Overexpression Of Twist Research Articles

Prognostic and clinicopathological value of Twist expression in breast cancer: A meta-analysis.

BackgroundDespite initial indications that the transcription factor Twist could be used as a breast cancer prognostic marker, there still exists some controversy about its reliability. Thus, the aim of the present study was to assess the relationship between Twist expression and prognosis in breast carcinoma.Materials and methodsWe identified eligible studies that reported an association between Twist expression and breast cancer prognosis by searching the literature in PubMed, Embase, the Cochrane Library, and Web of Science databases, through June 5, 2017. Studies investigating Twist protein or mRNA expression as well as reporting survival data in breast cancer were included. The pooled hazard ratio (HR) and odds radio (OR) with a 95% confidence interval (95% CI) were used to estimate associations.ResultsA total of 2,671 patients from seven included studies were assessed, and the data indicated that increased Twist expression significantly correlated with poor overall survival (OS) (HR, 1.15; 95% CI, 1.00–1.33; P = 0.04) in breast cancer. In addition, we also observed a significant correlation of elevated Twist expression with larger tumor size (OR, 1.92; 95% CI, 1.31–2.81; P = 0.0009), lymph node involvement (OR, 3.81; 95% CI, 1.16–12.54; P = 0.03), higher nuclear grade (OR, 1.45; 95% CI, 1.06–2.00; P = 0.02), and positive human epidermal growth factor receptor 2 (HER2) status (OR, 1.49; 95% CI, 1.06–2.09; P = 0.02). However, no correlation between Twist expression and disease-free survival (DFS), age, estrogen receptor (ER) status, and progesterone receptor (PR) status was observed.ConclusionsOur results demonstrate that Twist over-expression is a statistically significant indicator of OS in breast cancer. In addition, our meta-analysis shows that increased Twist expression is significantly associated with larger tumor size, lymph node involvement, higher nuclear grade, and positive HER2 status.

TWIST mediates resistance to paclitaxel by regulating Akt and Bcl-2 expression in gastric cancer cells

The transcription factor TWIST has been reported to play an important role in tumor progression as well as resistance to anti-cancer drugs. However, the role of TWIST in gastric cancer and the molecular mechanisms by which this protein elicits drug resistance remain poorly understood. We transfected gastric cancer cell lines with lentiviral vector to generate TWIST-overexpressing stable cell lines. Our study showed that overexpression of TWIST not only increased cell migration and invasion but also induced resistance to the anti-cancer drug paclitaxel in gastric cancer. Paclitaxel increased gastric cancer cell death in dose-dependent manner; this was decreased following TWIST overexpression. Furthermore, treatment with paclitaxel decreased Akt phosphorylation and Bcl-2 expression, whereas these effects were suppressed by TWIST overexpression. Treatment of cells with Akt inhibitor or small interfering RNA targeting for Bcl-2 led to increased paclitaxel-induced cell death, indicating that TWIST elicits resistance to paclitaxel via the regulation of the Akt and Bcl-2 pathway. Our results suggest an underlying mechanism for TWIST-mediated paclitaxel resistance and indicate that TWIST represents a potential target for overcoming paclitaxel resistance in gastric cancer cells.

MiR-3656 expression enhances the chemosensitivity of pancreatic cancer to gemcitabine through modulation of the RHOF/EMT axis

The highly refractory nature of pancreatic cancer (PC) to chemotherapeutic drugs is one of the key reasons contributing to the poor prognosis of this disease. MicroRNAs (miRNAs) are key regulators of gene expression and have been implicated in a variety of processes from cancer development through to drug resistance. Herein, through miRNA profiling of gemcitabine-resistant (GR) and parental PANC-1 cell lines, we found a consistent reduction of miR-3656 in GR PANC-1 cells. miR-3656 overexpression enhanced the antitumor effect of gemcitabine, whereas silencing of miR-3656 resulted in the opposite effect. By performing mechanistic studies using both in vitro and in vivo models, we found that miR-3656 could target RHOF, a member of the Rho subfamily of small GTPases, and regulate the EMT process. Moreover, enforced EMT progression via TWIST1 overexpression compromised the chemotherapy-enhancing effects of miR-3656. Finally, we found significantly lower levels of miR-3656 and higher levels of RHOF in PC tissues compared with adjacent noncancerous pancreatic tissues, and this was also associated with poor PC patients’ prognosis. Taken together, our results suggest that the miR-3656/RHOF/EMT axis is an important factor involved in regulating GR in PC, and highlights the potential of novel miR-3656-based clinical modalities as a therapeutic approach in PC patients.

