Abstract 4431: Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy
Authors: Altomare Emanuela , Rossi Marco , Caracciolo Daniele , Amodio Nicola , Gaspari Marco , Taverna Domenico , Scumaci Domenica , Botta Cirino , Gullà Annamaria , Morelli Eugenio , Critelli Paola , Arbitrio Mariamena , Tagliaferri Pierosandro , Tassone Pierfrancesco 
Published: August 2018

Abstract
Th17 cells play a critical role in Multiple Myeloma (MM) microenvironment by triggering osteoclastogenic activity, that leads to severe skeleton damage. We have recently found that Th17 cells directly promote osteoclast (OCL) differentiation and function via expression of RANKL. It has been demonstrated that miRNAs are involved in the differentiation of Th17 cells. Murugaiyan et al. demonstrated that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Indeed, we have been able to negatively modulate Th17 differentiation by a specific inhibitor of miR-21 (mir21i). The overall effect of mir-21 inhibition is the suppression of OCL resorptive activity, thus attenuating Th17 mediated multiple myeloma bone disease (MMBD). However, the molecular mechanisms underlying these biological effects have not been clarified. On these bases, we aimed to identify proteins involved in the effect of miR21i on Th17 cells. Quantitative phosphoproteomics technologies were applied to study the effects of miR21i in Th17 cells. To unveil the molecular targets responsible for this interaction, Ingenuity's IPA® software was interrogated. This approach allowed to identify several unknown miR-21 target proteins and the related signal transduction pathway.To perfom global phosphoproteome analysis, we performed a mass spectrometry study of phosphopeptides on protein extracts from miR21i- treated Th17 and control Th17 cells, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software. Among analyses of more than 1134 phosphorylation sites, 386 have shown significant changes, as compared to controls. Interestingly, IPA analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition including PI3K/AKT, 14-3-3 and GRANZYME A signaling. Moreover IPA analysis disclosed RANKL among the top upstream proteins. The most up regulated protein was CYTIP and the ones with enhanced activity were PDCD4, STAT1, MINK1, FOXO1, while ITGAL showed reduced activity. To validate these data, we performed WB analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of the master Th17 transcription factor, RORC, were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated by mir21i. As PIAS3 and PDCD4 negatively regulate STAT3, we checked phospho-STAT3 expression and found that it was indeed downregulated, as expected. Overall, this study defines a phosphoproteomic signature of miR-21i treated Th17 cells, providing new insights into molecular impairment of Th17 differentiation by miR21i and suggest innovative molecular targeted approaches to treat MMBD
Citation Format: Emanuela Altomare, Marco Rossi, Daniele Caracciolo, Nicola Amodio, Marco Gaspari, Domenico Taverna, Domenica Scumaci, Cirino Botta, Annamaria Gullà, Eugenio Morelli, Paola Critelli, Mariamena Arbitrio, Pierosandro Tagliaferri, Pierfrancesco Tassone. Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4431.