Figure 3.

SHANK2 deregulates Hippo pathway, transforms primary cells and promotes tumor growth. (A) Ectopic expression of SHANK2 reduced YAP phosphorylation at high cell density in 293T cells. (B) In Hippo-proficient cell line 293T, ectopic expression of SHANK2 caused YAP to remain in nucleus at high cell density. Scale bars: 10 μm. (C) Expression of SHANK2 transformed 293T cells and enabled growth on soft agar. Lower panel, data represent mean ± SEM from results of three independent experiments. P value was calculated by Student’s t test; ***P < 0.001. (D and E) Expression of SHANK2 promoted tumor growth in nude mice. 2 million of 293T cells expressing vector control or exogenous SHANK2 were transplanted into nude mice (n = 7) (D). 2 million of CommA-Dβ mouse mammary cells expressing vector control or exogenous SHANK2 were transplanted into the fat pad of breast of nude mice (n = 8) (E). Measurement of tumor growth started after 12 days (D) or 27 days (E) post injection. Data present mean ± SEM. P value was calculated by Student’s t test; **P < 0.01, *P < 0.05. (F and F’’) Expression of SHANK2 promoted liver tumor formation in the context of Myc and p53 mutation. Liver tumors were induced in mice by hydrodynamically injecting the transposon vector control or exogenous SHANK2 combined with p53R246S and Myc. p53R246S is the murine version of p53R249S, a hotspot mutation commonly observed in human liver cancer. Shown are images of mice liver tumors (F), the statistics of tumor number (n = 3) (F’) and the expression level of YAP transcriptional target genes CTGF and CYR61 in tumors (F’’). Data represent mean ± SEM. P value was calculated by Student’s t test; **P < 0.001, ***P < 0.001. (G) Correlation of SHANK2 and CTGF, CYR61 expression in uterine corpus endometrial carcinoma (UCEC). The TCGA UCEC datasets were used for this analysis. Correlations between CTGF, CYR61 with YAP and two established positive regulators of YAP (WBP2 and STK26) were also shown in comparison