Abstract B33: Tgif inactivation defines a synthetic lethal interaction among oncogenic Kras and Twist1 in pancreatic ductal adenocarcinoma

Abstract The homeodomain protein TGIF function as a potent inhibitor of transforming growth factor beta (TGF-β) cytostatic signaling, whose inactivation is deemed instrumental to the pathogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) driven by oncogenic Kras. Surprisingly, we found that deletion of Tgif in the context of mutant Kras (KrasG12D) expression culminated in the development of highly metastatic PDAC, despite leading to hyperactivation of TGF-(β/Smad) signaling. Mechanically, we found that TGIF associated with and antagonized Twist1 by a process involving Smad4, a tumor suppressor that is frequently inactivated in human PDAC. Along these lines, Twist1 overexpression synergized with KrasG12D to induce PDAC progression, whereas its ablation completely blocked the malignant phenotype driven by the combined KrasG12D expression and TGIF ablation. Thus, by demonstrating that TGIF functions as a tumor suppressor in PDAC owing to its ability to antagonize the pro-malignant transcription factor Twist1, our studies provide key insights into the role of Twist1 that could be exploited to design synthetic lethal therapeutics against oncogenic Kras, which has proven so far to be intractable. Citation Format: Parash Parajuli, Purba Singh, Zhe Wang, Santosh Kumar, Lena Li, Sailaja Eragamreddi, Thien Ly Nguyen, Subhi Talal Younes, Xu Zhang, Keli Xu, Mohammed Razzaque, Céline Prunier, Azeddine Atfi. Tgif inactivation defines a synthetic lethal interaction among oncogenic Kras and Twist1 in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B33.

Twist1 in Hypoxia-induced Pulmonary Hypertension through Transforming Growth Factor-β-Smad Signaling.

Pulmonary hypertension (PH) is a devastating pulmonary vascular disease characterized by aberrant muscularization of the normally nonmuscularized distal pulmonary arterioles. The expression of the transcription factor, Twist1, increases in the lungs of patients with pulmonary arterial hypertension. However, the mechanisms by which Twist1 controls the pathogenesis of PH remain unclear. It is becoming clear that endothelial-to-mesenchymal transition (EndMT) contributes to various vascular pathologies, including PH; Twist1 is known to mediate EndMT. In this report, we demonstrate that Twist1 overexpression increases transforming growth factor (TGF) β receptor2 (TGF-βR2) expression and Smad2 phosphorylation, and induces EndMT in cultured human pulmonary arterial endothelial (HPAE) cells, whereas a mutant construct of Twist1 at the serine 42 residue (Twist1S42A) fails to induce EndMT. We also implanted fibrin gel supplemented with HPAE cells on the mouse lung, and found that these HPAE cells form vascular structures and that Twist1-overexpressing HPAE cells undergo EndMT in the gel, whereas Twist1S42A-overexpressing cells do not. Furthermore, hypoxia-induced EndMT is inhibited in endothelial cells overexpressing Twist1S42A mutant construct in vitro. Hypoxia-induced accumulation of α-smooth muscle actin-positive cells in the pulmonary arterioles is attenuated in Tie2-specific Twist1 conditional knockout mice in vivo. These findings suggest that Twist1 serine 42 phosphorylation plays a key role in EndMT through TGF-β signaling and that modulation of Twist1 phosphorylation could be an effective strategy for managing PH.

