Figure 4. CCT6A specifically interacts with SMAD2 and suppresses the SMAD2-mediated transcriptional program.

(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.