Interleukin-6 (IL-6)-activated Signal Transducer and Activator of Transcription 3 (STAT3) facilitates survival in the multiple myeloma cell line INA-6 and therefore represents an oncogenic key player. However, the biological mechanisms are still not fully understood. In previous studies we identified microRNA-21 as a STAT3 target gene with strong anti-apoptotic potential, suggesting that noncoding RNAs have an impact on the pathogenesis of human multiple myeloma. Here, we describe five long noncoding RNAs (lncRNAs) induced by IL-6-activated STAT3, which we named STAiRs. While STAiRs 1, 2 and 6 remain unprocessed in the nucleus and show myeloma-specific expression, STAiRs 15 and 18 are spliced and broadly expressed. Especially STAiR2 and STAiR18 are promising candidates. STAiR2 originates from the first intron of a tumor suppressor gene. Our data support a mutually exclusive expression of either STAiR2 or the functional tumor suppressor in INA-6 cells and thus a contribution of STAiR2 to tumorigenesis. Furthermore, STAiR18 was shown to be overexpressed in every tested tumor entity, indicating its global role in tumor pathogenesis. Taken together, our study reveals a number of STAT3-induced lncRNAs suggesting that the interplay between the coding and noncoding worlds represents a fundamental principle of STAT3-driven cancer development in multiple myeloma and beyond.

Background: The survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6activated transcription factor STAT3. Although transcriptional analyses have revealed many genes regulated by STAT3, to date no protein-coding STAT3 target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding STAT3 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by STAT3, named STAiRs. Here, we focus on STAT3-induced RNA 18 (STAiR18), an mRNA-like, long ncRNA that is duplicated in the human lineage. One STAiR18 locus is annotated as the already well described LINC00152/CYTOR, however, the other harbors the MIR4435-2HG gene and is, up to now, barely described.Methods: CAPTURE-RNA-sequencing was used to analyze STAiR18 transcript architecture. To identify the STAiR18 and STAT3 phenotype, siRNA-based knockdowns were performed and microarrays were applied to identify their target genes. RNA-binding partners of STAiR18 were determined by Chromatin-Isolation-by-RNA-Purification (ChIRP) and subsequent sequencing. STAT3 expression in dependence of STAiR18 was investigated by immunoblots, chromatin-and RNA-immunoprecipitations. Results: As identified by CAPTURE-RNA sequencing, a complex splice pattern originates from both STAiR18 loci, generating different transcripts. Knockdown of the most abundant STAiR18 isoforms dramatically decreased INA-6 cell vitality, suggesting a functional role in myeloma cells. Additionally, STAiR18 and STAT3 knockdowns yielded overlapping changes of transcription patterns in INA-6 cells, suggesting a close functional interplay between the two factors. Moreover, Chromatin isolation by RNA purification (ChIRP), followed by genome-wide RNA sequencing showed that STAiR18 associates specifically with the STAT3 primary transcript. Furthermore, the knockdown of STAiR18 reduced STAT3 levels on both the RNA and protein levels, suggesting a positive feedback between both molecules. Furthermore, STAiR18 knockdown changes the histone methylation status of the STAT3 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.