Retinoids are employed as anti-neoplastic or chemopreventative agents in clinical trials including women with breast carcinoma. We found retinoids only inhibit the growth of estrogen receptor (ER)-positive cells. We have now found that breast carcinoma cells possess a unique isoform of the retinoic acid nuclear receptor-alpha (RAR-alpha) whose levels must be enhanced by estrogens to allow retinoids to inhibit growth.
Specific Aim 1 : Determine whether ER positivity in human breast carcinoma biopsy specimens results in increased RAR and RXR mRNA levels. ER negative breast carcinoma cell lines are refractory to growth inhibitory by RA, and possess decreased RAR-alpha mRNA levels, while ER- positive cell lines express high RAR-alpha mRNA level Specific Aim 2: Demonstrate that RA modulation of genes via RAR-alpha plays a vital role in RA inhibition of breast carcinoma growth. 2A) Determine if transection of RA-refractory ER-negative breast carcinoma cells with a RAR-alpha expression vector and subsequent increased expression of RAR- alpha results in RA-inhibition of growth. 2B) Demonstrate that inhibition of RAR-alpha function utilizing a selective RAR-alpha antagonist results in resistance to RA-inhibition of growth 2C) Selective inhibition of wild type RAR-alpha function utilizing a RAR-alpha dominant negative mutant results in cells refractory to RA-mediated inhibition of growth.
Specific Aim 3 : We found human breast cancer cell lines express a unique isoform of RAR-alpha which is transcriptionally regulated by estradiol in ER-positive cells. We will identify and characterize the RAR-alpha isoform(s) expressed in human breast carcinoma cells; isolate, sequence and functionally characterize the 5'-upstream regulatory region (the promoter region) of the RAR-alpha gene and identify the regulatory elements responsible for estrogen-mediated modulation of RAR-alpha gene expression.
Specific Aim 4 : Elucidate the mechanism(s) involved in estrogen regulation of RAR-alpha gene transcription in human breast carcinoma cells. Ascertain utilizing the isolated promoter whether estradiol enhances genes transcription by binding to the promoter via an estrogen response element (ERE) or stimulates or represses the levels of other factors which in turn modulate gene activity.
Specific Aim 5 : Study the mRNA levels of the specific RAR-alpha isoform(s) in breast carcinoma cells and the surrounding stroma in paraffin embedded infiltrating ductal carcinoma specimens obtained from patients, and correlate the ER-status of the specimen and the level of RAR-alpha mRNA.
Specific Aim 6 : Determine whether RA also inhibits the in vivo growth and/or metastatic potential of ER-transfected MDA-MB-231 cells.
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