Z Gastroenterol 2014; 52 - P_4_45
DOI: 10.1055/s-0033-1360998

Stabilization of protumorigenic FUSE binding proteins (FBPs) in hepatocarcinogenesis

J Samarin 1, I Stein 3, E Horwitz 3, C Ho 4, X Chen 4, E Pikarsky 3, D Calvisi 2, P Schirmacher 1, K Breuhahn 1
  • 1University Hospital Heidelberg, Institute of Pathology, Heidelberg, Germany
  • 2Ernst-Moritz-Arndt-University, Institute of Pathology, Greifswald, Germany
  • 3The Hebrew University-Hadassah Medical School, Department of Pathology and Lautenberg Center for Immunology, Jerusalem, Israel
  • 4UCSF, Department of Bioengineering and Therapeutic Sciences, San Francisco, USA

Background:

Far upstream element-binding proteins (FBPs) represent a family of transcription factors, which are highly overexpressed in the majority human hepatocellular carcinoma (HCC). Nuclear FBP accumulation in HCC cells support tumor cell proliferation (FBP-1/-3) and migration (FBP-3). Although, FBPs are promising therapeutic target structures, the underlying mode of dysregulation has not been defined so far.

Methods:

Genomic alterations of the fubp (FBP-1; chr. 1p31.1), khsrp (FBP-2; chr. 19.p13.3), and fubp3 (FBP-3; 9p34.11) gene loci in primary human HCCs were determined by matrix-CGH. In order to identify specific stimuli of FBP enrichment in HCC cells, different human cell lines were cultured under different cell density and hypoxic conditions or treated with growth factors (e.g., TGFß, IGF-II), chemical inhibitors (e.g., AG1478, SB203580, MEK1/2, sorafenib, and PI3K inhibitors), and gene-specific siRNAs (e.g., Akt1 – 3, rictor, raptor). Impact of treatments on FBP mRNA and protein levels was defined using western immunoblotting and real-time PCR. Findings were confirmed using HCC mouse models (e.g., Mdr2-/- animals) and a human HCC tissue micro-arrays (immunohistochemistry and western blotting).

Results:

Only very few genomic gains were found for all FBP family members in human HCC samples (e.g., 6% for fubp1). In vitro, inhibition of EGFR (by AG1478) or administration of the multi-kinase inhibitor sorafenib reduced FBP protein levels. Furthermore, inhibition of the PI3K/Akt/mTOR pathway revealed strong effects on FBP protein half-live without significant impact on FBP transcription. Other pathways or stimuli only caused minor effects on FBP expression (e.g., p38 or Raf1 inhibition). These findings were confirmed in different mouse HCC models with high-level expression of FBPs. In these animals administration of sorafenib or rapamycin significantly diminished FBP amounts. Reduction of FBP concentrations after inhibition of the PI3K/Akt/mTOR pathway was rescued after inhibition of caspase-3/-7 activity. Lastly, a significant correlation between pAkt and nuclear FBP expression was detected in human HCC tissues.

Conclusion:

Our data demonstrate that activation of the PI3K/Akt/mTOR signalling axis is responsible for increased stabilisation of FBPs in human HCC cells. Elevated FBP half-live is at least partly mediated through crosstalk between the PI3K/Akt/mTOR pathway and effector caspase activity. These data are important for a deeper understanding of how druggable upstream regulators may affect FBP activity in hepatocarcinogenesis.