Abstract Breast cancer is the second leading cause of cancer death in women worldwide. Despite recent advances in breast cancer treatment, tumor metastasis and recurrence remain the most significant clinical obstacles, largely due to the resistance of residual breast tumor cells to existing therapies. RNA alternative splicing is a fundamental mechanism for gene regulation and protein diversity. Despite the fact that nearly all human genes are regulated by alternative splicing, very limited studies have addressed the role of alternative splicing in tumorigenesis. Our lab uses the cell adhesion molecule CD44 as a model to investigate the role of alternative splicing in breast cancer metastasis. CD44 undergoes extensive alternative splicing, giving rise to the standard (CD44s) and variant (CD44v) isoforms. Previous work from our lab demonstrated that the CD44s isoform is required for Epithelial to Mesenchymal transition (EMT) and breast tumor metastasis. Mechanistically, we have shown that CD44s, but not CD44v, potentiates Akt signaling, promoting cell survival and chemoresistance in breast cancer cells. To better understand CD44s-mediated downstream pathways critical for breast tumor metastasis, we analyzed CD44s-dependent gene signature and identified Hyaluronan Synthase 2 (HAS2), an enzyme that produces hyaluronan (HA), as one of the most significantly upregulated genes in response to CD44s expression. Interestingly, HA is the major ligand for CD44, and HA/CD44 interaction promotes Akt activation. These results led us to test a hypothesis that a positive feedback loop, involving CD44s and HAS2, sustains Akt signaling and promotes tumor cell survival. Indeed, our results show that CD44s-mediated Akt activation stimulates HAS2 transcription. This increase in HAS2 transcription is mediated by Akt phosphorylation of FOXO1, a transcription repressor inactivated by phosphorylation. We further show that HAS2 and HA activate Akt signaling in a CD44s-dependent manner. Importantly, disruption of this feed-forward loop inhibits EMT and triggers cell death. PI3K/Akt signaling is hyperactive in more than 70% of breast tumors. Hence, the proteins involved in this positive feedback loop represent very attractive and promising drug targets for breast cancer therapy. Our work has demonstrated how aberrant expression of alternative spliced isoform in breast cancer, through a positive feedback loop, leads to sustained Akt signaling, enhanced cell survival, and a metastatic phenotype. Disrupting this positive feedback loop may provide a foundation for an innovative approach to treat metastatic breast cancer patients. Citation Format: Liu S, Cheng C. A positive feedback loop couples CD44s and HAS2 for sustained Akt activation and tumor cell survival [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-06.