Breast cancer is a heterogeneous disease that is best treated based on the expressed biomarker profile. Currently, the major clinical challenges are drug resistance and metastatic spread. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and disease progression despite therapy. We previously identified novel biomarkers that could potentially be used with novel therapeutic strategies that target CSCs. We demonstrated in a presurgical window biomarker study of 20 breast cancer patients with ER-positive disease that signaling through Notch receptors mediates expression of 18 genes. Twenty women with ER-positive tumors were treated with 14 days of ET (tamoxifen or letrozole), with the addition of the oral GSI MK-0752 on day 15 (3 days on, 4 days off, 3 days on). Definitive surgery was on day 25. Analysis of biomarkers using microarray and RT-PCR during ET combined with MK-0752 GSI revealed statistically significant modulation of 18 genes: pro-apoptotic DAXX and NOXA; a tumor suppressor LFNG; Notch signaling such as NOTCH1, NOTCH4, HEYL, HES1, and HEY2; as well as proliferation-associated transcripts MIK67, CCND1, CCNA2; stem cell markers RUNX1 and ALDH1; and novel genes such as RICTOR, RPTOR, MMP7, ADAM19, and PgR (Albain et al. Proc SABCS 2014). The goal of the current study was to identify whether Notch1 directly regulated the 18 identified genes. We measured binding of Notch1 to CBF-1 (CSL/RPBJκ) binding sites of the 18 genes using a Chromatin Immunoprecipitation assay (ChIP) to determine whether Notch1 directly regulates the 18 genes. We scanned and designed primers 5 kb upstream and downstream of transcription start sites of the 18 genes and demonstrated that Notch1 was recruited to CBF-1 sites of 16 out of 18 genes. Different distribution of Notch1 binding was observed across all of the genes. Classical Notch targets, HEY1 and HES1 were among the most responsive genes and used as positive controls. Notch1 was found to be highly enriched on CBF-1 regulatory elements for the DAXX and NOXA genes. Binding of Notch1 to DAXX promoter elements increased in response to estrogen deprivation and this increase was attenuated upon GSI treatment. The biological activity of most genes was measured using the mammosphere-forming assay as a surrogate for CSC survival. Estrogen deprivation increased mammosphere-forming efficiency of ER+ breast cancer cells more than 2 fold compared to estrogen treatment. GSI blocked mammosphere formation by 95%-98% in response to anti-estrogen treatment. DAXX expression, was found to be necessary for GSI-mediated blockade of mammosphere formation. ChIP and mammosphere forming data calculated by ANOVA were found to be statistically significant. These results demonstrated that Notch1 is a direct transcriptional regulator of 16 genes identified by the clinical trial and in particular, DAXX, a pro-apoptotic gene that could serve as a cancer stem cell biomarker for anti-Notch therapy in ER+ breast cancer. Supported by a grant from the Breast Cancer Research Foundation.
Citation Format: Andrei Zlobin, Debra Wyatt, Jeffrey C. Bloodworth, Susan Hilsenbeck, Suzanne Fuqua, Lucio Miele, Kathy S. Albain, Clodia Osipo. DAXX is a novel Notch-1 gene target and biomarker of GSI-sensitivity in ER+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-121.