Abstract P5-09-04: Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy

Abstract

Abstract Background: Breast cancer (BC) is one of the most diagnosed tumors in the United States. The mechanism of the disease development and progression is complex and still not fully understood. Dysregulation or change in the expression of transcription factors has been proposed as a possible cause of the disease severity and poor prognosis. The INhibitor of Growth family member 4 (ING4) is a tumor suppressor that has been connected to various cancer-related processes. Therefore, this study aims to assess the global gene regulation of BC cells by ING4 and identify the associated cellular processes and molecular pathways related to BC following ING4 loss. Methods: siRNA Knockdown and CRISPR Knockout technologies were used to deplete ING4 expression. Real-time PCR, RNA-Seq and Chip-Seq were used to identify gene expression alteration and ING4 enrichment on target promoters. Ingenuity Pathways Analysis (IPA®) was used to highlight candidates, networks and cellular processes. Results: Protein and RNA levels of ING4 were significantly reduced in MDA-231 knockdown and MCF7 knockout cells using siRNA and CRISPR technologies. RNA-Seq of BC cells revealed a large set of differentially expressed genes were regulated following ING4 loss. Key genes and biological networks related to cellular processes, including amino acid metabolism, cancer, cellular development, cellular growth and proliferation, were identified using IPA®. Chip-seq also revealed that ING4 was recruited to genes important in BC development and progression. Several genes were validated using the RT-qPCR method, and results were consistent with RNA-seq and Chip-seq data. Importantly, MDA-231 cells grown in stripped serum have shown an upregulation of the ribosomal protein S6 phosphorylation (PS6), an mTOR target protein, following ING4 knockdown. Treatment of MDA-231 cells with rapamycin blocked the phosphorylation of S6 induced by ING4 loss. An increase in S6 phosphorylation was also observed in MCF7 knockout cells grown either in regular or serum-free growth medium. The phosphorylation was reversed by amino acid transporter inhibitors JPH and BCH. Conclusion: The present study shows that ING4 regulates many genes involved in biological pathways and networks related to BC development and progression. Importantly, ING4 is a novel regulator of the mTOR pathway. Collectively, regulation of ING4 gene expression and its downstream molecules provides a novel approach in cancer treatment. Citation Format: Aymen Shatnawi, Travis Salisbury. Regulation of mTOR signaling pathway by ING4 in breast cancer: A new target in cancer therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-04.