CCNE1 amplification is associated with aggressive potential in endometrioid endometrial carcinomas

Abstract

The clinicopathological significance of amplification was investigated of the gene encoding cyclin E (CCNE1) and we assessed whether CCNE1 was a potential target in endometrioid endometrial carcinomas. CCNE1 amplification and CCNE1 or F-box and WD repeat domain-containing 7 (FBXW7) expression in endometrial endometrioid carcinoma was assessed by immunohistochemistry and fluorescence in situ hybridization. CCNE1 knockdown by small interfering RNA (siRNA) was used to assess the CCNE1 function. The results showed that CCNE1 amplification was present in 9 (8.3%) of 108 endometrial carcinomas. CCNE1 amplification was correlated with high histological grade (Grade 3; p=0.0087) and lymphovascular space invasion (p=0.0258). No significant association was observed between CCNE1 amplification and FIGO stage (p=0.851), lymph node metastasis (p=0.078), body mass index (p=0.265), deep myometrial invasion (p=0.256), menopausal status (p=0.289) or patient age (p=0.0817). CCNE1 amplification was significantly correlated with shorter progression-free and overall survival (p=0.0081 and 0.0073, respectively). CCNE1 protein expression or loss of FBXW7 expression in endometrial endometrioid carcinoma tended to be correlated with shorter progression-free and overall survival; however, this difference was not statistically significant. Multivariate analysis showed that CCNE1 amplification was an independent prognostic factor for overall survival but not for progression-free survival (P=0.0454 and 0.2175, respectively). Profound growth inhibition was observed in siRNA-transfected cancer cells with endogenous CCNE1 overexpression compared with that in cancer cells having low CCNE1 expression. CCNE1 amplification was independent of p53, HER2, MLH1 and ARID1A expression but dependent on PTEN expression in endometrial carcinomas. These findings indicated that CCNE1 amplification was critical for the survival of endometrial endometrioid carcinomas. Furthermore, the effects of CCNE1 knockdown were dependent on the CCNE1 expression status, suggesting that CCNE1-targeted therapy may be beneficial for patients with endometrial endometrioid carcinoma having CCNE1 amplification.

Figure 1

Dual-color fluorescence in situ hybridization (FISH) was used to detect amplification of the CCNE1 gene in endometrial endometrioid carcinomas. (A) FISH analysis revealed a homogeneously stained region in the carcinoma portion with CCNE1 amplification. (B and D) A case of endometrial endometrioid carcinoma with positive immunoreactivity for CCNE1 and FBXW7 in endometrial carcinoma tissues. (C and E) A case with negative CCNE1 and FBXW7 staining in endometrial carcinoma tissues.

Figure 2

CCNE1 amplification is correlated with shorter progression-free/overall survival in patients with endometrial endometrioid carcinoma. (A) Kaplan-Meier survival analysis showed that CCNE1 amplification (solid line, n=9) was associated with shorter progression-free survival than absence of CCNE1 amplification (dashed line, n=99) (P=0.0081, log-rank test). CCNE1 overexpression was not significantly associated with progression-free/overall survival in patients with endometrial endometrioid carcinoma. (B) Kaplan-Meier survival analysis showed that a high CCNE1 expression (solid line, n=44) was associated with shorter relapse-free survival than low CCNE1 expression; however, the difference was not statistically significant (dashed line, n=54; P=0.3148, log-rank test). (C) Kaplan-Meier survival analysis showed that CCNE1 amplification (solid line, n=9) was associated with shorter overall survival than absence of CCNE1 amplification (dashed line, n=99; P=0.0073, log-rank test). (D) Kaplan-Meier survival analysis showed that high CCNE1 expression (solid line, n=54) was associated with shorter overall survival than low CCNE1 expression; however, this difference was not statistically significant (dashed line, n=54; P=0.2205, log-rank test). Negative FBXW7 expression was not significantly associated with progression-free/overall survival in patients with endometrial endometrioid carcinoma. (E) Kaplan-Meier survival analysis showed that negative FBXW7 expression (solid line, n=63) was associated with shorter progression-free survival than positive FBXW7 expression; however, this difference was not statistically significant (dashed line, n=54; P=0.3148, log-rank test). (F) Kaplan-Meier survival analysis showed that negative FBXW7 expression (solid line, n=63) was associated with shorter overall survival than positive FBXW7 expression; however, this difference was not statistically significant (dashed line, n=54; P=0.2307, log-rank test).

Figure 3

Western blot analysis. (A) Western blot analysis showed that CCNE1 protein expression was higher in JHUC-1 and HEC108 cells than in other cell lines. (B) Western blot analysis showed that CCNE1 protein expression was significantly decreased in CCNE1 siRNA-transfected cells compared with control siRNA-transfected cells. (C) Effects of CCNE1 siRNA on cell proliferation. Cell numbers were determined at 72 h after transfection with CCNE1 siRNA or control siRNA. Endometrial carcinoma cells with CCNE1 overexpression were more sensitive to growth inhibition by CCNE1 siRNA than cells without CCNE1 overexpression. The mean and SD were obtained from three experiments.