Extracellular Vesicles From SDHB Deficient hPheo1 Cells Activate STAT3 in Wild-Type Cells

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that originate from the adrenal medulla and extra-adrenal paraganglia, respectively. Inactivating mutations in succinate dehydrogenase (SDHx) genes leads to succinate accumulation, increased HIF1-α levels, and uncontrollable growth of PPGLs. We hypothesized that small extracellular vesicles (EVs) released from progenitor cells derived from pheochromocytoma (hPheo1) with a shRNA mediated knockdown of SDHB are enriched in succinate metabolites that play a key role in the activation of various tyrosine dependent signaling pathways that are involved in turmorigenesis and proliferation. We isolated EVs from the conditioned media of human wild-type hPheo1 cells and hPheo1 cells with shRNA SDHB knockdown. The EVs from three separate preparations of each group were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting using antibodies against different types of EV and one non-EV marker. Our results show small EVs from the SDHB knockdown hPheo1 cells increased the activation of phosphotyrosine residues in wild-type cells compared to cells treated with control EVs from the same cell type. Additionally, our data show these EVs increase phospho-STAT3 compared to the control EVs (3843.10 +/- 1138.89 vs. 213.65+/- 40.75; p<0.05; n=3) in cultured wild-type hPheo1 cells. Protein tyrosine kinases (PTKs) control various cellular processes including growth, differentiation, and metabolism by activating various signaling pathways including STAT3. The significance of these findings is that in some cancers, elevated succinate from a SDHx mutation has been shown to activate STAT3 which may explain a possible pathway for tumorigenesis. Studies from other investigators have shown that STAT3 expression is elevated in malignant PPGL tissues. Through enriched EV analysis our findings have confirmed the role of STAT3 in SDHB deficient cells. Additional studies are needed to identify other metabolites that are enriched in EVs that regulate phosphorylation of tyrosine residues and STAT3 activation.