PO-146 Multiple myeloma-derived exosomes carry amphiregulin and are responsible for the uncoupled bone remodelling

Abstract

IntroductionAnnexins are frequently deregulated in many cancers (‘annexinopathies’). Annexin A1 (ANXA1) overexpression has been reported in gastric, pancreatic and hepatocellular carcinoma; however, it is markedly down-regulated in breast, prostate, esophageal and head and neck cancers. The contrasting patterns of ANXA1 expression in different tumours types is just one of the enigmas in deciphering the underlying regulatory mechanisms and phenotypic specificity of ANXA1. We have previously demonstrated that ANXA1 down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC).Material and methodsIn an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 protein expression and ANXA1 mRNA levels in HNSCC tissue specimens by immunofluoresce, in situ hybridization and RT-qPCR. Subsequently, the relative levels of miR-196a and miR-196b were quantified in paired HNSCC specimens using Taqman miRNA assays and compared to ANXA1 mRNA levels. Functional studies were performed in HNSCC cells by transfection with specific pre-miR precursors, and also ANXA1 promoter activity using deletion constructs from the 5’ ANXA1 promoter region.Results and discussionResults showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumours, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3’UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control.ConclusionThese findings demonstrate that ANXA1 down-regulation in head and neck squamous cell carcinomas occurs via transcriptional control with direct involvement of miR-196a/b.