Abstract P5-04-10: The role of DAXX on enrichment of breast cancer stem cells from ER+ breast cancer
Authors: Peiffer DS , Wyatt D , Zlobin AY , Piracha A , Robinson PA , Albain KS , Osipo C 
Published: May 2019

Background: Resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors) is a major cause of mortality for women with estrogen receptor-positive (ER+) breast cancer (BrC). One mechanism of ET-resistance is due to heterogeneity of breast tumors, comprised of differentiated cells plus undifferentiated breast cancer stem cells (BCSCs). Survival of BCSCs requires Notch signaling and thus determining how it is activated in response to ET may elucidate a new target of therapeutic significance. We found that expression of the transcriptional repressor, DAXX, was inversely correlated with Notch inhibition in a human pre-surgical biomarker trial of ER+ BrC patients (ClinTrials.gov NCT00756717). Further, it has been demonstrated that expression of a sumoylation enzyme, UBC9, is dependent on ERα activation, and that it sumoylates DAXX. We hypothesized that targeting ERα by ET depletes UBC9 causing destabilization of the DAXX protein, resulting in Notch activation and enrichment of BCSCs.

Methods: A panel of ER+ BrC cells were grown in conditions of 5nM estradiol (E2) or ET conditions of E2 deprivation. Expression levels of NOTCH4, other BCSC-associated genes, UBC9 and DAXX were quantified by real-time PCR (RTPCR). BCSC survival was assessed by the mammosphere forming assay. To test if ET increases BCSC by destabilizing DAXX, DAXX was depleted or overexpressed by cell transfection. To assess the role of DAXX in tumor initiating potential, an extreme limiting dilution assay (ELDA) was conducted by injection of normal or DAXX-depleted cells into the mammary fat pads of female mice. Nuclear levels of DAXX protein were quantified by cell fractionation and enrichment of DAXX at the promoter regions of BCSC-associated genes was assessed by chromatin immunoprecipitation (ChIP). Protein levels of DAXX following UBC9 depletion were determined by cell fractionation.

Results: ET or siRNA depletion of DAXX increased survival of BCSCs, and expression of BCSC-associated genes, including NOTCH4. Conversely, DAXX overexpression under ET-conditions inhibited BCSC-associated gene expression and survival. ELDA demonstrated that cells depleted of DAXX had a higher estimated stem cell frequency compared to DAXX-expressing cells. Cell fractionation indicated that DAXX protein was concentrated in the nucleus, and these levels were decreased by ET treatment. ChIP studies revealed an enrichment of DAXX binding at the promoter regions of BCSC-associated genes (NOTCH4, SOX2, OCT4, NANOG), which was lost following ET. UBC9, but not DAXX mRNA levels, are dependent on activation of ERα, and depletion of UBC9 resulted in a decrease in DAXX levels.

Conclusions: These results suggest that E2 activation of ERα increases UBC9 levels, allowing for stabilization of nuclear DAXX in ER+ BrC cells, where it represses expression of BCSC-associated genes. Conversely, ET depletes UBC9, destabilizing DAXX, ultimately relieving repression of BCSC-associated genes and promoting increased BCSC survival. This would potentially result in resistance to ET. Thus, new strategies to stabilize DAXX protein during ET may prevent induction of BCSC gene expression and enrichment of BCSCs, improving therapeutic outcomes in ER+ BrC.

Support: Breast Cancer Research Foundation (Kathy S. Albain & Clodia Osipo)

Citation Format: Peiffer DS, Wyatt D, Zlobin AY, Piracha A, Robinson PA, Albain KS, Osipo C. The role of DAXX on enrichment of breast cancer stem cells from ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-10.