCs) have a potential for the noninvasive decision-making. AXL, a receptor tyrosine kinase is associated with promoting cell proliferation, migration, epithelial-to-mesenchymal transition and drug resistance. We previously reported that the establishment of the detection method of AXL-expressing CTCs by an automated microcavity array (MCA) system incorporating mesenchymal marker vimentin (VM). Here we analyzed the clinicopathological correlation with VM-positive AXL-expressing CTCs in advanced non-small-cell lung cancer (NSCLC) patients. Methods: For detecting CTCs, 6 mL of peripheral blood collected from patients was divided into two portions and used for CTC identification in parallel incorporating markers cytokeratin (CK) and VM, respectively. An automated MCA system was used for enrichment and staining for CD45, DAPI, CK or VM with the additional staining for AXL. DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. All statistical analyses were carried out using GraphPad Prism 6 and Mann-Whitney U test, Fisher’s exact test, and rog-rank test were performed accordingly. A p value &amp;lt;0.05 was considered as significant. Results: Twenty advanced NSCLC were enrolled in this clinical study between May and October 2018 at Wakayama Medical University Hospital. Clinical data was tracked until May 2019. The patient characteristics were as follows: median age 70 years (range, 54-82); male 80%; stage III/IV, 10/90%; adenocarcinoma/ squamous cell carcinoma/ other, 65/30/5%. VM-positive single CTCs were detected in 95% of patients (median, 3; range, 0-54) and AXL-expressing VM-positive single CTCs were detected in 60% of patients (median, 1; range, 0-42). AXL-positivity rates in single VM-positive CTCs between patients with previous treatment and those without were compared and patients with previous treatment harbored significantly higher AXL-positivity (p &amp;lt; 0.05). We also assessed the impact of AXL-expressing CTCs on the following treatment in 16 patients from whom we obtained the response data. Cutoff value for segregating partial response + stable disease (n = 13) from progressive disease (n = 3) to the following treatment was 45% of AXL-positivity on VM-positive single CTCs according to receiver operating characteristic curve and p value was 0.071, suggesting the trend favoring AXL-positive CTCs for predicting progressive disease. Progression free survival of treatment following CTC detection was significantly shorter in patients with 45% or more AXL positivity in CTCs (n = 3; median, 41 days) than in those with less than 45% of them (n = 13; median, not reached) (p &amp;lt; 0.01). Conclusion: Our data indicate that detection of AXL-expressing CTCs may have a potential to be indicative of poor prognosis and acquired drug resistance. Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Koichi Sato, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Clinical significance of vimentin-positive AXL-expressing circulating tumor cells in advanced non-small-cell lung cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A032. doi:10.1158/1535-7163.TARG-19-A032

Post-translational modifications of vimentin reflect different pathological processes associated with non-small cell lung cancer and chronic obstructive pulmonary disease.

Introduction: Vimentin has shown to be highly implicated in cancer initiation and progression. Vimentin is often a target of post-translational modifications (PTMs) which can be disease specific, thus targeting these specific modifications can be of high biomarker potential. In this study we set out to evaluate the biological relevance and serum biomarker potential of citrullinated vimentin (VICM) and non-citrullinated vimentin (VIM) in non-small cell lung cancer (NSCLC) and chronic obstructive pulmonary disease (COPD).Methods: A competitive ELISA targeting VIM was developed and quantified in serum from patients with NSCLC and COPD. VIM was compared with levels of VICM in the same indications.Results: VIM was significantly increased in NSCLC (n = 100) compared to healthy controls (n = 67) in two independent cohorts (p = 0.0003 and p < 0.0001). Furthermore, VIM was highly increased in late stages of NSCLC (p = 0.001), however VIM was not increased in COPD patients (n = 10). Contrarily, serum levels of VICM was not increased in late stages of NSCLC, but highly elevated in patients with COPD (p < 0.0001).Conclusions: These findings suggest a biomarker potential of VIM in NSCLC. Our findings also indicate that PTMs of vimentin are highly relevant and that targeting these modifications can have differential biomarker potential.

