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Development of Genetically Engineered Aromatase Overexpressing and Aromatase Inhibitior Resistant Breast Cancer Cell Lines to Study Possible Therapeutic Implications of a Novel Antiprogestin
Title
Development of Genetically Engineered Aromatase Overexpressing and Aromatase Inhibitior Resistant Breast Cancer Cell Lines to Study Possible Therapeutic Implications of a Novel Antiprogestin
Creator
Gupta, Akash
Date
2012-05-07, 2012-05
Description
Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI....
Show moreAromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the antiprogestin CDB-4124 (Proellex) in aromatase overexpressing and Letrozole resistant human breast cancer cell lines. Mechanism of antiproliferative action of Proellex was also explored. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom cells for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantified by quantitative RTPCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student’s ‘t’ test or ANOVA followed by Bonferroni’s post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50%to 60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex or other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PRB / EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients. Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogeninduced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland and cultured in vitro, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC). Three to four week old female Balb/c mice were sensitized with estradiol (1mg) + progesterone (1mg) for 9 days. On the 10 th day animals were sacrificed and 2.5x10 4 T47D parental or T47D aromatase overexpressing cells were injected into the fourth pair of mammary glands. The glands were excised then cultured at 37C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS supplemented with Testosterone (1nM) and progesterone (1mM) and growth promoting hormones (5 μg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific CK18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu.
Ph.D. in Biology, May 2012
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