Endocrine Abstracts

Alternative splicing is a crucial mechanism of gene regulation and this process can be dysregulated in cancer. In pituitary tumors (PitNETs), alteration in the serine/arginine-rich splicing factors (SRSFs) has been reported. Newly synthetized SRSFs are phosphorylated by serine-arginine protein kinase 1 (SRPK1) to facilitate their nuclear import. In response to extracellular stimuli SRPK1 may translocate to the nucleus as well to hyperphosphorylate SRSFs and favor their interaction with mRNA and the initiation of alternative splicing. SRPK1 is considered a proto-oncogene and its inhibition by small molecule inhibitors have shown antitumoral effects via the SRPK1-SRSF1-VEGF-A pathway modulation in different cancer types, however, so far, no studies have been carried out in the pituitary. This project aims to explore the role of SRPK1 inhibitors SRPIN340 and SPHINX31 in pituitary somato-lactotroph tumoral cells (GH4 and MMQ cells) by looking at the VEGF-A alternative splicing regulation, and to investigate SRPK1 protein expression levels in GH-secreting PitNETs. First, an abolishment of the EGF-mediated SRPK1 nuclear translocation in GH4 cells upon 10µM SRPIN340 incubation was observed by immunofluorescence experiments. This data, along with immunoblot results showing a reduction of phosphorylation of SRSFs induced by SRPIN340 and SPHINX31, demonstrated the efficacy of these compounds in impairing SRPK1 activity. SRPIN340 reduced cell proliferation (-38.9(43.9)%, p 0<0.05 vs basal at 1µM), cell migration (-65(46.3)%, P<0.001 vs basal at 10µM) and induced cell apoptosis (+40.5(26.6)%, P<0.05 vs basal at 10 µM), in GH4 cells. Similar results were obtained in MMQ cells and comparable effects between SRPIN340 and SPHINX31 were observed in both cell lines. Moreover, both SRPIN340 and SPHINX31 reduced PRL secretion in MMQ cells (-30.2(49)% P<0.05 vs basal, -39.2(18)% P<0.001 vs basal, at 10µM SRPIN340- and SPHINX31-treated cells, respectively). In addition, the pro-angiogenic and pro-mitotic VEGF-A_164a transcript isoform was significantly decreased by both inhibitors in GH4 cells (-61(33.5)% P<0.05 vs basal, -55(20.5)% P<0.05 vs basal, in SRPIN340- and SPHINX31-treated cells, respectively). Finally, SRPK1 was expressed at variable levels in 14 GH-secreting PitNETs. Interestingly, an overexpression of SRPK1 was found in GH-secreting PitNETs tissues whose corresponding primary cultures were in vitro resistant to octreotide in terms of GH suppression (0.09(0.31) vs 0.02(0.03), P<0.05 vs in vitro responder group). In conclusion, SRPK1 inhibition may represent a novel strategy to exert antitumoral effects in somato-lactotroph tumoral cells via SRPK1-SRSF1-VEGF-A pathway regulation. Further studies are needed to investigate the role of SRPK1 as a potential marker of octreotide responsiveness in GH-secreting PitNETs.