Rhizoma Pleionis inhibits of breast cancer epithelial-mesenchymal transition by VEGF-A related angiogenesis

Objective To investigate the Rhizoma Pleionis mediating vascular endothelial growth factor A (VEGF-A)-related angiogenesis in breast cancer cells to inhibit epithelial-mesenchymal transition (EMT) in breast cancer. Methods Firstly, the proliferation activity of MDA-MB-231 and 4T1 of breast cancer cells was detected by methyl thiazolyl tetrazolium (MTT) method at different concentrations (0.2 g/ml, 0.4 g/ml, 0.6 g/ml, 0.8 g/ml, 1.0 g/ml); secondly, the specific concentration of Rhizoma Pleionis was selected and tested by Transwell test for its effect on breast cancer cell metastasis and invasion. The formation of human umbilical vein endothelial cells (HUVEC) capillary wall was measured to detect the effect of Rhizoma Pleionis on cell angiogenesis. The EMT-related protein and angiogenesis-related molecules were detected by Western blot. Results MTT results showed that Rhizoma Pleionis of 0.8 g/ml and 1.0 g/ml can inhibit the MDA-MB-231 and 4T1 cells proliferation in a dose-dependent manner, with statistically significant difference (P<0.05). Rhizoma Pleionis can inhibit the invasion of breast cancer cells and inhibit the formation of blood vessel walls in HUVECs. Western blot confirmed that the expression level of EMT-related proteins: E-cadherin was significantly increased and the expression level of N-cadherin, Vimentin and Snail was significantly decreased after treatment with Rhizoma Pleionis (P<0.05); and the level of the related molecule hypoxia inducible factor-1α (HIF-1α), VEGF-A and vascular endothelial growth factor receptor-2 (VEGFR-2) was significantly reduced (P<0.05). Conclusions Rhizoma Pleionis inhibits the epithelial-mesenchymal transition of breast cancer by influencing the angiogenesis of VEGF-A, and inhibits the invasion and metastasis of breast cancer. Key words: CREMASTRA VARIABILIS; Vascular endothelial growth factor A; Breast neoplasms; Tumor cells, cultured; Epithelial-mesenchymal transition

LncRNA-DANCR promotes growth and metastasis of colorectal cancer via activating epithelial-mesenchymal transition process

Background: The aim of this study was to investigate the effects and underlying mechanism of long non- coding RNA-differentiation antagonizing non-protein coding RNA (lncRNA-DANCR) on colorectal cancer (CRC). Methods: The expression of lncRNA-DANCR in CRC and pericarcinous tissues from 40 CRC patients, and the expression in HT-29 cells and FHC cells, were determined by qRT-PCR. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The migration and invasion of CRC cells were detected by wound healing assay and transwell assay, respectively. HT-29 cells were transfected and divided into three groups: BLANK group, si-NC group and si-DANCR group. After transfection, the expression of lncRNA-DANCR was detected by qRT-PCR. The expression of E-cadherin and vimentin was detected by western blot and immunofluorescence. The mice model of xenograft tumor was established and histological changes of lung lobes sections were measured by hematoxylin-eosin (HE) staining. Results: The expression of lncRNA-DANCR in CRC tissues and HT-29 cells was significantly higher than that in non-CRC tissues and FHC cells. Silencing lncRNA-DANCR could significantly inhibit the proliferation, invasion and metastasis of HT-29 cells. Western blot showed that the expression of E-cadherin increased significantly and vimentin decreased significantly after silencing lncRNA-DANCR. The same results were observed in immunofluorescence experiment. Silence of lncRNA-DANCR markedly suppressed the growth and metastasis of CRC. Conclusions: LncRNA-DANCR may facilitate the growth and metastasis of CRC by regulating the epithelial-mesenchymal transition (EMT) process.

