Abstract P1-08-01: Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model
Authors: Mori Hitomi , Saeki Kohei , Chang Gregory , Wu Xiwei , Hsu Pei-Yin , Kanaya Noriko , Somlo George , Chen Shiuan 
Published: March 2020

Background: Estradiol (E2) is known to promote breast cancer. However, several small-scale prospective clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)- resistant cases of hormone receptor-positive postmenopausal breast cancer. Considering that the practicality and safety of E2 treatments are still undefined, the objective of this study is to uncover the mechanisms of E2-induced tumor regression, which will lead to a better design of E2 therapies.

Methods: While most of our ER+ breast cancer patient-derived xenograft (PDX) models are positively regulated by E2, an estrogen-suppressive model (named GS3) was established from an AI resistant ER+/PR−/HER2− brain metastatic breast cancer. E2 pellets (1mg each) or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC), RNA sequencing, and reverse phase protein array (RPPA) analyses of PDX specimens were conducted to decipher molecular changes after E2 treatments. Importantly, since the cancer tissue has a very heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3.

Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist (MPP), not by ERβ antagonist (PHTPP). IHC showed that PR expression appeared, Ki-67 and CEACAM5 (CEA) expressions decreased, and the number of apoptotic cells significantly increased after E2 treatment. RNA sequencing indicated that estrogen-regulated genes, e.g., PGR, EGR3, PDZK1, and GREB1, were up-regulated, while tumor growth was repressed by E2. Single cell RNAseq analysis demonstrated that cells from E2-treated and Placebo-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes (HVGs). Importantly, after one-week, many cells of E2-treated PDXs were arrested within the G1 phase of the cell cycle (G1, 60%; S, 19%; G2M, 21%), whereas, cells of placebo-treated samples advanced to the S and G2M phases (G1, 47%; S, 27%; G2M, 26%). Gene Set Variation Analysis of cells from E2-treated PDXs revealed enrichment of genes associated with reactive oxygen species (ROS) mechanism. E2 increased cells expressing pro-apoptotic genes, such as TP53, BOK,IL24, and PGLYRP2. Gene expressions including mitogen-activated protein kinase (MAPK) signaling pathway, such as GADD45A, MAP3K5 (ASK1), MAPK9 (JNK2), and especially MAPKAPK2 were also increased in E2 treated PDXs.

Conclusions: E2/ERα-mediated suppression of GS3 tumor growth was accompanying the inhibition of the cell cycle progression and increased expression of pro-apoptotic genes. It is known that classical MAPK pathway induces cell proliferation, meanwhile, JNK MAPK pathway induces p53 signaling pathway and apoptosis. We hypothesize that E2 induces ROS and promotes apoptosis of GS3 due to JNK MAPK pathway activation. This could be an unexpected outcome of AI resistance. Using this valuable estrogen-down-regulated ER+ breast cancer PDX and informative integrative omics analyses, critical molecular mechanisms on E2-mediated apoptosis are being revealed in our laboratory. The ongoing research using E2-sensitive PDXs will identify key markers to select AI resistant patients who are expected to respond effectively to estrogen-induced apoptosis treatments as well as to evaluate the efficacy of drug combination with estrogen, including CDK 4/6 inhibitors.

Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest and apoptosis in aromatase inhibitor-resistant breast cancer: Insights from single cells analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-01.