Elevated PDK1 Expression Drives PI3K/AKT/MTOR Signaling Promotes Radiation-Resistant and Dedifferentiated Phenotype of Hepatocellular Carcinoma.

Oluwaseun Adebayo Bamodu,Hang-Lung Chang,Chi-Tai Yeh,Jiann-Ruey Ong,Jo-Ting Tsai,Wei-Hwa Lee

doi:10.3390/cells9030746

Abstract

Resistance to radiotherapy (IR), with consequent disease recurrence, continues to limit the efficacy of contemporary anticancer treatment for patients with hepatocellular carcinoma (HCC), especially in late stage. Despite accruing evidence implicating the PI3K/AKT signaling pathway in cancer-promoting hypoxia, cancerous cell proliferation and radiotherapy-resistance, it remains unclear which molecular constituent of the pathway facilitates adaptation of aggressive HCC cells to tumoral stress signals and drives their evasion of repeated IR-toxicity. This present study investigated the role of PDK1 signaling in IR-resistance, enhanced DNA damage repair and post-IR relapse, characteristic of aggressive HCC cells, while exploring potential PDK1-targetability to improve radiosensitivity. The study employed bioinformatics analyses of gene expression profile and functional protein–protein interaction, generation of IR-resistant clones, flow cytometry-based ALDH activity and side-population (SP) characterization, siRNA-mediated loss-of-PDK1function, western-blotting, immunohistochemistry and functional assays including cell viability, migration, invasion, clonogenicity and tumorsphere formation assays. We showed that the aberrantly expressed PDK1 characterizes poorly differentiated HCC CVCL_7955, Mahlavu, SK-HEP1 and Hep3B cells, compared to the well-differentiated Huh7 or normal adult liver epithelial THLE-2 cells, and independently activates the PI3K/AKT/mTOR signaling. Molecular ablation of PDK1 function enhanced susceptibility of HCC cells to IR and was associated with deactivated PI3K/AKT/mTOR signaling. Additionally, PDK1-driven IR-resistance positively correlated with activated PI3K signaling, enhanced HCC cell motility and invasiveness, augmented EMT, upregulated stemness markers ALDH1A1, PROM1, SOX2, KLF4 and POU5F1, increased tumorsphere-formation efficiency and suppressed biomarkers of DNA damage—RAD50, MSH3, MLH3 and ERCC2. Furthermore, the acquired IR-resistant phenotype of Huh7 cells was strongly associated with significantly increased ALDH activity, SP-enrichment, and direct ALDH1-PDK1 interaction. Moreover, BX795-mediated pharmacological inhibition of PDK1 synergistically enhances the radiosensitivity of erstwhile resistant cells, increased Bax/Bcl-2 apoptotic ratio, while suppressing oncogenicity and clonogenicity. We provide preclinical evidence implicating PDK1 as an active driver of IR-resistance by activation of the PI3K/AKT/mTOR signaling, up-modulation of cancer stemness signaling and suppression of DNA damage, thus, projecting PDK1-targeting as a putative enhancer of radiosensitivity and a potential new therapeutic approach for patients with IR-resistant HCC.

Highlights

Following our in-house mining of high through-put gene expression data from public repositories, to unravel the role of the PI3K/AKT/mTOR signaling axis, and PDK1 signaling in hepatocellular carcinoma (HCC), we performed in silico proteotranscriptomic analyses of selected molecular components of the pathway in question
Results of our Western blot analyses showed that compared to its non-expression in the normal adult liver epithelial THLE-2 cells, the expression of p-PI3K p85 (Tyr458)/p55 (Tyr199), PI3K, p-PDK1 (Ser 241), PDK1, p-AKT (Ser 473), AKT, p-mTOR
We showed that while (iii) aberrant PDK1 expression is implicated in the acquisition of IR-resistance and evasion of DNA damage by HCC cells, (iv) PDK1-dependent IR-resistance is associated with the enhanced metastatic and cancer stem cell-like phenotypes of HCC cells and that the (v) pharmacologic targeting of PDK1 resensitizes HCC cells to radiotherapy-induced apoptosis signals dose-dependently and significantly suppresses their oncogenicity

Summary

Introduction

Hepatocellular carcinoma, with an increasing annual incidence and arising mostly (90%) in the context of chronic liver disease, such as underlying liver cirrhosis and chronic hepatitis B or C, accounts for a significant 75% of all liver cancer incidence and is associated with very poor survival rates, especially as patients present in late stage, with comorbidities, micro- and/or macrovascular invasion, multicentric or multifocal large tumors, organ shortage and are inoperable [2,3,4]. The preferred curative modalities for patients with HCC are surgical resection and orthotopic liver transplantation (OLT), for patient not meeting the criteria for curative therapy, such as those with unresectable tumors, treatment options include systemic chemotherapy, molecularly targeted therapies, transarterial chemoembolization (TACE), percutaneous ethanol injection (PEI), cryoablation and various forms of IR, namely, microwave ablation (MWA), radiofrequency ablation (RFA), radioembolization, stereotactic body radiotherapy (SBRT) and external beam radiation therapy (EBRT) [4,5,6]; these are fraught with enhanced risk of severe drug-related adverse events (AEs), acquired resistance to anticancer therapeutics and IR, and relatively dismal survival benefits [5,6], necessitating concerted screening for or development of novel highly efficacious therapeutics and/or the discovery of new actionable molecular oncotargets, which inhibit disease progression, alleviate resistance to treatment and improve prognosis in patients with pancreatic ductal adenocarcinoma (PDAC)

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