Experimental study on down-regulation of long non-coding RNA KCNQ1 opposite strand transcript 1 expression for reversing cisplatin resistance in human osteosarcoma cells

Objective To investigate the role and possible mechanism of long non-coding RNA KCNQ1 opposite strand transcript 1 (KCNQ1OT1) in the development of cisplatin (DDP) resistance in osteosarcoma (OS). Methods The expression of KCNQ1OT1 in parental cells and drug-resistant cells was detected by real-time polymerase chain reaction (qPCR). The small interfering RNA (siRNA) targeting lncRNA-KCNQ1OT1 was synthetized and transfected into osteosarcoma cells. The cisplatin sensitivity half maximal inhibitory concentration (IC50), migration and invasion of OS cells were assessed with WST-1 assays and Transwell assays, respectively. The expression of epithelial-mesenchymal transition (EMT) associated molecules was detected by qPCR. Results The IC50 values of cisplatin-resistant MG63/DDP and 143B/DDP in osteosarcoma were (17.960±0.153) μmol/L and (7.725±0.183) μmol/L, respectively, which were significantly higher than the IC50 value of the parental cells [(5.074±0.159), (2.633±0.219) μmol/L] (t=101.148, 30.903, P<0.01). The relative expression levels of KCNQ1OT1 in MG63/DDP and 143B/DDP cells were 6.830±0.105 and 6.284±0.465, respectively, compared with parental cells (3.081±0.167, 2.014±0.216), the difference was statistically significant (t=22.917, 11.722, P<0.01). Knocking down the expression of KCNQ1OT1 can inhibit the migration and invasion of osteosarcoma DDP-resistant cells. After down-regulation of KCNQ1OT1, IC50 values of MG63/DDP and 143B/DDP were decreasedsignificantly. The expression of E-cadherin was down-regulated in the drug-resistant cells, and the expression of N-cadherin and Vimentin was up-regulated. After interference with KCNQ1OT1, the expression of E-cadherin was up-regulated and the expression of N-cadherin, Vimentin and Slug was down-regulated. Conclusion KCNQ1OT1 may promote osteosarcoma cisplatin resistance by regulating EMT. KCNQ1OT1 may be a potential therapeutic target for DDP-resistant osteosarcoma Key words: Long non-coding RNA; KCNQ1 opposite strand transcript 1; Osteosarcoma

Sex-biased differences in the correlation between epithelial-to-mesenchymal transition-associated genes in cancer cell lines.

There is a wide disparity in the incidence, malignancy and mortality of different types of cancer between each sex. The sex-specificity of cancer seems to be dependent on the type of cancer. Cancer incidence and mortality have been demonstrated as sex-specific in a number of different types of cancer, such as liver cancer, whereas sex-specificity is not noticeable in certain other types of cancer, including colon and lung cancer. The present study aimed to elucidate the molecular basis for sex-biased gene expression in cancer. The mRNA expression of the epithelial-to-mesenchymal transition-associated genes was investigated, including E-cadherin (also termed CDH1), vimentin (VIM), discoidin domain receptor 1 (DDR1) and zinc finger E-box binding homeobox 1 (ZEB1) in female- and male-derived cancer cell lines by reverse transcription (RT)-PCR and the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) database analysis. A negative correlation was observed between DDR1 and ZEB1 only in the female-derived cancer cell lines via RT-PCR analysis. A negative correlation between DDR1 index (defined by the logarithmic value of DDR1 divided by ZEB1, based on the mRNA data from the RT-PCR analysis) and an invasive phenotype was observed in cancer cell lines in a sex-specific manner. Analysis of the CCLE database demonstrated that DDR1 and ZEB1, which are already known to be sex-biased, were negatively correlated in female-derived liver cancer cell lines, but not in male-derived liver cancer cell lines. In contrast, cell lines of colon and lung cancer did not reveal any sex-dependent difference in the correlation between DDR1 and ZEB1. Kaplan-Meier survival curves using the transcriptomic datasets such as Gene Expression Omnibus, European Genome-phenome Archiva and The Cancer Genome Atlas databases suggested a sex-biased difference in the correlation between DDR1 expression pattern and overall survival in patients with liver cancer. The results of the present study indicate that sex factors may affect the regulation of gene expression, contributing to the sex-biased progression of the different types of cancer, particularly liver cancer. Overall, these findings suggest that analyses of the correlation between DDR1 and ZEB1 may prove useful when investigating sex-biased cancers.

