PAQR3 enhances Twist1 degradation to suppress epithelial–mesenchymal transition and metastasis of gastric cancer cells

Carcinogenesis, Apr 2016

Twist1 is an essential transcription factor required to initiate epithelial–mesenchymal transition (EMT) and promote tumor metastasis. PAQR3 is a newly found tumor suppressor that is frequently downregulated in many types of human cancers. Downregulation of PAQR3 is associated with accelerated metastasis and poor prognosis of the patients with gastric cancers. In this study, we demonstrate that PAQR3 is actively involved in the degradation of Twist1 and whereby regulates EMT and metastasis of gastric cancer cells. PAQR3 overexpression reduces the protein level but not the mRNA level of Twist1. The protein stability and polyubiquitination of Twist1 are altered by PAQR3. PAQR3 forms a complex with Twist1 and BTRC, an E3 ubiquitin ligase. PAQR3 enhances the interaction between Twist1 and BTRC. Twist1 is mobilized from the nucleus to a proteasome-containing structure in the cytoplasm upon overexpression of PAQR3 and BTRC, which is required for PAQR3-induced degradation of Twist1. The Twist1 box domain of the Twist1 protein is required for the interaction of Twist1 with both PAQR3 and BTRC, indispensable for PAQR3-mediated degradation of Twist1. Both BTRC and Twist1 are required for the inhibitory effects of PAQR3 on migration and EMT phenotype of gastric cancers cells. Importantly, Twist1 is indispensable for the inhibitory effect of PAQR3 on metastasis of gastric cancer cells in vivo . Collectively, these findings not only pinpoint that Twist1 mediates the modulatory function of PAQR3 on EMT and metastasis but also suggest that targeting Twist1 is a promising strategy to control metastasis of tumors with downregulation of PAQR3.

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PAQR3 enhances Twist1 degradation to suppress epithelial–mesenchymal transition and metastasis of gastric cancer cells

Carcinogenesis PAQR3 enhances Twist1 degradation to suppress epithelial-mesenchymal transition and metastasis of gastric cancer cells Weiwei Guo 1 Xue You 0 1 Daqian Xu 1 Yuxue Zhang 1 Zheng Wang 1 Kaiyang Man 0 1 Zhenzhen Wang 1 Yan Chen 0 1 0 School of Life Sciences and Technology, Shanghai Tech University , Shanghai 200031 , China 1 Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences , 320 Yueyang Road, New Life Science Building, A2214, Shanghai 200031 , China Twist1 is an essential transcription factor required to initiate epithelial-mesenchymal transition (EMT) and promote tumor metastasis. PAQR3 is a newly found tumor suppressor that is frequently downregulated in many types of human cancers. Downregulation of PAQR3 is associated with accelerated metastasis and poor prognosis of the patients with gastric cancers. In this study, we demonstrate that PAQR3 is actively involved in the degradation of Twist1 and whereby regulates EMT and metastasis of gastric cancer cells. PAQR3 overexpression reduces the protein level but not the mRNA level of Twist1. The protein stability and polyubiquitination of Twist1 are altered by PAQR3. PAQR3 forms a complex with Twist1 and BTRC, an E3 ubiquitin ligase. PAQR3 enhances the interaction between Twist1 and BTRC. Twist1 is mobilized from the nucleus to a proteasome-containing structure in the cytoplasm upon overexpression of PAQR3 and BTRC, which is required for PAQR3induced degradation of Twist1. The Twist1 box domain of the Twist1 protein is required for the interaction of Twist1 with both PAQR3 and BTRC, indispensable for PAQR3-mediated degradation of Twist1. Both BTRC and Twist1 are required for the inhibitory effects of PAQR3 on migration and EMT phenotype of gastric cancers cells. Importantly, Twist1 is indispensable for the inhibitory effect of PAQR3 on metastasis of gastric cancer ceinllvsivo. Collectively, these findings not only pinpoint that Twist1 mediates the modulatory function of PAQR3 on EMT and metastasis but also suggest that targeting Twist1 is a promising strategy to control metastasis of tumors with downregulation of PAQR3. - Introduction Epithelial–mesenchymal transition (EMT) is a critical process head mesenchyme, somites and limb buds ( 6). Twist1 has a very that allows epithelial cells to lose their junctions and apical–broad impact on the metastatic processes of tumor cells, as it basal polarity, leading to gain the mobility and invasive phe-no is essential for cell–cell adhesion via suppression of epithelial type of the cancer cells1(,2). The EMT program is initiated by marker such as E-cadherin and catenin as well as augmen-ta a set of key transcription factors such as Twist1, Snail and ZEB tion of mesenchymal markers such as vimentin and N-cadherin (1). These transcription factors execute EMT by repressing genes (7,8). The protein level of Twist1 is affected by multiple signaling encoding epithelial-like proteins and activating mesenchymal pathways. HIF-1α could directly regulate Twist1 expressio9n).( genes (3,4). Twist1 is a basic helix-loop-helix domain-contai-n MAPKs phosphorylate Twits1 at Ser68 to block ubiquitin-me-di ing transcription factor. Functional loss of Twist1 was found to ated degradation of the protein10( ). It was also reported that cause several diseases such as Saethre–Chotzen syndrome5)(. BTRC (also known asβ-Trcp or FBXW1) is the E3 ubiquitin ligase Twist1-null mouse embryos die from defective development in of Twist1 and involved in the degradation and the protei1n1)(. Abbreviations lentivirus-based method. AGS cells with stable expression of luciferase were infected with different shRNA-containing lentivirus. Lentivirus production and infection The knockdown of PAQR3, BTRC and Twist1 was performed by a lentivirusbased method. AGS cells with stable expression of luciferase were infected with different shRNA-containing lentivirus. The lentiviral vector of FG12 PAQR3-shRNA has an independent open reading frame from that of green Recent studies have indicated that Twist1 is associated with fluorescent protein. BTRC-shRNA and Twist1-shRNA vectors have an the invasive and metastatic phenotypes of many tumor types independent open reading frame from that of puromycin. These vectors (8,12). Twist1 was reported to be associated with the initiation, were transfected into HEK293T cell and the virus was harvested from the progression and metastasis of gastric cancer13(,14), breast supernatant after transfection for 48 andh7.2The lentivirus was purified cancer (8), hepatocellular carcinoma1(5), prostate cancer 1(6), by centrifugation at 2000 rpm (4°C, 120 min). Appropriate amounts of the esophageal squamous cell carcinoma 1(7), bladder cancer 1(8) lentivirus were added into the culture medium of AGS cells. Ath72after and pancreatic cancer 1(9). Our study revealed that Twis (...truncated)


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Guo, Weiwei, You, Xue, Xu, Daqian, Zhang, Yuxue, Wang, Zheng, Man, Kaiyang, Wang, Zhenzhen, Chen, Yan. PAQR3 enhances Twist1 degradation to suppress epithelial–mesenchymal transition and metastasis of gastric cancer cells, Carcinogenesis, 2016, pp. 397-407, Volume 37, Issue 4, DOI: 10.1093/carcin/bgw013