Reactivation of TWIST1 contributes to Ewing sarcoma metastasis.

Ewing sarcoma is a cancer of bone and soft tissue. Despite aggressive treatment, survival remains poor, particularly in patients with metastatic disease. Failure to treat Ewing sarcoma is due to the lack of understanding of the molecular pathways that regulate metastasis. In addition, no molecular prognostic markers have been identified for Ewing sarcoma to risk stratify patients. Ewing sarcoma patients were divided into high or low Twist1 gene expression and survival curves were generated using the R2 microarray-based Genomic Analysis platform (http://r2.amc.nl). Tumors from Ewing sarcoma patients were also evaluated for TWIST1 expression by immunohistochemistry. Ewing sarcoma xenografts were established to evaluate the role of TWIST1 in metastasis. The effects of Twist1 on migration and invasion were evaluated using migration and invasion assays in A673 and RDES cells. Twist1 expression was a negative prognostic marker for overall survival in a public Ewing sarcoma patient data set based on Twist1 mRNA levels and in patient tumor samples based on Twist1 immunohistochemistry. TWIST1 is detected in significantly higher percentage of patients with metastatic diseases than localized disease. Using Ewing sarcoma tumor xenografts in mice, we found that suppressing TWIST1 levels suppressed metastasis without affecting primary tumor development. Knockdown of Twist1 inhibited the migration and invasion capability, while overexpression of Twist1 promoted migration and invasion in Ewing sarcoma cells. These results suggest that TWIST1 promotes metastasis in Ewing sarcoma and could be used as a prognostic marker for treatment stratification; however, further validation is required in a larger cohort of patients.

Abstract 4118: The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer

Abstract Recent advances in the treatment of non-small cell lung cancer (NSCLC) stem from the paradigm shift of classifying patients into subtypes based upon the presence of distinct molecular drivers. Subsets of patients, such as those with EGFR mutations and ALK translocations, have dramatic responses in their tumors to tyrosine kinase inhibitors (TKIs) that specifically inhibit these oncogenic drivers. While many patients initially response to TKIs, therapeutic resistance is inevitable. For EGFR-mutant NSCLC, there are multiple described mechanisms of resistance to EGFR TKIs, including epithelial-mesenchymal transition (EMT). Previous studies have implicated the AXL kinase and ZEB1, an EMT transcription factor (EMT-TF), in EMT-mediated EGFR TKI resistance. We have previously demonstrated that the EMT-TF, TWIST1, is required for oncogene-driven NSCLC tumorigenesis, including those tumors with EGFR mutations. In this study, we investigated the role of TWIST1 in EMT-mediated resistance to EGFR TKIs. We have demonstrated that genetic or pharmacologic inhibition of TWIST1 resulted in growth inhibition in a panel of EGFR-mutant NSCLC cell lines and apoptosis in a subset of these lines. Interestingly, TWIST1 overexpression in EGFR-mutant NSCLC cell lines led to EGFR TKI resistance. Conversely, knockdown of TWIST1 in an erlotinib resistant EGFR-mutant NSCLC cell line restored erlotinib sensitivity. We found that TWIST1 mediates resistance to EGFR TKIs through suppression of apoptosis possibly through decreasing the expression of the pro-apoptotic Bcl-2 member, BCL2L11 (BIM). We observed that TWIST1 knockdown increased BIM levels, while TWIST1 overexpression decreased BIM expression. Furthermore, TWIST1-mediated resistance was overcome by treatment with the BCL-2/BCL-XL inhibitor, ABT-737. Knockdown of BIM recapitulated the resistance seen following TWIST1 overexpression, suggesting that TWIST1 suppression of BIM is a mechanism through which TWIST1 leads to EGFR TKI resistance. To explore the role of TWIST1 in modulating EGFR inhibitor sensitivity in vivo, we used an inducible EGFR-mutant transgenic mouse model, CCSP-rtTA/tetO-EGFRL858R (CE), which expresses EGFRL858R in the lung and a EGFR-mutant/Twist1 transgenic model, CCSP-rtTA/tetO-EGFRL858R/ Twist1- tetO7-luc (CET), which expresses both Twist1 and EGFRL858R in the lung. CET mice had a significantly increased tumor burden, decreased apoptosis and a decreased overall survival compared to CE mice following erlotinib treatment. In summary, we found that TWIST1 overexpression leads to EGFR TKI resistance by suppressing EGFR TKI-induced apoptosis through suppressing BIM expression. Future studies aim to establish the mechanisms of TWIST1 suppression of BIM expression and determine if our TWIST1 inhibitor, harmine, is effective in overcoming EMT-mediated resistance. Citation Format: Zachary A. Yochum, Hailun Wang, Jessica A. Cades, Susheel Khetarpal, Eric H. Huang, Phouc T. Tran, Timothy F. Burns. The EMT transcription factor TWIST1 mediates resistance to EGFR inhibitors in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2017-4118

