Abstract A5: microRNA response to hypoxic stress in soft tissue sarcoma cells
Authors: Gits Caroline M.M. , de Rijck Jonneke C.W.M. , van Kuijk Patricia F. , Jonkers Moniek B.E. , Verweij Jaap , Sleijfer Stefan , Wiemer Erik A.C. 
Published: June 2012

Many solid tumors encounter hypoxia; a state in which the tumor tissue is deprived of oxygen. Hypoxia is of great clinical importance since it promotes malignant progression by increasing genetic instability, angiogenesis, local invasion and distant metastases. Furthermore hypoxia may lower the susceptibility to apoptosis, and induce chemo/radiation therapy resistance. These alterations are in part mediated by hypoxia inducible transcription factors like HIF1 that affect gene expression. Less well studied in hypoxia is the relatively new level of post-transcriptional regulation by microRNAs. MiRNAs are small non-protein coding RNA molecules that regulate gene expression by binding to the 3′UTRs of target mRNAs, thereby causing translation inhibition and/or mRNA degradation. Several studies have identified hypoxia-regulated miRNAs (HRMs). However, the lack of a general HRM profile suggests cell type specific miRNA responses to hypoxic stress. In a pilot study with three cancer cell lines of different histogenetic origin we observed, in line with the current literature, strong hypoxic miRNA-responses in each cell line, but little overlap in HRMs among the cell lines. Therefore we decided to focus on the modulation of the hypoxic response by miRNAs in a single class of rare mesenchymal tumors, soft tissue sarcomas.

We cultured 13 sarcoma cell lines (derived from fibrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma and synovial sarcoma) under standard conditions in 20% oxygen (normoxia) or in 1 % oxygen (hypoxia) for 0h, 6h, 24h and 48h. Hypoxic conditions were verified by Hypoxia Inducible Factor 1α (HIF1α) upregulation at the protein level and by the upregulation of transcripts of HIF1 target genes CA-IX and VEGFA. MiRNA expression profiling by microarray technology using LNA™ modified oligonucleotides identified several soft tissue sarcoma specific HRMs. There was considerably more overlap in differentially expressed miRNAs among the various soft tissue sarcoma subtypes than among tumor types from more diverse origins. Although miR-210 expression was below the detection level on our array platform, RT-qPCR indicated a 2 to 24 fold upregulation of this commonly found HRM after 24h of hypoxia. The top three (p&lt;0.0002) differentially and overexpressed miRNAs among all sarcoma subtypes are miR-185*, miR-485-5p and miR-216a. Potential target genes of these miRNAs were identified by online target prediction algorithms (e.g. TargetScan). HIF1AN (predicted target of miR-216a and mir-485-5p) and HIF3α (11 predicted target sites for mir-485-5p) were selected as they both appear to play modulatory roles in the hypoxic response. We confirmed the downregulation of HIF1AN (a HIF1-inhibitor) under hypoxic conditions, which would fit the hypothesis that it is regulated by our HRMs. For HIF3α several transcripts with different 3′UTRs exist; only the longest 3′UTRs contain all eleven potential binding sites for miR-485-5p. The presence of these long transcripts is confirmed in all but one cell line. Therefore it is plausible that HIF3α is targeted by miR-485-5p. Luciferase reporter assays and transfection experiments, transiently over-expressing and down-regulating miR-485-5p and miR-216a, are being carried out for functional studies and target gene validation.

In Conclusion: We are the first to describe the miRNA response to hypoxia in soft tissue sarcomas and identified new HRMs. Furthermore we identified the hypoxia related genes HIF1 AN and HIF3α as potential targets of our HRMs. This research will further elucidate the role of miRNAs in the hypoxic response in soft tissue sarcomas and may ultimately lead to novel treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A5.