A cigarette compound-induced tumor microenvironment promotes sorafenib resistance in hepatocellular carcinoma via the 14-3-3η-modified tumor-associated proteome.

[BACKGROUND] Sorafenib resistance is the bottleneck in the clinical treatment of advanced hepatocellular carcinoma (HCC). Cigarette compounds accelerate HCC progression and chemotherapy resistance by forming a chemical microenvironment. Here, we aimed to further reveal the mechanisms by which the microenvironment formed by cigarette exposure contributes to sorafenib resistance.

[METHODS] HCC cell lines and xenografted nude mice were used as experimental models. A cohort of 119 patients enrolled from the First Affiliated Hospital of Huzhou University (between Mar 2019 to Jan 2024) and the Second Affiliated Hospital of Nanjing Medical University (between Jan 2018 to Jan 2024) with advanced HCC who had recurrent disease and were receiving sorafenib treatment was analyzed. The public data of the expression of genes and survival of HCC patients were all obtained from The Cancer Genome Atlas (TCGA) database, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted by Database for Annotation, Visualization and Integrated Discovery (DAVID). The phospho/angiogenesis antibody arrays were used to identify the key phosphorylated proteins/cytokines that have been differentially modified. Quantitative real-time polymerase chain reaction (qPCR), Western blotting, immunofluorescence and immunohistochemistry (IHC) were used to elucidate the role of key factors in regulating sorafenib resistance. Flow cytometry, TdT-mediated dUTP nick end labeling, caspase activity, enzyme-linked immunosorbent assay, and capillary tube formation assay were employed to investigate related biological functions.

[RESULTS] Chronic cigarette smoking exposure continuously activated 14-3-3η via an epigenetic accumulation of DNA demethylation. This "switch-like" factor, 14-3-3η, upregulated its key downstream pathway, B-Raf/extracellular regulated protein kinases (ERK). 14-3-3η and its regulated B-Raf/ERK protein work together, and then blocked the caspase cascade, enhancing the pro-survival ability of HCC cells. 14-3-3η/B-Raf/ERK activated nuclear factor kappa-B (NF-κB), which in turn transcriptionally upregulated multidrug resistance protein 1 (MDR1) and adenosine triphosphate (ATP) binding cassette transporter G2 (ABCG2), accelerating drug efflux. NF-κB also transcriptionally regulated vascular endothelial growth factor (VEGF) and granulocyte colony stimulating factor (G-CSF), inducing the neo-vascularization. These critical processes together resisted the HCC to sorafenib. Furthermore, these effects were blocked and sorafenib resistance was reversed when we targeted 14-3-3η with its novel chemical inhibitor, arsenic trioxide.

[CONCLUSIONS] A cigarette compound-formed tumor microenvironment resisted HCC to sorafenib by locking the adaptor protein 14-3-3η in a constitutively active state. This outcome provided a mechanistic rationale and a translational strategy to re-sensitize HCC patients exposed to cigarette to targeted therapy.