Abstract P3-06-07: ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer
Authors: Bhat Raksha , Abdulkareem Noor Mazin , Qin Lanfang , De Angelis Carmine , Yasser Hala , Thangavel Hariprasad , Torres Jennifer , Sethunath Vidyalakshmi , Mitchell Tamika , Nanda Sarmistha , Pereira Resel , Shea Martin , Schiff Rachel , Trivedi Meghana 
Published: March 2020

Background: Adhesion G protein-coupled receptors (GPCRs) form the second largest GPCR subfamily and control many cellular processes involved in cancer. ADGRF1 is an adhesion GPCR overexpressed and gene amplified in HER2+ and triple-negative breast cancer (BC). We have previously found a role of ADGRF1 in promoting tumorigenicity and metastatic processes in cell-based studies in HER2+ BC. The goal of this study was to evaluate the effects of ADGRF1 overexpression in vivo in HER2+ BC.

Methods: BT474 stable cell lines were generated using lentiviral system containing ADGRF1 cDNA using PHAGE system, overexpressing ADGRF1 in Doxycycline (Dox)-inducible manner. Mice (N=8/group) were injected with various numbers of cells with (+) or without (-) Dox (10K, 30K, 100K, 300K, 1 million) to assess for tumor initiation and growth. Transcriptomic and proteomic analysis of ADGRF1-overexpressing BT474 cells was also performed using RNAseq and RPPA analysis, respectively. The candidates were validated using western blotting and IHC staining. Cell cycle analysis was conducted by measuring DNA content with propidium iodide using flow cytometry.

Results: Dox-induced overexpression of ADGRF1 in mice led to inhibition of tumor growth compared to no Dox treated tumors. Furthermore, fewer mice injected with Dox-treated cells formed tumors. Our RNA-Seq data revealed downregulation of genes involved in cell cycle regulation, which included E2F targets, G2M checkpoint pathways, and MYC targets. Likewise in RPPA analysis, several proteins in the cell cycle pathway (FOXO1, AuroraA, Ki67, Ezh2, CHAF1, DEPTOR, Rb1, Phospho-Rb (S780), and Hexokinase II) were downregulated by ADGRF1 overexpression. Validation of RNAseq and RPPA candidates demonstrated lower number of Ki67+ cells and reduced NF-kB expression, and inhibition of STAT3 phosphorylation. Furthermore, we found that ADGRF1 overexpression by Dox also led to cell cycle arrest at GO/G1 phase.

Conclusion: Our results suggest that ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer. These results are in contrast to the in vitro data demonstrating a role of ADGRF1 in promoting tumorigenicity and metastatic processes in HER2+ BC. Understanding the mechanism of this difference is critical in investigating ADGRF1 as a drug target in breast cancer.

Citation Format: Raksha Bhat, Noor Mazin Abdulkareem, Lanfang Qin, Carmine De Angelis, Hala Yasser, Hariprasad Thangavel, Jennifer Torres, Vidyalakshmi Sethunath, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Martin Shea, Rachel Schiff, Meghana Trivedi. ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer [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 P3-06-07.