Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin.

Amber M D’Souza,Ruhi Gulati,Maria Rivas,James Geller,Meenasri Kumbaji,David Smithrud,Michael Johnston,Ashley Cast,Nikolai Timchenko

doi:10.3389/fphar.2021.580722

Amber M D’Souza, Ruhi Gulati + Show 7 more

Open Access

https://doi.org/10.3389/fphar.2021.580722

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Abstract

Objective: Relapsed hepatoblastoma (HBL) and upfront hepatocellular carcinoma (HCC) are notoriously chemoresistant tumors associated with poor outcomes. Gankyrin (Gank) is a known oncogene that is overexpressed in pediatric liver cancer and implicated in chemo-resistance. The goal of this study was to evaluate if the Gank-tumor suppressor axis is activated in chemoresistant hepatoblastoma patients and examine if an inhibitor of Gank, Cjoc42, might improve the chemosensitivity of cancer cells. Methods: Expression of Gank and its downstream targets were examined in fresh human HBL samples using immunostaining, QRT-PCR, and Western Blot. Cancer cells, Huh6 (human HBL) and Hepa1c1c7 (mouse HCC) were treated with Cjoc42 and with Cjoc42 in combination with cisplatin or doxorubicin. Cell proliferation, apoptosis, and chemoresistance were examined. To examine activities of Cjoc42 in vivo, mice were treated with different doses of Cjoc42, and biological activities of Gank and cytotoxicity of Cjoc42 were tested. Results: Elevation of Gank and Gank-mediated elimination of TSPs are observed in patients with minimal necrosis after chemotherapy and relapsed disease. The treatment of Huh6 and Hepa1c1c7 with Cjoc42 was not cytotoxic; however, in combination with cisplatin or doxorubicin, Cjoc42 caused a significant increase in cytotoxicity compared to chemotherapy alone with increased apoptosis. Examination of Cjoc42 in WT mice showed that Cjoc42 is well tolerated without systemic toxicity, and levels of tumor suppressors CUGBP1, Rb, p53, C/EBPα, and HNF4α are increased by blocking their Gank-dependent degradation. Conclusions: Our work shows that Cjoc42 might be a promising adjunct to chemotherapy for the treatment of severe pediatric liver cancer and presents mechanisms by which Cjoc42 increases chemo-sensitivity.

Highlights

Hepatoblastoma (HBL) is the most common primary pediatric hepatic malignancy (Aronson and Meyer, 2016)
Elevation of Gankyrin is observed in hepatoblastoma samples with advanced disease Our previous studies with a repository of frozen hepatoblastoma samples (51 samples) showed that Gank was 3-5-fold elevated in the majority of HBL samples
Since Gank triggers the degradation of tumor suppressor proteins in patients with HBL (Figure 1B), we examined if this activity of Gank is inhibited in Huh6 cells treated by doxorubicin and cjoc42

Summary

Introduction

Hepatoblastoma (HBL) is the most common primary pediatric hepatic malignancy (Aronson and Meyer, 2016). Intensive investigations of Gank have identified multiple pathways by which this oncogene might promote liver cancer (Timchenko and Lewis, 2015). These pathways include the elimination of tumor suppressors (Wang et al, 2010; D’Souza et al, 2018; Valanejad et al, 2018; Higashitsuji et al, 2000; Dawson, 2008) activation of beta-catenin/WNT signaling (He et al, 2016; Liu et al, 2019), and activation of stem-cell markers and several key liver cancer-related genes such as OCT4 and c-myc (Qian et al, 2012; Valanejad et al, 2017; Liu et al, 2019). Gank degrades TSPs in different liver diseases leading to increased liver proliferation and development of liver cancer, NAFLD, and fibrosis (Lewis et al, 2017; Nguyen et al, 2018; Cast et al, 2019)

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