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

Abstract

Esophageal squamous cell carcinoma (ESCC)ranks the 8thmost common cause of cancer mortality in Hong Kong and its overall survival rate remains dismal. In order to identify better molecular markers for early detection and therapeutic targeting, our lab has previously performed transcriptome sequencing on three pairs of ESCC samples and their non-tumor counterpart and successfully identified significantly differentially expressed genes at a global level, including overexpression of follistatin-like 1 (FSTL1). This observation was further validated in a larger cohort of samples by real-time qPCR, immunohistochemistry and ELISA analyses where mRNA and proteomic (endogenous / secretory) FSTL1 were found to be frequently overexpressed in ESCC. Clinically, upregulation of FSTL1 closely correlated with poor overall survival rate (p=0.0021). FSTL1 showed a good diagnostic potential as analyzed by receiver operating characteristic (ROC). Serum FSTL1 level of 70.45ng/ml showed70.5% sensitivity and 72.2% specificity towards ESCC diagnosis. Moreover, since FSTL1 is mapped on the hotspot of chromosomal amplification in ESCC, we performed fluorescence in situ hybridization (FISH) on ESCC cell lines and clinical samples. Result revealed that FSTL1 gene and/or chromosome 3q amplification were frequently found in ESCC. More importantly, FSTL1 copy number was positively correlated with higher proteomic FSTL1 expression (p<0.0001), suggesting that FSTL1 gene and/or chromosome 3q amplification may critically contribute to the preferential upregulation of FSTL1 in ESCC. Functional role of FSTL1 was further examined in vitro and in vivo using both lentiviral-based overexpression and shRNA knockdown approaches. Results demonstrated that FSTL1 can significantly promote proliferation, clonogenicity, migration, invasion, self-renewal and cisplatin resistance of ESCC cells in vitro as well as tumorigenicity and distant metastasis in vivo. Likewise, co-culture of FSTL1-containing conditioned medium or recombinant FSTL1 in low FSTL1-expressing ESCC cells enhanced the cells’ ability to proliferate, migrate and invade in vitro as well as form tumor in vivo, demonstrating the prominence of both endogenous and secretory FSTL1 in mediating oncogenic activities in ESCC. Importantly, administration of FSTL1 neutralizing antibody resulted in significant reduction in ESCC motility and invasiveness in vitro. Mechanism by which FSTL1 drives ESCC aggressiveness was subsequently elucidated. Agilent microarray profiling was performed in hope to identify deregulated gene expression in ESCC cells with or without FSTL1 expression modulated. By Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), we found NFκB pathway to be activated in FSTL1-overexpressing ESCC cells. This observation was further substantiated by Western blotting analysis where FSTL1-overexpresing ESCC cells showed enhanced phosphorylation of two key players of the canonical NFκB pathway, namely IκBα and p65. Further, FSTL1 is known for its antagonistic relationship with BMP pathway. Consistently, BMP pathway was also found to be silenced in FSTL1-overexpresingESCC cells, as evidenced by suppressed SMAD1 phosphorylation. We believe BMP and NFκB pathways may crosstalk to drive aggressive cancer features in FSTL1-mediated ESCC. Taken together, both endogenous and secretory FSTL1 plays important roles in mediating oncogenic and metastatic activities in ESCC via canonical NFκB pathway activation and BMP pathway attenuation. FSTL1 may potentially serve as a diagnostic biomarker and therapeutic target in ESCC.