TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis.

BACKGROUND: To investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis. METHODS: The protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis. RESULTS: The TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells. CONCLUSIONS: The results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

Circulating Tumor Cells With Epithelial-to-mesenchymal Transition Phenotypes Associated With Inferior Outcomes in Primary Breast Cancer.

BACKGROUND/AIM: Circulating tumor cells (CTCs) comprise a heterogeneous population of cancer cells with different clinical and biological value. The aim of this study was to evaluate the prognostic value of CTCs with an epithelial-mesenchymal transition (EMT) phenotype in primary breast cancer (PBC) patients. PATIENTS AND METHODS: This study included 427 primary breast cancer patients. RNA extracted from CD45-depleted peripheral blood mononuclear cell (PBMCs) was evaluated for the expression of EMT transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) by quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: In total, CTC EMT was detected in 77 (18.0%) patients. Patients without detectable CTC EMT in peripheral blood had significantly longer disease-free survival than patients with detectable CTC EMT. The prognostic value of CTC EMT was demonstrated in all subgroups of patients. CONCLUSION: CTCs with an EMT phenotype have a prognostic value in primary breast cancer.

Epithelial­mesenchymal transition was identified as a potential marker for breast cancer aggressiveness using reverse transcription­quantitative polymerase chain reaction.

The primary cause of breast cancer­associated mortality is the formation of distant metastasis. During the metastatic process, single tumor cells dissolve from the primary tumor site and undergo various changes in cell adhesion and motility properties. The tumor cells invade the blood stream and travel to different sites of the body, where they may initiate outgrowth. These cells are referred to as circulating tumor cells (CTCs). The process of changing cellular properties is known as epithelial to mesenchymal transition (EMT). As a different set of genes is upregulated during EMT, such genes may serve as marker genes for the detection of CTCs based on reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Therefore, EMT­ and breast cancer­related genes were selected as RT­qPCR markers. These genes were tested for performance in a model system of blood samples from healthy donors, to which a number of various breast cancer cell lines were added. The genes with optimal performance were subsequently used in RT­qPCR with 35 breast cancer patient samples. The genes which showed the highest and most consistent increase in gene expression with the increase in the number of cancer cell line cells added were CK19, Snail, FoxC2 and Twist. Following RT­qPCR for all patient samples, two subgroups were arranged: One group in which all genes were downregulated and the second group with at least one gene indicated an upregulation of gene expression. Comparisons were made between the tumour characteristics from these two groups. Results suggested that carcinomas of the first group exhibited a less aggressive tumor biology compared with those in the second group. The present study indicated a novel RT­qPCR based test for tumor malignancy.

Serum and tissue expression of gelatinase and Twist in breast cancer.

OBJECTIVE: This study aimed to investigate the levels of matrix metalloproteinases (MMP)-2, MMP-9 and Twist in tumor tissue and serum from 46 cases of breast cancer patients and 31 cases of benign breast diseases patients by immunohistochemical staining and enzyme-linked immunosorbent assay, respectively. The association of gelatinase and Twist expression with clinicopathological factors was also analyzed in the present study. PATIENTS AND METHODS: The studied population consisted of 46 breast cancer patients and 31 benign breast disease patients. Serum concentrations of MMP-2, MMP-9 and Twist were measured by using human enzyme-linked immunosorbent assay. The protein expression of Twist, MMP-2 and MMP-9 were determined by immunohistochemical. RESULTS: The results show that the levels of MMP-2, MMP-9 and Twist expression were significantly increased in tissue and serum from breast cancer group, compared to the group of benign breast lesions diseases (p < 0.05). The pre-operative serum levels of MMP-2, MMP-9 and Twist were positively correlated with their expression in breast cancer tissues, respectively (p < 0.05). We, then, correlated serum and tissue levels of MMP-2, MMP-9 and Twist in breast cancer samples with patients' clinicopathologic characteristics. Compared to low expression, high serum and tissue levels of MMP-2 and Twist were associated with lymph node metastasis and higher TNM stage, high tissue MMP-9 levels were associated with lymph node metastasis and higher TNM stage, and high serum MMP-9 levels were associated with c-erbB-2 expression. CONCLUSIONS: These data suggest that serum levels of MMP-2, MMP-9 and Twist could be as potential biomarkers for diagnosis and predicting metastasis of breast cancer.

Prognostic value of changes in the expression of stem cell markers in the peripheral blood of patients with colon cancer.

Cancer stem cells play an important role in carcinogenesis and resistance to treatment and may lead to metastasis. The isolation of circulating stem cells involves cell sorting based on the presence of cell surface markers. Many surface markers such as CD133, c-Kit, SOX, OCT4 and TWIST have been reported. In the present study, we determined the expression of different stem cell markers and their variation in expression at different stages of the treatment process. Samples of EDTA blood were collected from metastatic colorectal cancer patients, and circulating cancer stem cells were isolated for the analysis of the expression of stem cell markers using RT-PCR. These findings were correlated with the response to therapy. All statistical analyses were performed using the GraphPad Prism 5.03 software. Significant differences were found in the expression levels of the markers CD133, SOX2, OCT4 and TWIST1. No differences were found in c-Kit expression. Correlation in the expression levels of most of the markers was observed. Expression of CD133, OCT4, SOX2 and TWIST1 had a predictive value for colon cancer behavior. Evaluation of this stem cell gene expression panel may be useful for predicting the response during the process of treatment, and the relative easy access to samples facilitates this method. Moreover the correlation between CD133 and TWIST1 expression may be associated with tumor regrowth and metastatic relapse.