Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK

Xiao Xiang,Stephen D Baird,Anna Blinder,Tyson E Graber,Stéphanie Langlois,Tommy Alain,Steffany A L Bennett,Marie-Eve St-Pierre,Stephanie L Fowler,Kyle N Cowan,Philippe Charron

doi:10.1038/s41388-020-01623-2

Abstract

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.

Highlights

Supplementary information The online version of this article contains supplementary material, which is available to authorized users.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood [1]
Our work has shown that PANX1 levels are low in Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its
We found that PANX1 expression is downregulated in patientderived RMS cell lines and tumor specimens when compared to differentiated skeletal muscle cells and tissue [10]

Summary

Introduction

As ectopic PANX1 effectively alleviates RMS tumor growth, deciphering its downstream signaling pathways would bring insight into the molecular mechanism by which PANX1 reduces RMS malignant properties while offering an opportunity to identify potential new therapeutic targets. Knockdown of AHNAK in PANX1-expressing Rh18 and Rh30 cells abrogated the PANX1-mediated reduction in cell viability, migration, and increase in anoikis, suggesting that PANX1 regulation of RMS tumor malignant properties involves its interaction with AHNAK. Using data generated through these genome-wide unbiased approaches, we have generated the first PANX1 transcriptomic and proteomic public searchable databases for easy access to our entire RNA-seq and BioID data, which may foster new research avenues identifying pathways regulating PANX1 and its functions, as well as potential clinical translation toward novel therapeutic strategies

Results

Discussion

Material and methods

Compliance with ethical standards