Der Transkriptionsfaktor CUX1 beeinflusst epigenetisch induzierte zelluläre Stressreaktionen im hepatozellulären Karzinom

Hepatocellular carcinoma is the second most worldwide cancer-related death. Due to the absence of symptoms in the early stage of the disease and the late diagnosis, a curative treatment seems to be mostly ineffetctive. The multi-kinase-inhibitors Sorafenib (Nexavar) and Lenvatinib (Lenvima) are the only evidence-based drugs with a first-line use approved by the European Medicines Ageny (EMA) for systemic treatment of advanced local or even metastatic stage of hepatocellular carcinoma within the Child-Pugh-Score A. Despite many attempts of the current investigations to identify molecular-targeting drugs beside sorafenib, the results have been unsatisfying so far. Hopefully, the induction of epigenetic modifications could represent a promising approach for the development of a new systemic therapy. The application of deacetylase inhibitors (DACis) results in an increase of endoplasmatic-reticulum stress and autophagic process, which trigger liver cancer cell death. The study here proposed focused on the role exerted by the highly evolutionary conserved transcription factor CUX1 in DACi-mediated endoplasmic reticulum stress/autophagic mechanisms in HCC cells. CUX1 is responsible for promoting cell motility, invasion and cell cycle progression as well as for causing cell death resistance. Clinical studies associate a high level of CUX1 transcript in several cancers. The aim of this study was to elucidate the oncogenic and/or tumor suppressive potential of CUX1 in HCC cells. CUX1 transient knockdown was performed in two human HCC-cell lines Hep3B (p53-deficient) and HepG2 (p53-wildtype). After a 6 hour treatment (RT-qPCR) and 24 hour treatment (RT-qPCR and Western Blot) with Thapsigargin and the DACis Trichostatin A (TSA), SAHA and Panobinostat the expression of autophagic markers was detected by RT-qPCR and Western Blot. Fluorescent microscopy was performed to visualize the autophagosome maturation after autophagy induction via Panobinostat-treatment in the absence of CUX1. The effect of transient CUX1-knockdown on cell proliferation was detected by real time cell viability assays. CUX1 transient knockdown reduced the efficacy of short-term (6h) administration of especially thapsigargin and panobinostat in promoting the expression of ER-stress-markers. These findings could reveal oncogenic potential of CUX1-regulation regarding ER-Stress-mediated drug resistance. The real time cell viability assay pointed out an altered cell proliferation with the most significant effect in the absence of CUX1 and the administration of panobinostat in Hep3B cells. Regarding a possible resistance to apoptosis, CUX1 exerts an oncogenic function. Decreased mRNA-expression of autophagic markers after CUX1-Knockdown suggests impaired autophagic mechanism, depending on the time of administration with the DACis and the p53-levels of the examined cell lines. The measurement of increased Beclin1 and MAP1LC3B protein levels in the Western Blot analysis contributed to a reduced efficacy of the compounds to perform autophagy after CUX1-silencing. Autophagy, as a catabolic process, leads to a massive protein degradation. An increase of the protein levels within a reduction of the mRNA expression of autophagic markers therefore supports an impairment of autophagy in the absence of CUX1. A reduced maturation of the autophagosome vesicles (Hep3B-cells) as well as impaired autophagosome-lysosome-fusion (HepG2-cells) was observed under fluorescence microscopy in the absence of CUX1 after the administration of panobinostat. These findings suggest a tumor suppressive potential of CUX1 on HCC cells performing autophagic cell death after DACi treatment. Regarding to current studies implicating the contribution of CUX1 to resistance towards apoptosis highlights a possible interaction between autophagic-related cell death and apoptosis. CUX1 could either inhibit, promote or necessitate apoptosis and autophagy. While showing its pro-tumorigenic potential in HCC, CUX1 simultaneously promotes autophagy-related cell death and therefore exerts anti tumorigenic role too. Clinical studies analysing the effect of a combined treatment of sorafenib and panobinostat in metastatic stage of HCC and might provide a base for further clinical investigations regarding the influence of CUX1.