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

TWIST1-WDR5-Hottip Regulates Hoxa9 Chromatin to Facilitate Prostate Cancer Metastasis.

TWIST1 is a transcription factor critical for development that can promote prostate cancer metastasis. During embryonic development, TWIST1 and HOXA9 are coexpressed in mouse prostate and then silenced postnatally. Here we report that TWIST1 and HOXA9 coexpression are reactivated in mouse and human primary prostate tumors and are further enriched in human metastases, correlating with survival. TWIST1 formed a complex with WDR5 and the lncRNA Hottip/HOTTIP, members of the MLL/COMPASS-like H3K4 methylases, which regulate chromatin in the Hox/HOX cluster during development. TWIST1 overexpression led to coenrichment of TWIST1 and WDR5 as well as increased H3K4me3 chromatin at the Hoxa9/HOXA9 promoter, which was dependent on WDR5. Expression of WDR5 and Hottip/HOTTIP was also required for TWIST1-induced upregulation of HOXA9 and aggressive cellular phenotypes such as invasion and migration. Pharmacologic inhibition of HOXA9 prevented TWIST1-induced aggressive prostate cancer cellular phenotypes in vitro and metastasis in vivo This study demonstrates a novel mechanism by which TWIST1 regulates chromatin and gene expression by cooperating with the COMPASS-like complex to increase H3K4 trimethylation at target gene promoters. Our findings highlight a TWIST1-HOXA9 embryonic prostate developmental program that is reactivated during prostate cancer metastasis and is therapeutically targetable. Cancer Res; 77(12); 3181-93. ©2017 AACR.

CXCL11 mediates TWIST1-induced angiogenesis in epithelial ovarian cancer.

To investigate the role of TWIST1 in tumor angiogenesis in epithelial ovarian cancer and to identify key molecules involved in angiogenesis. TWIST1 small interfering RNA was transfected into A2780 cells, while a complementary DNA vector was transfected into non-malignant human ovarian surface epithelial cells to generate a TWIST1-overexpressing cell line. To evaluate how this affects angiogenesis, human umbilical vein endothelial cell tube formation assays were performed using the control and transfected cell lines. An antibody-based cytokine array was used to identify the molecules involved in TWIST1-mediated angiogenesis. After knockdown of TWIST1 via transfection of TWIST1 small interfering RNA into A2780 cells, the number of tubes formed by human umbilical vein endothelial cells significantly decreased in a tube formation assay. In a cytokine array, TWIST1 downregulation did not significantly decrease the secretion of the common pro-angiogenic factor, vascular endothelial growth factor, but instead inhibited the expression of the CXC chemokine ligand 11, which was confirmed by both an enzyme-linked immunosorbent assay and western blotting. In contrast, TWIST1 overexpression resulted in increased secretion of CXC chemokine ligand 11. Conversely, CXC chemokine ligand 11 downregulation did not inhibit the expression of TWIST1. Furthermore, the ability of TWIST1-expressing A2780 cells to induce angiogenesis was found to be inhibited after CXC chemokine ligand 11 knockdown in a tube formation assay. TWIST1 plays an important role in angiogenesis in epithelial ovarian cancer and is mediated by a novel pro-angiogenic factor, CXC chemokine ligand 11. Downregulation of CXC chemokine ligand 11 can inhibit tumor angiogenesis, suggesting that anti-CXC chemokine ligand 11 therapy may offer an alternative treatment strategy for TWIST1-positive ovarian cancer.

