Abstract 4806: Spry1 as a potential new hypoxia modulator in cutaneous melanoma
Authors: Montico Barbara , Giurato Giorgio , Guerrieri Roberto , Salvati Annamaria , Colizzi Francesca , Baboci Lorena , Sigalotti Luca , Covre Alessia , Maio Michele , Steffan Agostino , Nyman Tuula A. , Weisz Alessandro , Mongiat Maurizio , Andreuzzi Eva , Fratta Elisabetta 
Published: April 2023

Abstract
Introduction: At present, the precise role of Spry proteins in tumor progression is still debated. Our previous study showed that Spry1 knockdown (Spry1KO) in BRAFV600-mutant cutaneous melanoma (CM) promoted cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and reduced tumor growth in vivo. Furthermore, we observed that Spry1 loss impaired angiogenesis in vivo. Hypoxia has been recognized as one of the crucial features of CM. Hypoxia results in the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which represents a master regulator of angiogenesis. Hence, the present study was designed to explore a possible role of Spry1 in modulating angiogenesis and hypoxia in CM.
Material and method: SPRY1 gene was knocked-out using the CRISPR-strategy in the BRAF wild-type (wt) Mel 313 and in the BRAF-mutant Mel 593 cell lines. Angiogenic potential was assessed through in vivo CD31 staining and in vitro tube formation assays. By using in vitro and in vivo models, the effects of Spry1KO on hypoxia-related genes were investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass-spectrometry (IP-MS) was employed.
Results and discussion: Consistent with our previous data, tumors arising from Mel 313 and Mel 593 Spry1KO clones were significantly smaller than those from their corresponding parental cells. Tumor growth inhibition was accompanied by a substantial reduction of angiogenesis, as assessed by the immunofluorescence staining of CD31 in vivo and the tube formation assay in vitro. Accordingly, RNA-seq analysis indicated that the vascular endothelial growth factor A was significantly down-regulated following Spry1 silencing. Notably, Ingenuity Pathway Analysis identified “HIF-1α Signaling” as the most significant molecular and cellular function affected in vivo by Spry1 silencing regardless of the BRAF mutational status. Analyses of tumor tissues confirmed that Spry1KO significantly reduced HIF1-α protein abundance. Interestingly, HIF-1α mRNA levels were minimally affected, thus suggesting that Spry1KO might impact on HIF-1α expression post-translationally. IP-MS identified about 50 Spry1 protein partners, and most of them were mitochondrial. Importantly, western blot analysis of cytosolic and mitochondrial proteins revealed that Spry1 was largely expressed in CM cell lines irrespective of BRAF mutation.
Conclusions: Altogether, these data lead us to speculate that Spry1 might play a role in mitochondria homeostasis, thus impacting on hypoxia and angiogenesis in CM.
Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula A. Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 as a potential new hypoxia modulator in cutaneous melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4806.