Overexpression of Raf-1 kinase inhibitor protein inhibits cell invasion and migration in lung cancer cells through suppressing epithelial-mesenchymal transition

Background: Raf-1 kinase inhibitor protein (RKIP) is a small evolutionary conserved protein that was associated with the Raf-MEK-ERK pathway. However, whether RKIP would alter the invasion and metastasis of non-small cell lung cancer (NSCLC) and play the role through suppressing epithelial-mesenchymal transition (EMT) remains to be explored. Methods: A549 cells were transfected with RKIP-GV141 plasmid for overexpression of RKIP. Colony formation assay and MTT assay were performed to measure the effects of RKIP on the proliferation and cell viability assay of A549 cells. Transwell, Migration Assay and wound healing assay were performed to analyze the effects of RKIP on the invasion and metastasis of A549 cells. E-cadherin and vimentin were measured by Western blot to conform RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Results: RKIP is downregulated in NSCLC tissues compared to adjacent normal lung tissues by IHC. Decreased expression of RKIP contributes to poor prognosis in lung adenocarcinoma patients. Age and pTNM stage were independent prognostic factors for adenocarcinoma patients. Overexpression of RKIP reduces the cell viability and limits the proliferation, invasion, and migration of A549 cells in vitro . Wound healing assay showed the degressive ability of metastasis. High expression of E-cadherin and low expression of vimentin indicated that RKIP affects invasion and metastasis of NSCLC via inhibiting EMT. Conclusions: RKIP is decreased in NSCLC tissues. Overexpression of RKIP in A549 cells would decrease the cellular proliferation, viability, invasion ability, and metastasis ability probably via inhibiting EMT through upregulating E-cadherin expression and downregulating vimentin expression.

Oral sarcomatoid squamous cell carcinoma: a retrospective study based on 14 cases.

The treatment outcomes for oral sarcomatoid squamous cell carcinoma (OSSCC) are far from satisfactory in our hospital. The aim of this study was to retrospectively summarize the OSSCC cases admitted to our department. From 2003 to 2017, 14 patients were hospitalized and diagnosed with OSSCC. We summarized and analysed the medical histories, diagnostic examinations, treatment strategies, and clinical outcomes of the involved cases. Of the 14 cases, 8 were located in the gingiva. The imageological diagnosis identified the existence of a mass with an infiltrative morphology pre-operatively. The cytopathologic features revealed a malignant neoplasm with a mixture of squamous cell carcinoma (SCC) components and spindle cell neoplastic components. To confirm the diagnosis of OSSCC, the use of the immunohistochemical markers AE1/AE3 and Vimentin were more indicative. Complete follow-up data were available for 12 patients, and at the last follow-up, all 12 of the patients had died. The median overall survival for these patients was 11.67 months (range: 3-24 months). OSSCC patients respond poorly to the strategies solely referring to experiences from oral squamous cell carcinoma (OSCC) treatment. The effective diagnosis and treatment of OSSCC at an early stage is necessary. The treatment for OSSCC still poses a great challenge for clinical oncologists.

KIF18A promotes head and neck squamous cell carcinoma invasion and migration via activation of Akt signaling pathway

Background: KIF18A has been shown to participate in the development of various human malignancies. However, the role of KIF18A in head and neck squamous cell carcinoma (HNSCC) remains unknown. This study investigated the function of KIF18A in HNSCC as well as its possible mechanisms. Methods: In this study, we conducted in vitro experiments. First, we examined the effect of KIF18A on Tu686 and 6-10B cells via determining cell viability, colony formation ability and cell motility. And then, we examined that whether the carcinogenic effect of KIF18A is associated with Akt activation. Results: Our current study demonstrated that KIF18A expression was increased in HNSCC patients and its cell lines. Knockdown and overexpression of KIF18A in HNSCC cells indicated that KIF18A promoted cancer cell proliferation, invasion and migration. Moreover, these bioactivity changes in HNSCC cells were accompanied by enhanced Vimentin expression and suppressed E-cadherin expression induced by KIF18A. Further mechanistic analysis revealed that the carcinogenic effect of KIF18A is associated with Akt activation, and blocking the activity of Akt reversed the malignant progression caused by KIF18A overexpression in HNSCC cells. Conclusions: Together, our study reveals that KIF18A accelerates the progression of HNSCC and that targeting KIF18A may be a potential therapeutic strategy for the HNSCC.

