Keratinocyte growth factor (KGF) regulates estrogen receptor-alpha (ER-alpha) expression and cell apoptosis via phosphatidylinositol 3-kinase (PI3K)/Akt pathway in human breast cancer cells.
BACKGROUND
It is suggested that the phosphatidylinositol 3-kinase (PI3K/Akt) pathway may lead to tamoxifen (Tam) resistance in the estrogen receptor-alpha (ER-alpha)-positive breast cancer cell line, MCF-7. Our previous results demonstrated that keratinocyte growth factor (KGF) down-regulates ER-alpha expression and increases Tam resistance in MCF-7 cells. Therefore, we hypothesized that a possible mechanism for developing Tam resistance could be the regulation of ER-alpha and Bcl-2 family proteins through modulation of Akt activity.


METHODS
MCF-7 cells were treated with KGF, LY294002 (LY), a PI3 kinase inhibitor, 4-OH-Tam, KGF with LY, KGF with LY and 4-OH-Tam, or vehicles as control for 24 hours. Total RNA was extracted from MCF-7 cells and real-time PCR was employed to identify the ER-alpha expression in response to KGF. To determine that the resistance to 4-OH-Tam-inducing cell killing after the KGF treatment was due to the inactivation of the apoptotic pathway, low molecular weight DNA was isolated from cells of different treatments and inter-nucleosomal DNA fragmentation was investigated. The phosphorylation of signaling intermediates Akt, Bad, the activation of caspase-9, and the expression of ER-alpha, Bcl-2, Bcl-xL, and Bax were evaluated by immunoblot analysis for the study of KGF signaling effects. To determine the involvement of PI3K/Akt pathway in the survival effect of KGF, the growth rate of MCF-7 cell was measured by non-radioactive cell proliferation assay after treatments of KGF, LY, 4-OH-Tam, KGF with LY, KGF with LY and 4-OH-Tam, or vehicles as control for 3 days. The results of real-time PCR and cell proliferation assay were analyzed by Student's t-test and p-values of less than 0.05 were considered statistically significant.


RESULTS
Our results showed that in MCF-7 cells KGF increased Akt phosphorylation and induced ER-alpha mRNA expression which could be blocked by a PI3K/Akt pathway inhibitor, LY. KGF treatment also induced apoptosis based on the observation of the suppression of DNA fragmentation, variable increase in the expression of the Bcl-2 and Bcl-xL proteins and the decrease of the active form of caspase-9 protein, whereas LY blocked the anti-apoptotic effects of KGF. In the cell proliferation assay, KGF maintained MCF-7 cell survival in the presence of 4-OH-Tam which could be blocked by LY.


CONCLUSIONS
We confirmed the regulation of ER-alpha by KGF in human breast cancer cells at both mRNA and protein levels. We further demonstrated that KGF may play an inhibitory role in the induction of breast cancer cell apoptosis, conferring resistance against anticancer drugs on breast cancer cells.
Chang
Hsiang-Lin
HL
Laboratory of Reproductive and Molecular Endocrinology, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.
Sugimoto
Yasuro
Y
Liu
Suling
S
Wang
Li-Shu
LS
Huang
Yi-Wen
YW
Ye
Weiping
W
Lin
Young C
YC
D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D051523:Fibroblast Growth Factor 7 / Q000494:pharmacology*; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018931:Antineoplastic Agents, Hormonal; D047628:Estrogen Receptor alpha; D051523:Fibroblast Growth Factor 7; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D013629:Tamoxifen; C506487:ESR1 protein, human