Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.
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 ERalpha and ERbeta, 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 ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta 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.
Tu
Zheng
Z
Department of Gynecology, Peking University People's Hospital, 11 Xizhimen South Street, Beijing 100044, China.
Gui
Liming
L
Wang
Jianliu
J
Li
Xiaoping
X
Sun
Pengming
P
Wei
Lihui
L
D000818:Animals; D048708:Cell Growth Processes / Q000235:genetics; D016889:Endometrial Neoplasms / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D011905:Genes, ras; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation; D018631:ras Proteins / Q000235:genetics*
C117307:KRAS protein, human; D011960:Receptors, Estrogen; D008156:Luciferases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins