Heparin-Binding Protein 17/Fibroblast Growth Factor-Binding Protein-1 Knockout Inhibits Proliferation and Induces Differentiation of Squamous Cell Carcinoma Cells

Abstract

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.

Keywords: CRISPR/Cas9; FGF-2; HBp17/FGFBP-1; cell differentiation; squamous cell carcinoma.

Figure 1

Identification of A431-HBp17-KO and HO-1-N-1-HBp17-KO cells. Cellular protein and CM from A431-HBp17-KO1, -KO2, A431-WT, HO-1-N-1-HBp17-KO1, -KO2, and HO-1-N-1-WT cells were Western blotted against HBp17 goat polyclonal antibody. The absence of the band seen in A431-WT and HO-1-N-1-WT samples confirmed the deletion of HBp17 expression in A431-HBp17-KO (A) and HO-1-N-1-HBp17-KO (B) cells. Recombinant human HBp17 (2 ng) was used as a positive control (PC).

Figure 2

Decreased FGF-2 expression in HBp17-KO cell CM. (A) Western blot of FGF-2 in total cellular protein of A431-HBp17-KO1, -KO2, A431-WT, HO-1-N-1-HBp17-KO1, -KO2, and HO-1-N-1-WT cells. (B) ELISA of FGF-2 secreted into CM decreased for A431-HBp17-KO and HO-1-N-1-HBp17-KO cells compared to their WT counterparts. Each bar represents the mean + SD. * p < 0.05; n = 3.

Figure 3

Knockout of HBp17 inhibits cell proliferation and colony formation. (A) Cell proliferation and (B) colony formation were determined in A431-HBp17-KO1, -KO2, A431-WT, HO-1-N-1-HBp17-KO1, -KO2, and HO-1-N-1-WT cells. Experiments were performed in triplicate with data presented as means ± SD. * p < 0.05.

Figure 4

Knockout of HBp17 inhibits mobility of A431 and HO-1-N-1 cells. A431-HBp17-KO1, -KO2, and A431-WT (A) and HO-1-N-1-HBp17-KO1, -KO2, and HO-1-N-1-WT (B) cells were added to Transwell inserts. Cells migrating across the insert were counted after incubation for 24 h at 37 °C. Experiments were performed in triplicate, with the data presented as means ± SD. * p < 0.05.

Figure 5

HBp17 knockout suppresses A431 tumor growth in immunodeficient mice. A431-HBp17-KO1, -KO2, and A431-WT cells were inoculated into the flanks of male nude mice, and tumor volume was measured twice a week. Experiments were performed with five mice. Data are presented as mean tumor volume + SD. * p < 0.05.

Figure 6

cDNA microarray and proteomic analyses of A431-HBp17-KO2 and A431-WT cells. (A) cDNA microarray analysis found 11 cDNAs with greater than 2.5-fold higher expression in A431-HBp17-KO2 cells than in A431-WT cells. (B) Seven proteins were found to be expressed at least five times higher in A431-HBp17-KO2 cells than in A431-WT cells. Abbreviations: aldo-keto reductase family 1 member C3 (AKR1C3), keratin1 (KRT1), aldo-keto reductase family 1 member C2 (AKR1C2), carbonic anhydrase 2 (CA2), fatty acid-binding protein 5 (FABP5), secretory leukocyte protease inhibitor (SLPI), serine proteinase inhibitor clade B member 3 (SERPINB3), plasma membrane calcium-transporting ATPase 4 (ATP2B4), S100 calcium-binding protein A8 (S100A8), S100 calcium-binding protein A9 (S100A9), small proline-rich protein 1B (SPRR1B), and small proline-rich protein 1A (SPRR1A).

Figure 7

Knocking out HBp17 induces the expression of cornified envelope-related proteins. (A) Upregulation of FABP5, SPRR-A, SPRR-B, IVL, LOR, and FLG mRNAs in A431-HBp17-KO2 cells was evaluated by qRT-PCR. (B) In HO-1-N-1-HBp17-KO1, the expression of FABP5, IVL, and FLG mRNAs was elevated. GAPDH levels were used as an internal control. Experiments were performed in triplicate, with the data presented as means ± SD. * p < 0.05. Increased cornified envelope-related protein expression in A431-HBp17-KO2 (C) and HO-1-N-1- HBp17-KO1 cells (D) was evaluated by Western blot. (E) Immunofluorescence staining for IVL (green) in A431-HBp17-KO2 and HO-1-N-1-HBp17-KO1, A431-WT, and HO-1-N-1-WT cells. Cell nuclei were stained with DAPI (blue). Abbreviations: fatty acid-binding protein 5 (FABP5), small proline-rich protein 1A (SPRR1A), small proline-rich protein 1B (SPRR1B), Involucrin (IVL), Loricrin (LOR), Filaggrin (FLG).

Figure 7

Knocking out HBp17 induces the expression of cornified envelope-related proteins. (A) Upregulation of FABP5, SPRR-A, SPRR-B, IVL, LOR, and FLG mRNAs in A431-HBp17-KO2 cells was evaluated by qRT-PCR. (B) In HO-1-N-1-HBp17-KO1, the expression of FABP5, IVL, and FLG mRNAs was elevated. GAPDH levels were used as an internal control. Experiments were performed in triplicate, with the data presented as means ± SD. * p < 0.05. Increased cornified envelope-related protein expression in A431-HBp17-KO2 (C) and HO-1-N-1- HBp17-KO1 cells (D) was evaluated by Western blot. (E) Immunofluorescence staining for IVL (green) in A431-HBp17-KO2 and HO-1-N-1-HBp17-KO1, A431-WT, and HO-1-N-1-WT cells. Cell nuclei were stained with DAPI (blue). Abbreviations: fatty acid-binding protein 5 (FABP5), small proline-rich protein 1A (SPRR1A), small proline-rich protein 1B (SPRR1B), Involucrin (IVL), Loricrin (LOR), Filaggrin (FLG).