Abstract
Simple SummaryFibroblast growth factor (FGF) plays an important role in tumor growth by inducing angiogenesis in addition to promoting the proliferation of squamous cell carcinoma (SCC) and oral squamous cell carcinoma (OSCC) cells. Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) purified from A431 cell-conditioned media based on its capacity to bind to FGF-1 and FGF-2 is recognized as a pro-angiogenic molecule as a consequence of its interaction with FGF-2. In this study, we have examined the functional role of HBp17/FGFBP-1 in A431 and HO-1-N-1 cells using the CRISPR/Cas9 technology. Our results showed that HBp17/FGFBP-1 knockout inhibited cell proliferation, colony formation, and cell motility compared to control. The amount of FGF-2 was decreased in culture medium conditioned by HBp17/FGFBP-1 knockout cells compared to control. We performed cDNA/protein expression analysis followed by Gene Ontology and protein–protein interaction analysis. The results demonstrate that both gene and protein expression related to epidermal development, cornification, and keratinization were upregulated in HBp17/FGFBP-1-knockout A431 and HO-1-N-1 cells.Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) has been observed to induce the tumorigenic potential of epithelial cells and is highly expressed in oral cancer cell lines and tissues. It is also recognized as a pro-angiogenic molecule because of its interaction with fibroblast growth factor (FGF)-2. In this study, we examined the functional role of HBp17/FGFBP-1 in A431 and HO-1-N-1 cells. Originally, HBp17/FGFBP-1 was purified from A431 cell-conditioned media based on its capacity to bind to FGF-1 and FGF-2. We isolated and established HBp17/FGFBP-1-knockout (KO)-A431 and KO-HO-1-N-1 cell lines using the clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing technology. The amount of FGF-2 secreted into conditioned medium decreased for A431-HBp17-KO and HO-1-N-1-HBp17-KO cells compared to their WT counterparts. Functional assessment showed that HBp17/FGFBP-1 KO inhibited cell proliferation, colony formation, and cell motility in vitro. It also inhibited tumor growth in vivo compared to controls, which confirmed the significant difference in growth in vitro between HBp17-KO cells and wild-type (WT) cells, indicating that HBp17/FGFBP-1 is a potent therapeutic target in squamous cell carcinomas (SCC) and oral squamous cell carcinomas (OSCC). In addition, complementary DNA/protein expression analysis followed by Gene Ontology and protein–protein interaction (PPI) analysis using the Database for Visualization and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins showed that both gene and protein expression related to epidermal development, cornification, and keratinization were upregulated in A431-HBp17-KO and HO-1-N-1-KO cells. This is the first discovery of a novel role of HBp17/FGFBP-1 that regulates SCC and OSCC cell differentiation.
Highlights
Fibroblast growth factor (FGF) is a cell signaling protein originally isolated from the pituitary gland as a growth factor that promotes fibroblast proliferation [1,2]
Two kinds of pCas-Guide-Heparin-binding protein 17 (HBp17) guide RNAs and the functional cassette were co-transfected into A431 and HO-1-N-1 cells by electroporation
We have examined the functional role of HBp17 in A431 and HO-1-N-1 cells using the clusters of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology
Summary
Fibroblast growth factor (FGF) is a cell signaling protein originally isolated from the pituitary gland as a growth factor that promotes fibroblast proliferation [1,2]. Fibroblast growth factor is believed to play an important role in tumor growth and invasion by inducing angiogenesis, in addition to promoting the proliferation of squamous cell carcinoma (SCC) cells [4,5]. The expressions of FGF-1 and FGF-2 increased with the transformation to SCC, and FGF-like activity is present in SCC cell and salivary gland tumor cell culture mediums. Heparin-binding protein 17 (HBp17) was originally co-purified with FGF-2 from culture media conditioned by A431 human epidermoid carcinoma cells, and the molecular weight of HBp17 is 17 kDa [8]. Liu et al reported that HBp17/FGFBP-1 complementary DNA (cDNA)-transfected A431-#4, a nontumorigenic A431 clonal variant that does not express HBp17/FGFBP-1, forms palpable tumors in nude mice but parental cells do not [12]
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