Filaments and phenotypes: cellular roles and orphan effects associated with mutations in cytoplasmic intermediate filament proteins.

Cytoplasmic intermediate filaments (IFs) surround the nucleus and are often anchored at membrane sites to form effectively transcellular networks. Mutations in IF proteins (IFps) have revealed mechanical roles in epidermis, muscle, liver, and neurons. At the same time, there have been phenotypic surprises, illustrated by the ability to generate viable and fertile mice null for a number of IFp-encoding genes, including vimentin. Yet in humans, the vimentin ( VIM) gene displays a high probability of intolerance to loss-of-function mutations, indicating an essential role. A number of subtle and not so subtle IF-associated phenotypes have been identified, often linked to mechanical or metabolic stresses, some of which have been found to be ameliorated by the over-expression of molecular chaperones, suggesting that such phenotypes arise from what might be termed “orphan” effects as opposed to the absence of the IF network per se, an idea originally suggested by Toivola et al. and Pekny and Lane.

The opposite effects of angiotensin 1-9 and angiotensin 3-7 in prostate epithelial cells

There is growing evidence that renin-angiotensin system (RAS) components have been involved in the development of various types of cancers, including prostate cancer. This article for the first time reports the impact of Ang1-9 and Ang3-7 on viability and proliferation, migration and invasion of epithelial prostate cells. The results of this study clearly show that Ang1-9 and Ang3-7 exert different/opposite effects on in vitro biological properties of prostate cells. It appears that Ang1-9 has pro-cancer activities via the ability to induce cell divisions, enhance cell motility and stimulate the expression of such genes as vascular endothelial growth factor (VEGF), hypoxia-inducible factors (HIF-1), vimentin (VIM) and REL proto-oncogene, NF-kB subunit (REL). On the contrary, Ang3-7 did not show any mitogenic activity. Furthermore, this peptide hormone limited the migration of PNT1A cells probably by downregulation of VEGF and VIM expression. Finally, it is worth noting that both angiotensins have the ability to modulate gene expression for angiotensin receptors. Unfortunately, we could not unequivocally identify the type of angiotensin receptor responsible for signal transduction pathway involved in PNT1A cell survival and proliferation. Undoubtedly, further research and testing in this area are necessary.

Mechanism of YAP1-JUN promoting proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells

Objective To investigate the role of Yes-associated protein 1 (YAP1)-JUN in promoting the proliferation and epithelial mesenchymal transitions (EMT) transformation of laryngeal squamous cell carcinoma. Methods The expression and subcellular localization of YAP1 were detected by tissue immunofluorescence assay. The effects of YAP1 on the proliferation of laryngeal squamous cell were examined by cell clone formation experiment. Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the effect of YAP1 on the expression and transcriptional level of EMT-related molecular markers. The interaction between YAP1 and JUN was detected by immunocoprecipitation (CoIP) and Western blot assay, which regulated the expression of downstream genes to control the EMT process. Results The expression of YAP1 in laryngeal squamous cell carcinoma tissue increased and transferred from cytoplasm to nucleus. The number of clones increased significantly after YAP1 up regulation (P<0.01). The expression level of key gene E-cadherin in epithelial cells was significantly inhibited after YAP1 up (P<0.01), while the expression level of the key genes of interstitial cells, β-catenin, vimentin and N-cadherin was significantly up (P<0.01). YAP1 was interacted with nuclear transcription factor JUN, and the proliferation ability of YAP1 decreased significantly (P<0.01) after the inhibition of JUN expression (P<0.01), and the expression of EMT related molecular markers decreased significantly (P<0.01). Conclusions YAP1 combined with JUN gene promotes the proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells. Key words: Laryngeal neoplasms; Carcinoma, squamous cell; Yes-associated protein 1; Cell proliferation; Epithelial-mesenchymal transition

