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

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 Cells 2020, 9, 746 2 of 19 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.

SOX2 and p53 Expression Control Converges in PI3K/AKT Signaling with Versatile Implications for Stemness and Cancer

“…This involves, for example, IR-resistant forms of hepatocellular carcinoma. In this indication, the appearance of poorly differentiated, stem cell like features coincides with enforced AKT signaling, a joint induction of SOX2 and p53 mRNAs, and enhanced repair mechanisms to resolve IR-imposed DNA lesions [ 149 ]. Taken together, the interplay of PI3K/AKT and p53 in cancer presents highly divergent.…”

Integrative Bioinformatics Study Reveals Tangeretin Targets and Molecular Mechanisms Against Metastatic Breast Cancer

“…PI3K/Akt and mTOR signaling pathways are essential for maintaining the proliferation and survival of cancer cells (54). A recent study has shown that activation of PI3K/AKT/mTOR signaling increases hepatocellular carcinoma resistance to radiotherapy (55). On the one hand, activation of the PI3K/Akt pathway leads to the transcriptional activity of NFκB (56).…”

Hypoxia-Induced Cancer Cell Responses Driving Radioresistance of Hypoxic Tumors: Approaches to Targeting and Radiosensitizing

“…In addition to its important role in the Warburg effect, HK2 can directly interact with HIF-1α, and this mortalin-mediated interaction, occurring at the outer membranes of mitochondria, modulates the voltage-dependent anion-selective channel 1 (VDAC1) activity to preserve hypoxia-stressed cancer cells from apoptosis [ 40 ]. As a result of hypoxia-induced energy metabolism reprogramming, the augmented PDK1 expression in hypoxic tumors can drive the PI3K/Akt/mTOR signaling that promotes the EMT and formation of the radioresistant CSC-like phenotype, as it was shown for PDK1-overexpressing hepatocellular carcinoma [ 120 ]. Another glycolytic enzyme, fructose-bisphosphate aldolase A, was shown to be induced by hypoxia in colorectal cancer cells and correlated to their chemo- and radioresistance and, also, to a poor prognosis [ 121 ].…”