Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor

Abstract

Objective. To investigate the tumorigenesis of mutant [ 12Asp]-K-ras in endometrial carcinoma and its relationship with ER. Methods. We constructed pcDI-[ 12Asp]K-ras4B by inserting full-length [ 12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[ 12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [ 12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [ 3H]thymidine incorporation. After transfected with pcDI-[ 12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERα and ERβ, respectively. Results. [ 12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection ( P < 0.05). More colonies were grown 10 days after incubating pcDI-[ 12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[ 12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERα and ERβ was observed in pcDI-[ 12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERα and ERβ expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[ 12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. Conclusions. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.