The predictive value of CTCs characterization for time to castration resistance of high-volume metastatic castration sensitive prostate cancer

Objective To explore the predictive value of circulating tumor cells (CTCs) characterization for time to castration resistance of newly diagnosed high volume metastatic castration sensitive prostate cancer (mCSPC) patients. Methods Newly diagnosed high volume mCSPC patients were prospectively enrolled in this study from September 2015 to February 2017. The inclusion criteria include that the patients' age should be between 18 to 85 years old. The Prostate cancer should be diagnosed by biopsy or cytopathology. No endocrinological therapy, radiative therapy or chemotherapy was used before the study. High-volume metastatic lesion was confirmed by imaging. Those patients who accepted previous endocrinological therapy, radiative therapy or chemotherapy were excluded in this study. Those patients combined with concomitant tumor were also excluded. The health males were enrolled in the control group. All patients received androgen deprivation therapy (ADT) with goserelin plus bicalutamide (goserelin 3.6 mg subcutaneous injection, once a month plus bicalutamide 50mg orally, once a day). CanPatrol system was used to count CTCs in peripheral blood of patients and characterize CTCs based on expressions of epithelial markers(EpCAM and CK8/18/19) and mesenchymal markers(vimentin and twist). Primary endpoint was time to castration resistance. Survival analysis was conducted using Kaplan-Meier method and log-rank test was used to assess the difference of survival between groups, and univariate and multivariate analyses of prognostic factors were conducted using the Cox proportional hazards model. Results A total of 108 newly diagnosed high volume mCSPC patients were enrolled in this study. The median age of enrolled patients was 68 years old (ranging 51-85 years old), and median PSA was 196.2 ng/ml(ranging 5.8-5 011.9 ng/ml). The median level of hemoglobin was 32 g/L(ranging 9-172 g/L). The median level of LDH was 179 U/L(ranging 49-630 U/L). The ECOG scores was 0-1 score in 94 cases(87.0%), 2 scores in 14 cases (13.0%). The Gleason scores was 6-7 in 20 cases (18.5%) and more than 8 in 88 cases (81.5%). All patients had bone metastatic lesions, among which 41 (38.0%) patients had more than 10 metastatic lesions and 6 (5.6%) patients with visceral metastasis, 30(27.8%) patients with limb bone metastasis. The median CTCs count was four, and ranging 0-35. Mesenchymal CTCs positive and negative (negative included CTCs negative, epithelial CTCs positive and biophenotypic CTCs positive) patients were 58(53.7%) and 50, respectively. There was no correlation between CTCs characterization with age, baseline PSA, Gleason score, ALP and other clinical parameters (P>0.05). In control group, the mean age was 26 years old (ranging 20-31 years old). No CTCs were detected among those people. After a median follow-up of 24 months (ranging 18-32 months), 90 patients (83.3%) progressed to castration resistant prostate cancer (CRPC). The median time to CRPC for patients of mesenchymal CTCs positive and negative was (10.5±1.4) and (14.0±3.4) months, respectively(P<0.001). Univariate analysis revealed CTCs characterization(HR=1.647, P=0.003), the number of metastatic lesions (HR=1.624, P=0.025)and limb bone metastasis(HR=1.706, P=0.019) were prognostic factors of time to CRPC; further multivariate analysis showed that only baseline mesenchymal CTCs positive (HR=1.562, P=0.008) was independent prognostic factors of unfavorable time to CRPC. Conclusions CTCs characterization can predict time to CRPC of newly diagnosed high volume mCSPC patients receiving ADT, and patients of baseline mesenchymal CTCs positive are more likely to progress to CRPC. Key words: Prostatic neoplasms; Castration sensitive prostate cancer; Androgen deprivation therapy; Circulating tumor cells; Epithelial mesenchymal transition; Castration resistant prostate cancer