Abstract
We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHT(R))) to tamoxifen showed significant (>1.8-fold) up-regulation of eight miRNAs and marked down-regulation (>50%) of seven miRNAs in OHT(R) cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27(Kip1), a known target of miR-221/222, was reduced by 50% in OHT(R) cells and by 28-50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27(Kip1) in the resistant OHT(R) cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.
Highlights
New cases of invasive breast cancer was diagnosed in the United States in 2007, and 40,460 women will die of this cancer
Because the levels of these two miRNAs were significantly elevated in HER2/neu-positive breast tumors, we further explored their role in acquiring resistance to endocrine therapy. miR-221 and miR-222 were ectopically expressed in tamoxifen-sensitive MCF-7 cells (MCF-7/221/222), and miR221/222-overexpressing cells were selected using G418
Altered expression of miRNAs in primary human cancers has been used for tumor diagnosis and prognosis, the potential involvement of miRNAs in induction of drug resistance, tamoxifen resistance, has not been explored
Summary
Cell Culture and Tissue Procurement—Tamoxifen-sensitive MCF-7 cells and 4-hydroxytamoxifen-resistant (OHTR) cells were obtained from Dr Kenneth P. MCF-7 and OHTR cells were cultured in growth medium (minimum essential medium with 2 mmol/liter L-glutamine, 0.1 mmol/liter nonessential amino acids, 50 units/ml penicillin, 50 mg/ml streptomycin, 6 ng/ml insulin, and 10% fetal bovine serum) in the absence of 4-hydroxytamoxifen for 7 days. ZR75.1 cells were obtained from Dr Brett Hall (Ohio State University) and maintained in RPMI 1640 medium containing 10% fetal bovine serum. MiRNA Microarray—The miRNA microarray was performed at the Ohio State University Comprehensive Cancer Center Microarray Core Facility (see supplemental “Experimental Procedures” for details). TaqMan Reverse Transcription (RT)-PCR for miRNA Quantification—Total RNA was isolated from formaldehyde-fixed paraffinembedded unsectioned primary human breast cancer tissue cores with a RecoverAllTM total nucleic acid isolation kit (Ambion) and from the cell lines with TRIzolTM (Invitrogen), reverse-transcribed using a TaqManTM microRNA reverse transcription kit, and subjected to real-time PCR using a TaqManTM microRNA assay kit (Applied Biosystems). Statistical Analysis—Statistical significance was analyzed by unpaired Student’s t test, and p Յ 0.05 was considered to be statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
