Lipocalin 2 is a Yap‐beta‐catenin target and a biomarker of disease burden in a murine model of hepatoblastoma

Hepatoblastoma is the most common type of pediatric liver malignancy. Despite advances in therapy, metastatic disease is still associated with a poor prognosis. Aberrations in the Wnt/b‐catenin signaling pathway due to mutations in CTNNB1 are the most prevalent mutations findings in these tumors. Recently mutations in the nuclear translocation of the Yes‐Associated‐Protein Pathway (YAP) has also been reported in hepatoblastomas. In fact b‐catenin‐Yap co‐activation was observed in 80% of hepatoblastoma patients. Hydrodynamic tail vein injection of active upregulated‐beta‐catenin and active‐YAP plasmids in mice also led to hepatoblastoma tumors. Gene array analysis using mRNA isolated from these mice tumors from tumor‐bearing livers had identified lipocalin 2 as one of the upregulated genes, with increased binding sites to both TCF4 and TEAD, which are transcription factors for beta‐catenin and YAP respectively. Lipocalin 2 is a small molecule protein being increasingly studied in the field of cancer. Previously we had shown that indeed human hepatoblastoma tumors showed notable lipocalin‐2 (LCN2) expression by immunohistochemistry. To address if the increased LCN2 expression observed in humans correlated with the mouse model of hepatoblastoma, liver lysates from wild type livers and mouse hepatoblastoma tumors were analyzed. The tumor‐bearing livers were found to have a 40‐fold increased lipocalin 2 expression by real time PCR. We next used ELISA to quantify the amount of serum lipocalin 2 in these animals. We saw increased values of Lipocalin 2 serum levels in mice with tumors compared to the wild type. In fact, in fact we found that serum lipocalin 2 levels was an indicator of the tumor burden in these mice. The liver weight to body weight ratio of the mice had a direct positive correlation with serum lipocalin 2. To address that lipocalin 2 was regulated by Wnt and Yap signaling, we preformed western blot analysis on human hepatoblastoma HepG2 cells, that had been transfected with beta‐catenin or Yap silencing RNA. Protein levels of lipocalin 2 were decreased in following beta‐catenin knockdown of both, although it was more pronounced upon beta‐catenin suppression of cells. Additionally, western blot preformed on liver lysates from hepatocyte specific beta‐catenin knock out mice, also showed decreased levels of lipocalin 2. In conclusion, we have shown lipocalin 2 to be regulated by beta‐catenin and Yap signaling in hepatoblastoma and may be useful as a biomarker of the disease burden in a hepatoblastoma mouse model. There is an implication that this may be a potential serum marker in human patients as well. The fluctuations of lipocalin 2 levels in the serum may be a direct result of alterations in beta‐catenin signaling and could be participating to oncogenesis.