Supplementary Material for: Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER<sup>+</sup> Breast Cancer Cells

<b><i>Background/Aims:</i></b> Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-<i>α</i> positive (ER<sup>+</sup>) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, <i>H19</i>, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER<sup>+</sup> breast epithelial cells. We hypothesized that <i>H19</i> expression is also important for the proliferation and survival of ETR cells<i>.</i> <b><i>Methods:</i></b> Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of <i>H19</i> function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered <i>H19</i> expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). <b><i>Results:</i></b> Here we report that treating ETR cells with Tam or Fulvestrant increases <i>H19</i> expression and that it’s decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, <i>H19</i> expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an <i>H19</i>-dependent manner in these cells. Lastly, we demonstrate that <i>H19</i> regulates <i>ERα</i> expression at the transcript and protein levels in the ETR cells and that <i>H19</i> protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and <i>H19</i> expression in the ETR cells. <b><i>Conclusion:</i></b> In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, <i>H19</i> expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.