Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA transfactor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was "rescued" by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. Implications: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer.

HIF1 (Hypoxia-inducible Factor 1) is a transcription factor that plays a crucial role in the hypoxia stress response. Its primary function is to return the cell to its homeostatic state following oxygen deprivation. However, chronic hypoxia exposure can cause irreversible physiological changes that can lead to pulmonary hypertension (PH) and the need for therapeutics to ameliorate these conditions is great and unmet. Previous studies in our lab have demonstrated that CPEB2 (cytoplasmic polyadenylation element binding protein 2) is a translational repressor of one of the HIF1 subunits: HIF1α. Our lab demonstrated that the alternatively spliced CPEB2A isoform of CPEB2 is a repressor of translation, while the CPEB2B isoform is a translational activator of HIF1α during hypoxia, suggesting a major regulatory role for CPEB2 AS in the pulmonary hypoxic response. Although it is well established that during hypoxia, HIF1α levels are dramatically upregulated due to a decrease in the degradation of this factor, we propose that during chronic hypoxia, the expression of HIF1α is maintained via a translational mechanism, likely alongside a decrease in proteolytic degradation. In this study we demonstrate that depletion of the CPEB2B splice isoform has an inhibitory effect on the translation of nascent HIF1α protein during chronic hypoxia, but not the acute phase. We further demonstrate that this pathway is dependent on the initiation factor eIF3H. Finally, we show data which indicate that CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences depending on surrounding sequence context. These findings are important, since they provide evidence for potential of CPEB2 to act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.