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Opposing Effects of Runx2 and Estradiol on Breast Cancer Cell

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Opposing Effects of Runx2 and Estradiol on Breast Cancer Cell Published OnlineFirst December 6, 2011; DOI: 10.1158/1078-0432.CCR-11-1530 Clinical Cancer Predictive Biomarkers and Personalized Medicine Research Opposing Effects of Runx2 and Estradiol on Breast Cancer Cell Proliferation: In Vitro Identification of Reciprocally Regulated Gene Signature Related to Clinical Letrozole Responsiveness Nyam-Osor Chimge1,4, Sanjeev K. Baniwal2,4, Jingqin Luo7,8, Simon Coetzee4, Omar Khalid1,4, Benjamin P. Berman5,6, Debu Tripathy3,6, Matthew J. Ellis8, and Baruch Frenkel1,2,4 Abstract Purpose: To assess the clinical significance of the interaction between estrogen and Runx2 signaling, previously shown in vitro. Experimental Design: MCF7/Rx2dox breast cancer cells were treated with estradiol and/or doxycycline to induce Runx2, and global gene expression was profiled to define genes regulated by estradiol, Runx2, or both. Anchorage-independent growth was assessed by soft-agar colony formation assays. Expression of gene sets defined using the MCF7/Rx2dox system was analyzed in pre- and on-treatment biopsies from hormone receptor–positive patients undergoing neoadjuvant letrozole treatment in two independent studies, and short-term changes in gene expression were correlated with tumor size reduction or Ki67 index at surgery. Results: Reflecting its oncogenic property, estradiol strongly promoted soft-agar colony formation, whereas Runx2 blocked this process suggesting tumor suppressor property. Transcriptome analysis of MCF7/Rx2dox cells treated with estradiol and/or doxycycline showed reciprocal attenuation of Runx2 and estrogen signaling. Correspondingly in breast cancer tumors, expression of estradiol- and Runx2-regulated genes was inversely correlated, and letrozole increased expression of Runx2-stimulated genes, as defined in the MCF7/Rx2dox model. Of particular interest was a gene set upregulated by estradiol and downregulated by Runx2 in vitro; its short-term response to letrozole treatment associated with tumor size reduction and Ki67 index at surgery better than other estradiol-regulated gene sets. Conclusion: This work provides clinical evidence for the importance of antagonism between Runx2 and E2 signaling in breast cancer. Likely sensing the tension between them, letrozole responsiveness of a genomic node, positively regulated by estradiol and negatively regulated by Runx2 in vitro, best correlated with the clinical efficacy of letrozole treatment. Clin Cancer Res; 18(3); 1–11. Ó2011 AACR. Introduction monal carcinogenesis is difficult to discern. Although genes such as c-Myc, cyclin D,andcyclin E have been The mitogenic effect of estrogens in breast epithelial implicated in estrogen-driven breast cancer (1), there is cells is one of the best examples of hormonal carcino- limited understanding of why some are better correlated genesis. Which of the many primary and secondary estro- than others with disease progression and drug respon- gen-responsive genes in breast epithelial cells drive hor- siveness. Recent high-throughput gene expression studies have led to the identification of gene groups, whose Authors' Affiliations: Departments of 1Biochemistry & Molecular Biology, baseline mRNA levels and hormone responsiveness cor- 2 3 4 Orthopaedic Surgery, and Medicine, Institute for Genetic Medicine, related well with clinical and histologic features of disease 5USC Epigenome Center and 6Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, progression (2–6). Interestingly, first-generation prognos- California; and 7Division of Biostatistics, 8Siteman Cancer Center Breast tic gene signatures were enriched for proliferation- and Cancer Program, Washington University, St Louis, Missouri cell-cycle–related genes and were particularly valid for Note: Supplementary data for this article are available at Clinical Cancer estrogen receptor alpha (ERa)-positive tumors (7, 8). Research Online (http://clincancerres.aacrjournals.org/). Better understanding of the biological significance of such Corresponding Authors: Baruch Frenkel, Institute for Genetic Medicine, gene signatures will potentially lead to novel pharmaco- Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, CSC/IGM240, Los Angeles, CA 90033. Phone: 323-442- logic approaches targeting key molecular nodes that reg- 1144; Fax: 323-442-2764; E-mail: [email protected]; and Nyam-Osor ulate breast cancer progression. Chimge, E-mail: [email protected] The Runx family of mammalian transcription factors doi: 10.1158/1078-0432.CCR-11-1530 plays fundamental roles in the differentiation of osteoblasts Ó2011 American Association for Cancer Research. and chondrocytes (Runx2; ref. 9), hematopoietic cells www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst December 6, 2011; DOI: 10.1158/1078-0432.CCR-11-1530 Chimge et al. subline that conditionally express Runx2, we employed the Translational Relevance recently described lentivirus-based pSLIK vector system, which allows tight doxycycline (dox)-inducible, RNA We identified, in an unbiased manner, a gene set PolII-mediated transcription of a gene of interest (36). upregulated by estradiol and downregulated by Runx2 Lentiviral particles encoding dox-inducible Flag-Runx2 in an MCF7 breast cancer culture model, whose short- (MASN isoform, type-2) and the Hygromycin B selection term response to letrozole in tumor biopsies correlated marker were constructed and packaged as previously with future tumor growth better than the responsiveness described (20). Transduced MCF7/Rx2dox cells were of hormone-regulated gene sets defined regardless of selected in Dulbecco’s modified Eagle’s medium (DMEM) Runx2; initial decrease in the expression of these genes containing 10% FBS and 100 mg/mL Hygromycin B in patients receiving letrozole therapy correlated most (GIBCO). significantly with Ki67 index and tumor size reduction assessed months later. Many of the genes encoded com- Western blot analysis ponents of the mitotic polo-like kinase pathway. Recip- Proteins were separated on 10% SDS-PAGE and West- rocal antagonism between the mitogenic properties of ern blotting was performed using mouse monoclonal estradiol and the antimitogenic properties of Runx2 anti-FLAG M2 (Sigma), mouse monoclonal anti-Runx2 should be further studied as a potential basis for the (Invitrogen), and goat anti-GAPDH (glyceraldehyde-3- development of predictive strategies and identification phosphate dehydrogenase, V-18; Santa Cruz Biotechn., of novel therapeutic targets for breast cancer. Inc.) as primary antibodies. After incubation with either horseradish peroxidase–conjugated goat anti-mouse sec- ondary antibodies (sc2031) or donkey anti-goat anti- bodies (sc2020) from Santa Cruz Biotech., Inc., proteins (Runx1; ref. 10, 11) and neurons (Runx3; ref. 12). Runx were visualized with enhanced Chemiluminescence proteins are also increasingly implicated in cancer progres- Plus Western blotting detection kit (GE Healthcare UK sion, both positively and negatively (13, 14). Contrasting limited). their prometastatic role, which was mostly studied in advanced breast and prostate cancer (14–20), Runx proteins Immunofluorescence are well known for their tumor suppressor properties. Runx2 and ERa were visualized with the respective pri- Runx3 is a bona fide tumor suppressor gene, whose meth- mary antibodies and secondary antibodies conjugated to ylation contributes to gastric cancer (21–23), ablation of either rhodamine or fluorescein, respectively. Cells were Runx1 activity leads to leukemia (24, 25), and Runx2 viewed using an LSM 510 Zeiss confocal microscope at 60Â inhibits cell-cycle progression in osteoblasts and prostate magnification. cancer cells (20, 26–28). Runx2 physically interacts with the DNA-binding Soft-agar colony formation assay domains of both ERa and the androgen receptor. These MCF7/Rx2dox cells were suspended in 0.4% agarose pre- interactions usually result in reciprocal inhibition of the pared in DMEM containing 10% charcoal-stripped serum respective transcriptional activation activities (29–31), (CSS) and plated over 0.5% base agar at a density of 5,000 although different outcomes have been reported (32–35). cells per well in 6-well plates. Cells were incubated at 37C The physical interaction between ERa and Runx2 (29), and with media change every 2 days and stained with 0.005% our more recent observation that E2 independently regu- Crystal violet after 21 days of incubation. Colonies were lates half of the Runx2-responsive genes in breast cancer counted using a dissecting microscope. cells (see herein), led to the speculation that the oncogenic potential of estradiol in breast epithelial cells might be High-throughput gene expression analyses related in part to antagonizing Runx2. In this study, we Two days before treatment MCF7/Rx2dox cells were first define gene sets coregulated by estrogen and Runx2 switched to phenol red-free DMEM containing 5% CSS. signaling in breast cancer cells in vitro. Then, by correlating Cells were treated with 0.5 mg/mL dox to induce Runx2 expression pattern of these genes in breast cancer tumor expression and/or with 10 nmol/L E2. After 48 hours biopsies with clinical response to aromatase inhibition of treatment, RNA was extracted using Aurum Total therapy, we show that genes upregulated by E2 and down- RNA Mini Kit (BioRad) and submitted to the Southern regulated by Runx2 are more informative than those California Genotyping
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