Abstract 5531: Significance and functions of estrogen receptor beta (ESR2) isoforms in glioblastoma
Authors: Liu Jinyou , Sareddy Gangadhara Reddy , Zhou Mei , Viswanadhapalli Suryavathi , Brenner Andrew , Tekmal Rajeshwar R. , Vadlamudi Ratna K. 
Published: July 2017

Background: Glioblastoma (GBM) are the deadliest form of primary brain neoplasms. Several lines of evidence suggest tumor suppressive role of female sex hormones on brain tumors. However, mechanisms by which estrogens mediate protection against the GBM remains unknown. GBM preferentially express estrogen receptor beta (ESR2). Emerging evidence suggests that ESR2 is expressed as multiple isoforms, (ESR2-1 to 5, with variation at the C-terminal domain); however, much of the published information is focused primarily on ESR2-1. Little is known about the expression and functions of other ESR2 isoforms in GBM. The objective of this study is to examine the expression and determine the functions of ESR2 isoforms in GBM cells.

Methods: Expression of ESR2 isoforms was profiled using 10 different patients derived and 5 established GBM cells. To study the functions of individual ESR2 isoforms in GBM cells, we have generated ESR2 knockout (ESR2-KO) cells using CRISPR/Cas9 system and then established GBM model cells expressing individual ESR2-1, 2, and 5 using lentiviral transduction in the ESR2-KO background GBM cells. IPMS analysis was used to identify binding proteins of each ESR2 isoforms. Effect of each isoform on the growth, apoptosis, cell cycle progression, migration and invasion was analyzed using established methods. Mechanistic studies were conducted using reporter gene assays, RT-qPCR and signaling analysis.

Results: RT-qPCR results demonstrated that ESR2-5 is highly expressed in majority of primary and established GBM cells compared to ESR2-1 and ESR2-2, with ESR2-4 is the least expressed. Expression of ESR2-1 but not ESR2-5 significantly reduced proliferation of GBM cells. Further, ESR2-KO cells exhibited high migratory and invasive potential compared to parental GBM cells and re-expression of ESR2-1, but not ESR2-5 resulted in reduction in migratory and invasive potential of GBM cells. ESR2-KO GBM cells exhibited decreased levels of cell cycle arrest and apoptosis proteins p27, p21 and PUMA compared to parental GBM cells and re-expression of ESR2-1 or ESR2-5 in ESR2-KO cells rescued the phenotype. IPMS studies identified several common and unique proteins that bind to each of the isoforms. Both ESR2-1 and ESR2-5 interacted with mTOR, while ESR2-5 uniquely interacted with several proteins related to DNA repair, immunomodulatory and apoptosis pathways. Overexpression of ESR2-1 reduced the activation of mTOR signaling molecules including p-mTOR, p-S6K and p-S6 in GBM cells compared to ESR2-KO cells, while ESR2-5 enhanced mTOR downstream signaling.

Conclusion: Using ESR2KO cells, we have provided genetic evidence for the role of ESR2-1 in GBM tumor suppression. Our results also discovered that ESR2-5 is highly expressed in GBMs. Unlike ESR2-1, ESR2-5 has lesser tumor suppression ability and its interactions with mTOR, DNA repair, and apoptotic pathways may have important implications in GBM progression.

Citation Format: Jinyou Liu, Gangadhara Reddy Sareddy, Mei Zhou, Suryavathi Viswanadhapalli, Andrew Brenner, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. Significance and functions of estrogen receptor beta (ESR2) isoforms in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2017-5531