Abstract P5-05-07: An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer

Abstract BACKGROUND: FASN expression is associated with a more aggressive breast cancer phenotype and is both transcriptionally and post-transcriptionally regulated downstream of receptor tyrosine kinase signaling pathways. Moreover, 2nd and 3rd generation FASN inhibitors are showing promise for the treatment of breast cancer in clinical trials. Lipogenic transcripts, such as FASN, can be post-transcriptionally regulated through pre-mRNA splicing mediated through serine arginine rich protein kinases (SRPKs) and their respected substrates, serine-arginine rich splicing factors (SRSFs). Recent work has highlighted the mTORC1 signaling pathway as an upstream inducer of lipogenic pre-mRNA splicing, however, the role of extracellular environmental cues, such as growth factors and their respected receptors have not been explored. Here, an IGF-1-mTORC1-SRPK2 axis is demonstrated in the FASN regulation through SRSF-1 in breast cancer. METHODS: MDA-MB-231, MCF-7 breast cancer or MCF-10A non-transformed cells were exposed to IGF-1 followed by siRNA knockdown of IGF-1R, SRPK2, or SRSF-1. FASN expression was quantified by RT-qPCR or western blot analysis and de novo palmitate was measured by U-13C glucose incorporation followed by GS-MS. For mRNA stability, cells were pretreated with actinomycin-D with either vehicle or SRPK2 inhibitor for various timepoints followed by RT-qPCR for lipogenic and glycolytic mRNA abundance. eGFP-SRSF-1 was transfected in MDA-MB-231 and MCF-7 to visualize SRSF-1 localization in response to mTORC1 inhibition and/or SRPK2 knockdown and visualized by fluorescence microscopy. For intron retention, RT-PCR was performed with FASN intron and exon specific primers and resolved on a 2.5% agarose gel. RESULTS: Both IGF-1R and SRPK2 RNAi mediated knockdown significantly reduced FASN mRNA and protein and de novo synthesized palmitate levels. Similar results were obtained with mTORC1 inhibition. IGF-1 promoted the stabilization of FASN mRNA as well as reduced intron retention. This reduction of intron retention upon IGF-1 was abolished by SRPK2 knockdown. Additionally, IGF-1 contributed to a more diffuse localization in the nucleoplasm of SRSF-1, which become more retained in nuclear speckles upon both SRPK2 knockdown and mTORC1 inhibition. CONCLUSION: These current findings establish a potential IGF-1-mTORC1-SRPK2 axis in breast cancer that contributes to metabolic programming through FASN. More specifically, SRSF-1 is the potential mediator of FASN expression through this pathway, which could be a potential therapeutic target for breast cancers that overexpress FASN and components of the IGF-1R signaling axis. Citation Format: Bryan Mcclellan, Paul Gries, Brittany Harlow, Andrew J Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. An IGF-1R-mTORC1-SRPK2 signaling axis contributes to FASN regulation in breast cancer [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 P5-05-07.