Abstract
Simple SummaryPatients with advanced intrahepatic cholangiocarcinoma (iCCA) have a very poor prognosis, and no targeted therapy is approved for advanced iCCA. A therapeutic strategy for wild-type p53 cancers is the reactivation of p53 by inhibition of its the negative regulators, MDM2, and WIP1. In the present study, we used HDM201 (an MDM2-p53 binding antagonist) to increase p53 stabilization and upregulate the expression of downstream targets (p21 and MDM2) in RBE and SK-Hep-1 liver adenocarcinoma cell lines. The survival rate and clonogenicity decreased after HDM201 treatment in a dose-dependent manner. Combined treatment with HDM201 and GSK2830371 (WIP1 inhibitor) increased p53 phosphorylation, leading to sustained p53 activation. This combination treatment resulted in G2/M phase arrest and promoted cytotoxicity compared with MDM2 inhibitor monotherapy. Furthermore, increased expression of p53 signaling pathway target genes were identified following combination treatment with HDM201 and GSK2830371, suggesting potential roles for this combination strategy in iCCA therapy.Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA.
Highlights
Cholangiocarcinoma (CCA) consisting of intrahepatic cholangiocarcinomaand extrahepatic cholangiocarcinoma is a relatively rare but aggressive biliary tract cancer
To determine the growth inhibitory activity of GSK2830371 in liver adenocarcinoma cell lines, RBE and SK-Hep-1 cells were treated with GSK2830371 alone at different concentrations up to a maximal concentration of 10 μM, and cell proliferation was measured by CCK-8 assay
Minimal growth inhibitory activity was observed with 0–10 μM GSK2830371 (WIP1 inhibitor) in 0.5% DMSO for 72 and 96 h
Summary
Cholangiocarcinoma (CCA) consisting of intrahepatic cholangiocarcinoma (iCCA)and extrahepatic cholangiocarcinoma (eCCA) is a relatively rare but aggressive biliary tract cancer. ICCA is the second most common primary liver cancer. It has aggressive biological behavior and is typically diagnosed at an advanced stage with poor prognosis [5]. Pemigatinib (an FGFR2 inhibitor) demonstrated activity in previously treated advanced cholangiocarcinoma based on a phase 2 trial and was approved by the US FDA in 2020 [10], but only 10–15% of patients with iCCA have the FGFR2 fusion gene [11]. Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination
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