MicroRNA-610 suppresses osteosarcoma oncogenicity via targeting TWIST1 expression.

Osteosarcoma is the most frequent primary bone tumor affects adolescents and young adults. Recently, microRNAs (miRNAs) are short, non-coding and endogenous RNAs that played as important roles in the initiation and progression of tumors. In this study, we try to explore the biological function and expression of miR-610 in the osteosarcoma. We showed that miR-610 expression was downregulated in the osteosarcoma tissues and cell lines. Elevated expression of miR-610 suppressed the osteosarcoma cell proliferation, cell cycle, invasion and EMT program. Moreover, overexpression of miR-610 increased sensitivity of MG-63 and U2OS cells to cisplatin. Twist1 was identified as a direct target gene of miR-610 in the osteosarcoma cell. Furthermore, we demonstrated that Twist1 was upregulated in the osteosarcoma tissues and cell lines. The expression of Twist1 was negatively associated with the expression of miR-610 expression in the osteosarcoma tissues. Ectopic expression of Twist1 inhibited the sensitivity of miR-610-overexpressing MG-63 cells to cisplatin. We also showed that overexpression of Twist1 increased the proliferation and invasion of miR-610-overexpressing MG-63 cells. These data indicated that ectopic expression of miR-610 suppressed the osteosarcoma cell proliferation, cell cylce, invasion and increased the sensitivity of osteosarcoma cells to cisplatin through targeting the Twist1 expression.

Skp2 deficiency restricts the progression and stem cell features of castration-resistant prostate cancer by destabilizing Twist.

Castration-resistant prostate cancer (CRPC) remains a major clinical challenge due to the lack of effective targeted therapy for its treatment. The mechanism underlying how CRPC gains resistance towards hormone depletion and other forms of chemotherapy is poorly understood. Research on understanding the factors that drive these processes is desperately needed to generate new therapies to cure the disease. Here, we discovered a fundamental role of S-phase protein kinase 2 (Skp2) in the formation and progression of CRPC. In transgenic adenocarcinoma mouse prostate model, Skp2 depletion leads to a profound repression of prostate tumor growth and distal metastasis and substantially prolonged overall survival. We revealed that Skp2 regulates CRPC through Twist-mediated oncogenic functions including epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) acquisitions. Mechanistically, Skp2 interacted with Twist and promoted the non-degradative ubiquitination of Twist. Consequently, Skp2 stabilized Twist protein expression by preventing proteasomal degradation of Twist by β-TrCP. We found that Twist overexpression augments CSC self-renewal and population and that Skp2 inhibition reverts Twist’s effects on CSC regulation. Furthermore, genetically depleting or pharmacologically inactivating Skp2 synergistically re-sensitized CRPC cells towards chemotherapies such as paclitaxel or doxorubicin. Together, the current study uncovering Skp2-mediated Twist stabilization and oncogenic functions in CRPC offers new knowledge on how CRPC progresses and acquires chemoresistance during tumor progression. It provides prove-of principle that Skp2 targeting is a promising approach to combat metastatic CRPC by targeting Twist and CSCs.

Combination Twist1 and CA15-3 in axillary lymph nodes for breast cancer prognosis.

