Autophagy-Related Chemoprotection against Sorafenib in Human Hepatocarcinoma: Role of FOXO3 Upregulation and Modulation by Regorafenib

Abstract

Early acquisition of sorafenib resistance is responsible for the dismal prognosis of advanced hepatocarcinoma (HCC). Autophagy, a catabolic process involved in liver homeostasis, has been associated with chemosensitivity modulation. Forkhead box O3 (FOXO3) is a transcription factor linked to HCC pathogenesis whose role on autophagy-related sorafenib resistance remains controversial. Here, we unraveled the linkage between autophagy and sorafenib resistance in HCC, focusing on the implication of FOXO3 and its potential modulation by regorafenib. We worked with two HepG2-derived sorafenib-resistant HCC in vitro models (HepG2S1 and HepG2S3) and checked HCC patient data from the UALCAN database. Resistant cells displayed an enhanced basal autophagic flux compared to HepG2, showing higher autophagolysosome content and autophagy markers levels. Pharmacological inhibition of autophagy boosted HepG2S1 and HepG2S3 apoptosis and subG1 cells, but reduced viability, indicating the cytoprotective role of autophagy. HCC samples displayed higher FOXO3 levels, being associated with shorter survival and autophagic genes expression. Consistently, chemoresistant in vitro models showed significant FOXO3 upregulation. FOXO3 knockdown suppressed autophagy and caused resistant cell death, demonstrating that overactivation of such pro-survival autophagy during sorafenib resistance is FOXO3-dependent; a cytoprotective mechanism that the second-line drug regorafenib successfully abolished. Therefore, targeting FOXO3-mediated autophagy could significantly improve the clinical efficacy of sorafenib.

Effects of fasting on FOXO3 expression as an anti-aging biomarker in the liver

“…In normal conditions, they maintain the satellite cell pool so that it could actively replicate while remaining in its undifferentiated state [ 23 ]. However, in pathological conditions, overexpression of FOXO3 is associated with the development and progression of several diseases, such as Alzheimer's disease [ 24 ] and cancer [ [25] , [26] , [27] ]. The pathogenesis of Alzheimer's disease involves activation of the FOXO signaling pathway, which induces neuronal cell death [ 24 ].…”

“…The pathogenesis of Alzheimer's disease involves activation of the FOXO signaling pathway, which induces neuronal cell death [ 24 ]. Moreover, activation of FOXO3 stimulates chemotherapy drug resistance in glioblastoma multiforme [ 25 ] and hepatocellular carcinoma [ 26 , 27 ]. Therefore, the protective effect of FOXO3 mainly plays a role in normal cells.…”

Hepatocellular carcinoma cells loss lenvatinib efficacy in vitro through autophagy and hypoxia response-derived neuropilin-1 degradation

“…8 ). Previous research has described a context-dependent role of autophagy in drug response in HCC, either enhancing lenvatinib efficacy [ 40 ], or promoting sorafenib failure [ 25 ]. Altogether, liver tumor cells could modulate autophagy as part of a cellular response to decrease therapeutic efficacy.…”

“…After the corresponding treatments, cells were washed with cold PBS and scraped in a homogenization buffer, following our previous protocol [ 11 , 25 ]. We employed the primary antibodies HIF-1α (rabbit polyclonal IgG, 1:500, ab2185, Abcam), NRP1 (rabbit monoclonal IgG, 1:1000, ab81321, Abcam), sequestosome-1 (p62/SQSTM1) (rabbit polyclonal, 1:1000, #5114, Cell Signaling) and microtubule-associated proteins 1 A/1B light chain 3B (LC3-II) (rabbit monoclonal IgG, 1:1000, #12741, Cell Signaling), as well as the β-actin antibody (A3854, Sigma-Aldrich) as loading control.…”

“…Immunofluorescence and laser confocal microscopy Immunocytochemistry was conducted as previously described [25]. For overnight incubation at 4 °C we employed primary antibodies against Ki67 (1:200, sc-23900, Santa Cruz Biotechnology, Dallas, TX, USA) or NRP1 (1:250, ab81321, Abcam, Cambridge, UK).…”