Background: Cancer‐associated fibroblasts (CAFs) play an important role in the induction of chemo‐resistance. This study aimed to clarify the mechanism underlying CAF‐mediated resistance to two tyrosine kinase inhibitors (TKIs), sorafenib and lenvatinib, and to identify a novel therapeutic target for overcoming TKI resistance in hepatocellular carcinoma (HCC). Methods: We performed a systematic integrative analysis of publicly available gene expression datasets and whole‐transcriptome sequencing data from 9 pairs of CAFs and para‐cancer fibroblasts isolated from human HCC and para‐tumor tissues, respectively, to identify key molecules that might induce resistance to TKIs. We then performed in vitro and in vivo experiments to validate selected targets and related mechanisms. The associations of plasma secreted phosphoprotein 1 (SPP1) expression levels before sorafenib/lenvatinib treatment with progression‐free survival (PFS) and overall survival (OS) of 54 patients with advanced HCC were evaluated using Kaplan‐Meier and Cox regression analysis. Results: Bioinformatic analysis identified CAF‐derived SPP1 as a candidate molecule driving TKI resistance. SPP1 inhibitors reversed CAF‐induced TKI resistance in vitro and in vivo. CAF‐derived SPP1 activated rapidly accelerated fibrosarcoma (RAF)/mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) through the integrin‐protein kinase C‐alpha (PKCα) signaling pathway and promoted epithelial‐to‐mesenchymal transition (EMT). A high plasma SPP1 level before TKI treatment was identified as an independent predictor of poor PFS ( P = 0.026) and OS ( P = 0.047) in patients with advanced HCC after TKI treatment. Conclusions: CAF‐derived SPP1 enhances TKI resistance in HCC via bypass activation of oncogenic signals and EMT promotion. Its inhibition represents a promising therapeutic strategy against TKI resistance in HCC. Moreover, plasma SPP1 level before TKI treatment represents a potential biomarker for treatment response prediction.

This study investigated the diagnostic potential of serum small extracellular vesicle‐derived long noncoding RNAs (EV‐lncRNAs) for hepatocellular carcinoma (HCC). Driver oncogenic lncRNA candidates were selected by a comparative analysis of lncRNA expression profiles from two whole transcriptome human HCC datasets (Catholic_LIHC and TCGA_LIHC). Expression of selected lncRNAs in serum and small EVs was evaluated using quantitative reverse transcription PCR. Diagnostic power of serum EV‐lncRNAs for HCC was determined in the test (n = 44) and validation (n = 139) cohorts. Of the six promising driver onco‐lncRNAs, DLEU2, HOTTIP, MALAT1, and SNHG1 exhibited favorable performance in the test cohort. In the validation cohort, serum EV‐MALAT1 displayed excellent discriminant ability, while EV‐DLEU2, EV‐HOTTIP, and EV‐SNHG1 showed good discriminant ability between HCC and non‐HCC. Furthermore, a panel combining EV‐MALAT1 and EV‐SNHG1 achieved the best area under the curve (AUC; 0.899, 95% CI = 0.816–0.982) for very early HCC, whereas a panel with EV‐DLEU2 and alpha‐fetoprotein exhibited the best positivity (96%) in very early HCC. Serum small EV‐MALAT1, EV‐DLEU2, EV‐HOTTIP, and EV‐SNHG1 may represent promising diagnostic markers for very early‐stage HCC.

Early detection of cancer provides effective treatment and saves lives. The objective of this study was to determine whether serum gastrokine 1 (GKN1) protein is a gastric cancer‐specific diagnostic biomarker. The serum concentration of GKN1 in healthy individuals (median: 6.34 ng/μL, interquartile range (IQR): 5.66‐7.54 ng/μL) was significantly higher compared with the levels in gastric cancer patients (median: 3.48 ng/μL, IQR: 2.90‐4.11 ng/μL; P < .0001). At the optimum cutoff (4.94 ng/μL) of serum GKN1 protein, the sensitivity and specificity were 91.2% and 96.0%, respectively, for gastric cancer. Using serum GKN1 protein as the diagnostic reference, the ROC curve showed a satisfactory diagnostic efficacy with an AUC value of 0.9954 (95% CI 0.9919‐0.9988) and Youden index of 0.8740. In addition, the diagnostic accuracy of the serum GKN1 protein at the optimum cutoff was 0.9675. Interestingly, serum GKN1 concentrations in patients with advanced gastric cancer (AGC; median: 3.11 ng/μL, IQR: 2.72‐3.72 ng/μL) were lower than in patients with early gastric cancer (EGC; median: 4.31 ng/μL, IQR: 3.88‐4.88 ng/μL). The diagnostic accuracies at the optimum serum GKN1 cutoff were 0.8912 and 0.9589 for EGC and AGC, respectively. Furthermore, the serum GKN1 concentrations robustly discriminated the patients with gastric cancer from the patients with colorectal, liver, lung, breast, pancreatic, ovary, and prostatic cancers with AUC values greater than 0.94. These data suggest that serum GKN1 is a promising and highly specific diagnostic biomarker for the prompt detection of early and advanced gastric cancers.

Cancer-associated fibroblasts (CAFs) contribute to tumor progression, and microRNAs (miRs) play an important role in regulating the tumor-promoting properties of CAFs. The objectives of this study were to clarify the specific miR expression profile in CAFs of hepatocellular carcinoma (HCC) and identify its target gene signatures. Small-RNA-sequencing data were generated from nine pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively. Bioinformatic analyses were performed to identify the HCC-CAF-specific miR expression profile and the target gene signatures of the deregulated miRs in CAFs. Clinical and immunological implications of the target gene signatures were evaluated in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA_LIHC) database using Cox regression and TIMER analysis. The expressions of hsa-miR-101-3p and hsa-miR-490-3p were significantly downregulated in HCC-CAFs. Their expression in HCC tissue gradually decreased as HCC stage progressed in the clinical staging analysis. Bioinformatic network analysis using miRWalks, miRDB, and miRTarBase databases pointed to TGFBR1 as a common target gene of hsa-miR-101-3p and hsa-miR-490-3p. TGFBR1 expression was negatively correlated with miR-101-3p and miR-490-3p expression in HCC tissues and was also decreased by ectopic miR-101-3p and miR-490-3p expression. HCC patients with TGFBR1 overexpression and downregulated hsa-miR-101-3p and hsa-miR-490-3p demonstrated a significantly poorer prognosis in TCGA_LIHC. TGFBR1 expression was positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages in a TIMER analysis. In conclusion, hsa-miR-101-3p and hsa-miR-490-3p were substantially downregulated miRs in CAFs of HCC, and their common target gene was TGFBR1. The downregulation of hsa-miR-101-3p and hsa-miR-490-3p, as well as high TGFBR1 expression, was associated with poor clinical outcome in HCC patients. In addition, TGFBR1 expression was correlated with the infiltration of immunosuppressive immune cells.