Twist1 overexpression is involved in epithelial-mesenchymal transition resulting in migration and metastasis of breast cancer. Carcinoma antigen 15–3 (CA15-3) is widely used to monitor the prognosis for patients after treatment. However, the significance of Twist1 in axillary lymph nodes (ALN) and CA15-3 for co-examination for survival rates remains to be elucidated. The present study aimed to explore the role of the combination of Twist1 expression in metastasized ALN and the serum level of CA15-3 in evaluating the prognosis of patients with breast cancer. cluster of differentiation (CD)44, CD24, aldehyde dehydrogenase (ALDH)1 and Twist1 expression in normal and metastasized ALN from 102 patients with breast cancer were detected using laser confocal microscopy and the expression of the genes evaluated by reverse transcription-quantitative polymerase chain reaction; E-cadherin, N-cadherin and vimentin expression was also tested by western blotting. The serum concentrations of CA15-3 prior to and following surgery were analyzed by chemiluminescence immunoassay. The expression of CD44, ALDH1 and Twist1 mRNA in the primary breast cancer tissues and involved ALN was upregulated compared with the normal ALN (P<0.05). The proteins N-cadherin and vimentin of the involved ALN were poorly expressed compared with breast cancer tissues, however E-cadherin protein expression was higher in metastasized and normal ALN compared with primary cancer tissues (P<0.05). Of the 102 patients, the serum CA15-3 levels of the patients in stages I and II were significantly lower compared with stages III and IV (P<0.05). Twist1+/CA15-3+, HER2-negative/Twist1+/CA15-3+ and Triple-receptor negative/Twist1+/CA15-3+ groups displayed a shorter progression-free survival compared with others. The results of the present study demonstrated that CD44, ALDH1 and Twist1 were significantly overexpressed in involved ALN. The serum levels of CA15-3 in those patients were clearly increased and the survival rates decreased, which suggested that a combination of Twist1 in ALN and CA15-3 may function as an indicator for the prognosis of patients with breast cancer.

Exposure to TNF-α combined with TGF-β induces carcinogenesis in vitro via NF-κB/Twist axis

Persistent human papilloma virus (HPV) infection induces chronic inflammation resulting in human cervical cancer. However, the mechanisms underlying carcinogenesis via chronic inflammation remain largely unclear. We investigated the role of pro-inflammatory factors in epithelial-mesenchymal transition(EMT) and cancer stem cell-like(CSCL) characteristics of HeLa cells exposed to TNF‑α with or without TGF‑β. We then determined the role of NF-κB/Twist signal axis in the pathogenesis of cervical cancer. We found that HeLa cells exposed to TNF‑α following chronic treatment with TGF‑β exhibited EMT, self-renewal and high mobility. Knockdown of NF-κBp65 inhibited NF-κB and Twist1 expression, and EMT and CSCL properties of HeLa cells following co-treatment with TNF‑α and TGF‑β. Conversely, overexpression of NF-κBp65 potentiated the above effects. However, knockdown or overexpression of Twist1 had no effect on NF-κBp65 expression, but inhibited or promoted EMT and CSCL features. Notably, overexpression of Twist1 rescued NF-κBp65 knockdown. Our results demonstrate the role of NF-κB/Twist signaling axis in which HeLa cells treated with TNF‑α following chronic exposure to TGF‑β induce EMT and CSCL properties. The NF-κB/Twist signal axis may represent an effective therapeutic target in cervical cancer.

