Sublethal heat stress-induced HSF4 upregulation via GRSF1 in an m6A-dependent manner in hepatocellular carcinoma

Sublethal heat stress exacerbates hepatocellular carcinoma (HCC) by promoting recurrence and metastasis. This study aimed to investigate the molecular mechanisms by which sublethal heat stress enhances HCC progression to improve therapeutic strategies and patient outcomes in HCC management.

Cell-based and mouse models of insufficient thermal ablation in HCC were established via sublethal heat stress treatment. Twenty-four BALB/c nude mice were randomly divided into four groups, with six mice per group. RNA sequencing was used for transcriptional profiling. Messenger RNA (mRNA) and protein levels were quantified using quantitative reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry. Metastatic lesions in mouse liver and lung tissues were identified by hematoxylin and eosin staining. Proliferative, migratory, and invasive characteristics of tumor cells were probed via colony formation, 5-ethynyl-2'-deoxyuridine incorporation, scratch wound healing, and Transwell assays. Interactions between related genes were determined using dual-luciferase reporter, chromatin immunoprecipitation, co-immunoprecipitation, and RNA immunoprecipitation assays. Methylated RNA immunoprecipitation was used to detect N6-methyladenosine (m6A) levels in heat shock transcription factor 4 (HSF4).

HSF4 was identified as a key gene that was upregulated in sublethal heat stress-treated cells (6.20-fold increase in Huh7 cells and 8.34-fold increase in Hep3B cells; P <0.001). HSF4 knockdown inhibited sublethal heat stress-induced cell proliferation by 61.09% in Huh7 cells and 47.28% in Hep3B cells (P <0.001 and P <0.01), migration by 31.64% in Huh7 cells and 17.01% in Hep3B cells (P <0.05), invasion by 29.68% in Huh7 cells and 47.03% in Hep3B cells (P <0.05 and P <0.001), and epithelial-mesenchymal transition (EMT). Furthermore, HSF4 knockdown repressed liver (reduced liver weight-to-body weight ratio by 32.46%, P <0.001) and lung (decreased metastatic nodules from 10.5 to 5.17 per section, P <0.001) metastases in vivo. Mechanistically, HSF4 increased cell cycle and apoptosis regulator 1 (CCAR1) levels and activated β-catenin pathway by binding to CCAR1 promoter. CCAR1 overexpression counteracted the suppressive effects of HSF4 knockdown on cell proliferation, migration, invasion, and EMT following sublethal heat stress. In addition, sublethal heat stress increased the HSF4 levels by upregulating guanine-rich RNA sequence binding factor 1 (GRSF1) (2.92-fold in Huh7 cells and 2.87-fold in Hep3B cells, P <0.001) in an insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2)-m6A-dependent manner. HSF4 overexpression blocked the inhibitory effects of GRSF1 knockdown on HCC cell proliferation and metastasis following sublethal heat stress.

Sublethal heat stress increased HSF4 expression by upregulating GRSF1 through IGF2BP2-mediated m6A modification, thereby increasing CCAR1 levels and activating β-catenin pathway, ultimately facilitating HCC proliferation and metastasis.