Beschreibung:
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. 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 Brenner, Stefano Tiziani, Christopher Jolly, Linda deGraffenried. Regulation of FASN expression through a novel IGF-1R-mTORC1-SRPK2-SRSF1 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC02-01.