CCT6A suppresses SMAD2 and promotes prometastatic TGF-β signaling
Zhe Ying, … , Jun Li, Mengfeng Li
Zhe Ying, … , Jun Li, Mengfeng Li
Published April 4, 2017
Citation Information: J Clin Invest. 2017;127(5):1725-1740. https://doi.org/10.1172/JCI90439.
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Research Article Oncology

CCT6A suppresses SMAD2 and promotes prometastatic TGF-β signaling

Abstract

Paradoxically, during early tumor development in many cancer types, TGF-β acts as a tumor suppressor, whereas in the advanced stages of these cancers, increased TGF-β expression is linked to high metastasis and poor prognosis. These findings suggest that unidentified mechanisms may function to rewire TGF-β signaling toward its prometastatic role in cancer cells. Our current study using non–small-cell lung carcinoma (NSCLC) cell lines, animal models, and clinical specimens demonstrates that suppression of SMAD2, with SMAD3 function intact, switches TGF-β–induced transcriptional responses to a prometastatic state. Importantly, we identified chaperonin containing TCP1 subunit 6A (CCT6A) as an inhibitor and direct binding protein of SMAD2 and found that CCT6A suppresses SMAD2 function in NSCLC cells and promotes metastasis. Furthermore, selective inhibition of SMAD3 or CCT6A efficiently suppresses TGF-β–mediated metastasis. Our findings provide a mechanism that directs TGF-β signaling toward its prometastatic arm and may contribute to the development of therapeutic strategies targeting TGF-β for NSCLC.

Authors

Zhe Ying, Han Tian, Yun Li, Rong Lian, Wei Li, Shanshan Wu, Hui-Zhong Zhang, Jueheng Wu, Lei Liu, Junwei Song, Hongyu Guan, Junchao Cai, Xun Zhu, Jun Li, Mengfeng Li

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Figure 4

CCT6A specifically interacts with SMAD2 and suppresses the SMAD2-mediated transcriptional program.

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CCT6A specifically interacts with SMAD2 and suppresses the SMAD2-mediate...
(A) Immunoprecipitation and subsequent mass spectroscopic analysis identified CCT6A as a highly specific SMAD2-interactive protein in PC9 cells. Representative image was derived from 3 independent experiments. (B) Immunoprecipitation in 293T cells that expressed Flag-tagged SMADs 1, 2, 3, 5, and 8 and HA-tagged CCT6A indicated that CCT6A specifically interacted with SMAD2. Representative blot was derived from 3 independent experiments. (C) Immunoprecipitation of truncated SMAD2 constructs indicated that CCT6A specifically interacted with the MH2 domain of SMAD2. Representative blot was derived from 3 independent experiments. (D) Western blot analysis of CCT6A in NSCLC cell lines using 2 primary lung epithelial cell lines (LE-1 and LE-2) and an immortalized lung epithelial cell line (Beas2B) as a control showed that CCT6A was highly expressed in NSCLC cell lines, particularly in highly metastatic lines (PC9, H1650, and 95D) (upper panel). Western blot analysis of CCT6A in NSCLC tissue using 2 normal lung tissue specimens as controls indicated that CCT6A was highly expressed in patient-derived NSCLC tumors (lower panel). Representative blots were derived from 3 independent experiments. (E) Microarray-based transcription profiling demonstrated that CCT6A overexpression significantly attenuated the expression of SMAD2-specific target genes, as well as target genes shared by SMAD2 and SMAD3 in A549 cells. Box and whiskers plots represent the mRNA levels. **P < 0.01, by Student’s t test. (F) qPCR analysis of the expression level of SMAD2- or SMAD3-specific and shared target genes in the indicated cells. Heatmaps represent the mRNA levels. (G) GSEA analysis indicated that the CCT6A levels were inversely associated with the expression levels of SMAD2-specific targets. The defined “high” and “low” expression levels of CCT6A were stratified by the median expression level.