The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. Conclusion: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression. (HEPATOLOGY 2014;60:1241-1250 A berrant activation, mutations, or dysregulation of transcriptional regulators are frequently detected in hepatocellular carcinoma (HCC). Tumor-relevant transcriptional regulators consist of DNA-interacting transcription factors (e.g., c-Myc, NF-jB) and transcriptional coactivators (e.g., b-catenin or YAP), which fine-tune initiation, progression, and termination of the transcriptional machinery to regulate the expression of target genes in a timedependent manner. Because transcriptional regulators Abbreviations: DN, dysplastic nodule; FBP, far upstream element binding protein; FIR, FBP-interacting repressor; FUSE, far upstream element; HCC, hepatocellular carcinoma.

Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination.

The far upstream element (FUSE)-binding proteins (FBP)- 1, FBP-2, and FBP-3 represent a family of single-strand nucleic acid binding factors, regulating transcriptional as well as post-transcriptional processes. Elevated expression and pro-tumorigenic functions of all FBPs have been described for human liver cancer [1, 2]. First data indicated that FBP-1 affects microtubule dynamics through regulating MT-destabilizing factors in non-small cell lung cancer (NSCLC) [3], however, comprehensive studies analyzing the expression and functional relevance of all FBPs in NSCLCs are missing so far. In order to define the expression of FBPs in lung cancer, FBP-transcript and protein levels were analysed in primary human NSCLC tissue samples. Semiquantitative real-time PCR as well as Tissue-Micro-Array (TMA) analyses revealed an elevated expression of FBP-1 and FBP-2 in most NSCLC tissue samples (>60%) in comparison to non-tumorous specimens. Interestingly, nuclear accumulation of FBP-1 significantly correlated with FBP-2 expression (r=0.33; p<0.01), suggesting common regulatory mechanisms. In vitro, transient inhibition of FBP-1 by gene-specific siRNAs in NSCLC cell lines (Calu-6) was associated with decreased tumor cell viability (-76%; MTT assay), proliferation (-83%; BrdU assay), and increased apoptosis (2.8-fold; Nicoletti FACS assay). In contrast, inhibition of FBP-2 reduced cell viability of Calu-1 cells (-32%; MTT assay) but predominantly reduced tumor cell migration (-62%; two-dimensional scratch assay) as well as tumor cell invasion (-81%; sprouting assay) in all analyzed NSCLC cell lines (Calu-1, Calu-6, and A549), suggesting that both FBP isoforms facilitate partly distinct tumor-supporting effects. Surprisingly, efficient single-gene inhibition of FBP-1 or FBP-2 in A549 cells did not affect tumor cell viability. In contrast, the concomitant knock down of both FBP isoforms resulted in significantly decreased cell viability (-69%), suggesting that some FBP family members may compensate the loss of other members. Actually, FBP-2 negatively regulates FBP-1 expression in A549 cells, resulting in increased FBP-1 transcript and protein levels after FBP-2 inhibition. Therefore, functional compensation prevents A549 cells from anti-tumorigenic effects after FBP-2 knock down. In summary, this study provides evidence that coordinated overexpression of FBP-1 and FBP-2 is a frequent event in NSCLCs and that both factors are support tumor growth and NSCLC cell dissemination. Interestingly, partial functional redundance and mutual negative regulation of FBPs indicate that these factors may fine-tune the oncogenic behavior of NSCLC cells. References: [1] M Malz et al., (2009) Hepatology; [2] A Brauckhoff et al., (2011) J. Hepatol. [3] S Singer et al., (2009) Cancer Res Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1300. doi:1538-7445.AM2012-1300