Genomic pathways modulated by Twist in breast cancer

BackgroundThe basic helix-loop-helix transcription factor TWIST1 (Twist) is involved in embryonic cell lineage determination and mesodermal differentiation. There is evidence to indicate that Twist expression plays a role in breast tumor formation and metastasis, but the role of Twist in dysregulating pathways that drive the metastatic cascade is unclear. Moreover, many of the genes and pathways dysregulated by Twist in cell lines and mouse models have not been validated against data obtained from larger, independant datasets of breast cancer patients.MethodsWe over-expressed the human Twist gene in non-metastatic MCF-7 breast cancer cells to generate the estrogen-independent metastatic breast cancer cell line MCF-7/Twist. These cells were inoculated in the mammary fat pad of female severe compromised immunodeficient mice, which subsequently formed xenograft tumors that metastasized to the lungs. Microarray data was collected from both in vitro (MCF-7 and MCF-7/Twist cell lines) and in vivo (primary tumors and lung metastases) models of Twist expression. Our data was compared to several gene datasets of various subtypes, classes, and grades of human breast cancers.ResultsOur data establishes a Twist over-expressing mouse model of breast cancer, which metastasizes to the lung and replicates some of the ontogeny of human breast cancer progression. Gene profiling data, following Twist expression, exhibited novel metastasis driver genes as well as cellular maintenance genes that were synonymous with the metastatic process. We demonstrated that the genes and pathways altered in the transgenic cell line and metastatic animal models parallel many of the dysregulated gene pathways observed in human breast cancers.ConclusionsAnalogous gene expression patterns were observed in both in vitro and in vivo Twist preclinical models of breast cancer metastasis and breast cancer patient datasets supporting the functional role of Twist in promoting breast cancer metastasis. The data suggests that genetic dysregulation of Twist at the cellular level drives alterations in gene pathways in the Twist metastatic mouse model which are comparable to changes seen in human breast cancers. Lastly, we have identified novel genes and pathways that could be further investigated as targets for drugs to treat metastatic breast cancer.

Analysis of Epithelial-Mesenchymal Transition Induced by Overexpression of Twist.

Breast cancer, the most common malignancy among women worldwide, is a heterogeneous disease, and it therefore has remarkably different biological characteristics and clinical behavior. Breast cancer has been divided into several different molecular subtypes based on the status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 (HER2, also named as ErbB2) status. Her2 is a member of EGFR family of transmembrane tyrosine kinase-type receptors, and is involved in the activation of its downstream signaling cascades, which could promote cell proliferation, metastasis, and angiogenesis in tumors. In addition, Twist, a transcriptional factor has been shown to associate with ErbB2 signaling to increase the proliferation and the number of cells, and to induce epithelial-mesenchymal transition. Deregulated cell proliferation can result in hyperplasia and even malignancies. Actually, the proliferative or survival ability of cells can be measured by a variety of methods. Clonogenic assay and CCK8 assay can serve as useful tools to test whether the clonogenic survival ability of tumor cells can be enhanced or reduced upon stimulation of appropriate mitogenic signals or a given cancer therapy respectively. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. Moreover, migration and invasion assay, in some degree, represents the potential for EMT promotion. Here, we introduce colony formation assay; CCK8 proliferation assay; soft agar; and migration and invasion assay using overexpression of ErbB2 and EGFR receptors as an example.

Aqueous Huaier Extract Suppresses Gastric Cancer Metastasis and Epithelial to Mesenchymal Transition by Targeting Twist.

Trametes robiniophila Murr. (Huaier) is a widely used anti-cancer agent in China. Strong evidence for the anti-proliferative activity of Huaier has been reported; however, its anti-metastatic potential against gastric cancer (GC) as well as its underlying mechanism of action are unknown. Here, we show that treatment with an aqueous Huaier extract over a range of concentrations significantly suppressed both the invasiveness and migratory ability of GC cells. Huaier could also partly reverse the epithelial-mesenchymal transition (EMT), as characterized by increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal markers N-cadherin and vimentin. In addition, Huaier-treated cells expressed lower levels of Twist compared to untreated controls, and overexpression of Twist via transfection could partially abolish the anti-metastatic activity of Huaier. Furthermore, elevated Twist expression was correlated with an advanced TNM stage, a high rate of lymph node metastasis, and reduced disease-free survival in GC patients. These findings reveal a novel anti-metastatic mechanism for Huaier, which inhibits the EMT by targeting Twist, suggesting its potential application against a GC relapse.

Overexpression of ID1 reverses the repression of human dental pulp stem cells differentiation induced by TWIST1 silencing.

TWIST1 and ID1 expression in the DSPCs was modulated by lentivirus transduction. Genes expression was assessed with qRT-PCR. The proteins level was evaluated by Western blot. The DPSCs differentiation was assessed with the proliferation, alkaline phosphatase (ALP) activity, and calcium concentration assays. TWIST1 silencing suppressed the expression of ID1 and both the early and late markers of odontoblasts' differentiation detected at the transcript and protein level. The forced overexpression of ID1 increased the expression of the late markers of odontoblasts differentiation but diminished the expression of the early markers. DPCSs with the silenced TWIST1 and subsequent ID1 overexpression displayed an increase in the expression of the late markers of odontoblasts differentiation. Cells with silenced TWIST1 and overexpressing ID1 had increased activity of ALP, higher calcium concentration and decreased proliferation rate. The high level of ID1 expression might be a critical factor stimulating DPSCs differentiation and it might compensate the repressed differentiation of DPSCs caused by TWIST1 silencing. The mutual correlation between the expression level of TWIST1 and ID1 might be a critical factor driving the process of the human odontoblasts' differentiation.

Twist1 Enhances Hypoxia Induced Radioresistance in Cervical Cancer Cells by Promoting Nuclear EGFR Localization.

Twist1 is a crucial transcription factor that regulates epithelial mesenchymal transition and involves in metastasis. Recent evidence suggests that Twist1 plays important role in hypoxia-induced radioresistance, but the underlying mechanism remains elusive. Here we investigated the change of Twist1 expression in human cervical squamous cancer cell line SiHa after hypoxia treatment. We also explored the role of Twist1 in radioresistance by manipulating the expression level of Twist1. We observed that hypoxia treatment elevated the expression of Twist1 in SiHa cells. Knockdown of Twist1 with siRNA increased the radiosensitivity of SiHa cells under hypoxia condition, accompanied by reduced levels of nuclear Epidermal Growth Factor Receptor (EGFR) and DNA-dependent protein kinase (DNA-PK). Conversely, overexpression of Twist1 led to increased radioresistance of SiHa cells, which in turn increased nuclear EGFR localization and expression levels of nuclear DNA-PK. Moreover, concomitant high expression of hypoxia-inducible factor-1α (HIF-1α) and Twist1 in primary tumors of cervical cancer patients correlated with the worse prognosis after irradiation treatment. Taken together, these data provide new insights into molecular mechanism underlying hypoxia-induced radioresistance in cervical cancer cells, and suggest that Twist1 is a promising molecular target to improve the efficacy of cancer radiotherapy.

Expression and significance of Twist, estrogen receptor, and E-cadherin in human breast cancer cells and tissues.

Breast cancer is one of the most common malignancies in women, and the tumor cells' invasion and metastasis is the main cause of death. Recent reports showed that Twist, a transcription factor, plays multiple roles in breast cancer initiation, progress, and metastasis. However, the underlying mechanisms of Twist in tumor invasion and metastasis of breast cancer still remain unclear. Here, we examined the correlation between Twist, E-cadherin, and estrogen receptor (ER) in promoting invasion and metastasis in breast cancer cells and tissues. The mRNA and protein expression of Twist, E-cadherin, and ER in breast cancer cell lines (MCF-7, MDA-MB-435, MDA-MB-231, and ZR-75-30) and human invasive ductal carcinoma (IDC) tissues from 32 patients were detected by reverse transcription-polymerase chain reaction and immunohistochemistry (IHC), respectively. Expression of Twist in cells with high ability of invasion and metastasis was higher than that in MCF-7 cell line which has low ability of invasion and metastasis, while the expression of ER and E-cadherin was much more higher in MCF-7 cell line than in other cells. IHC showed that the expression rate of Twist in IDC tissues and adjacent tissues was 84.38% and 31.25% and the positive expression of E-cadherin and ER was 21.88% and 40.63% in IDC tissues and 81.25% and 84.38% in adjacent tissues, respectively. Interestingly, overexpression of Twist promoted cellular invasion and metastasis and decreased the expression of E-cadherin, ER, AKT, and p-AKT in HEK-293 cells. Taken together, these findings demonstrated that Twist was upregulated in high invasion and metastasis cell lines as well as IDC tissues companioned with downregulated expression of E-cadherin and ER, which provides important clues for the deeper study of breast cancer.