Functional characterization, clinical relevance and therapeutic implication of follistatin-like 1 (FSTL1) in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) ranks the third most common cause of cancerrelated mortality in Hong Kong. Despite advances in diagnosis and treatment strategies, the prognosis of HCC remains dismal. Thus, novel therapeutic options are still urgently awaited. Over 80% of HCC cases are presented with a background of fibrosis and/or cirrhosis, a pathological state where chronic inflammation and hepatocellular regeneration in the presence of enriched stromal myofibroblasts are commonly observed. Throughout the past decade, much emphasis has been placed on the role of tumor microenvironment (TME) in cancer development. Numerous studies conducted have proved that cancer cells are regulated by signals generated from fibroblasts in the TME. Thus, it is logical to speculate that targeting signaling factors within the TME may shed light to novel therapeutic options. Follistatin-like 1 (FSTL1) has been widely reported as a pro-inflammatory mediator in different fibrosis-related and inflammatory diseases. Here, we found FSTL1 to be up-regulated during liver regeneration and HCC development in our partial hepatectomy and HCC mice models. Moreover, we found FSTL1 to either be absent or expressed at very low levels in human HCC cells, but preferentially secreted from the myofibroblasts of peri-tumor liver and HCC tissue. By immunofluorescence and Person correlation analysis performed in human HCC clinical samples, fibrosis/HCC-related mouse models and expression data of an online HCC cohort (TCGA), we demonstrated FSTL1 to co-localize and positively correlate with α-SMA, a marker of myofibroblasts, confirming the source of FSTL1 as liver myofibroblasts including activated fibroblasts in both tumor and peri-tumor compartments. Clinically, high FSTL1 in FAP+ fibroblasts was significantly correlated with more advanced pathological TNM staging in HCC patients. As a secretory protein, it shows potential prognostic value to be adopted as a novel serological biomarker marker for HCC screening and/or diagnosis. Subsequent in vitro functional analyses found FSTL1 to induce HCC cell proliferation, metastasis and sorafenib resistance; while FSTL1 attenuation by a FSTL1 specific neutralizing antibody (nAb) elicited the opposite effects. Such oncogenic proliferative and metastatic effects were also observed in HCC patient-derived organoid cultures. Further, FSTL1 was also found to enhance HCC cell tumor formation and metastasis in in vivo models; while the administration of FSTL1 nAb exhibited opposing effects, thus suggesting a therapeutic efficacy of neutralizing FSTL1. RNA-sequencing was subsequently performed in hope to elucidate the dysregulated downstream molecular mechanism mediated by FSTL1. By Gene Set Enrichment Analysis (GSEA), we found the myc pathway to be activated in FSTL1-treated HCC cells. Western Blot analyses subsequently demonstrated enhanced phosphorylation of two key upstream players of the myc pathway, namely TAK-1 and p38 MAPK, which further substantiated the GSEA results. To conclude, myofibroblast-secreted FSTL1 mediates HCC progression and metastasis via a deregulated TAK-1/ p38 MAPK/ c-myc signaling cascade and anti-FSTL1 nAb holds therapeutic potential for HCC patients. We believe that FSTL1 could potentially serve as both a novel diagnostic/prognostic biomarker and therapeutic target in HCC.