Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

Generation of a Twist1 conditional null allele in the mouse

Twist1 is the mouse ortholog of TWIST1, the human gene mutated in Saethre-Chotzen syndrome. Previously, a Twist1 null allele was generated by gene targeting in mouse embryonic stem cells. Twist1 heterozygous mice develop polydactyly and a craniofacial phenotype similar to Saethre-Chotzen patients. Mice homozygous for the Twist1 null allele die around embryonic day 11.5 (E11.5) with cranial neural tube closure and vascular defects, hindering in vivo studies of Twist1 function at later stages of development. Here, we report the generation of a Twist1 conditional null allele in mice that functions like a wild-type allele but can be converted to a null allele upon Cre-mediated recombination.     

Insulin-like growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor

In this study we investigated the molecular mechanisms whereby insulin-like growth factor 1 (IGF-1) induced Twist gene expression and the role of Twist in the anti-apoptotic actions of the IGF-1 receptor. In NIH-3T3 fibroblasts overexpressing the human IGF-1 receptor (NWTb3), treatment with IGF-1 (10(-8) m) for 1 and 4 h increased the level of Twist mRNA as well as protein by 3-fold. In contrast, insulin at physiological concentrations did not stimulate Twist expression in NIH-3T3 fibroblasts overexpressing the human insulin receptor. The IGF-1 effect was specific for the IGF-1 receptor since, in cells overexpressing a dominant negative IGF-1 receptor, IGF-1 failed to increase Twist expression. Pre-incubation with the ERK1/2 inhibitor U0126 or expression of a dominant negative MEK-1 abolished the effect of IGF-1 on Twist mRNA expression in NWTb3 cells, suggesting that Twist induction by IGF-1 occurs via the mitogen-activated protein kinase signaling pathway. In vivo, IGF-1 injection increased the mRNA level of Twist in mouse skeletal muscle, the major site of Twist expression. Finally, using an antisense strategy, we demonstrated that a reduction of 40% in Twist expression decreased significantly the ability of IGF-1 to rescue NWTb3 cells from etoposide-induced apoptosis. Taken together, these results define Twist as an important factor involved in the anti-apoptotic actions of the IGF-1 receptor.     

Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition

Twist1 is highly expressed in primary and metastatic non-small cell lung cancer (NSCLC), and thus acts as a critical target for lung cancer chemotherapy. In the current study, we investigated the underlying mechanism initiated by silencing of Twist1 that sensitizes NSCLC cells to cisplatin. Silencing of Twist1 triggered ATP depletion, leading to AMP-activated protein kinase (AMPK)-activated mammalian target of rapamycin (mTOR) inhibition in NSCLC cells. AMPK-induced mTOR inhibition, in turn, resulted in downregulation of ribosome protein S6 kinase 1 (S6K1) activity. Downregulation of mTOR/S6K1 reduced Mcl-1 protein expression, consequently promoting sensitization to cisplatin. Overexpression of Mcl-1 reduced PARP cleavage induced by cisplatin and Twist1 siRNA, suggesting that this sensitization is controlled through Mcl-1 expression. Interestingly, cells treated with Twist1 siRNA displayed upregulation of p21^Waf1/CIP1, and suppression of p21^Waf1/CIP1 with specific siRNA further enhanced the cell death response to cisplatin/Twist1 siRNA. In conclusion, silencing of Twist1 sensitizes lung cancer cells to cisplatin via stimulating AMPK-induced mTOR inhibition, leading to a reduction in Mcl-1 protein. To our knowledge, this is the first report to provide a rationale for the implication of cross-linking between Twist1 and mTOR signaling in resistance of NSCLC to anticancer drugs.     

Modulation of Oxidative Stress by Twist Oncoproteins

Expression of developmental genes Twist1 and Twist2 is reactivated in many human tumors. Among their oncogenic activities, induction of epithelial to mesenchymal transition is believed to increase cell motility and invasiveness and may be related to acquisition of cancer stem cell phenotype. In addition, Twist proteins promote malignant conversion by overriding two oncogene-induced failsafe programs: senescence and apoptosis. Reactive oxygen species (ROS) are also important mediators of apoptosis, senescence and motility and are tightly linked to disease, notably to cancer. We report here that Twist factors and ROS are functionally linked. In wild type cells both Twist1 and Twist2 exhibit antioxidant properties. We show that Twist-driven modulation of oncogene-induced apoptosis is linked to its effects on oxidative stress. Finally, we identify several targets that mediate Twist antioxidant activity. These findings unveil a new function of Twist factors that could be important in explaining their pleiotropic role during carcinogenesis.     

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple‐negative breast cancer

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.