FCGBP functions as a tumor suppressor gene in head and neck squamous cell carcinoma

Abstract

Purpose: The pathogenesis of head and neck squamous cell carcinoma (HNSCC) was complex and the overall survival was not satisfying. It was urgent to uncover novel molecules that play vital role in HNSCC for disease monitoring and drug development.

Methods: Distinguished expression of FCGBP mRNA in HNSCC was analyzed by TCGA-HNSC and three GEO datasets, the relationship between FCGBP and clinical stage and survival was analyzed by GEPIA 2, the immune infiltration pattern analysis was conducted by TIMER 2.0, pathways affected by FCGBP was conducted by GSEA and GO/KEGG. In vitro experiments (including qRT-PCR, siRNA transfection, CCK8, transwell assay and flow cytometry) were conducted to confirm bioinformatic analysis.

Results: FCGBP was down-regulated in tumor samples compared with normal tissues at both mRNA and protein levels, and positively correlated with survival in HNSCC. Genes co-expressed with FCGBP were mainly enriched in immune-related biological processes and pathways. GSEA indicated that FCGBP was associated with activated immune reaction and inhibiting well-known pro-tumor pathways. GSE41613 validated FCGBP as an independent prognostic marker for HNSCC and FCGBP was down-regulated in HNSCC cell lines by qRT-PCR. Migration and invasion of SCC9 and CAL27 were enhanced by FCGBP-targeting siRNAs, the ratio of cytotoxic T lymphocytes were down-regulated while the ratio of myeloid-derived suppressor cells were increased by FCGBP-targeting siRNAs.

Conclusion: FCGBP was a tumor suppressor gene and was an independent prognostic marker for better survival. The underlying mechanism may be that FCGBP inhibited tumor migration and invasion and activated immune response against tumor cells.

Keywords: FCGBP; HNSCC; Immune infiltration; Prognostic marker; Tumor suppressor gene.

Fig. 1

FCGBP expression level and correlation with clinical stage and survival in HNSCC. a Differential FCGBP mRNA expression in tumor and normal tissues by pan-cancer analysis downloaded from TIMER 2.0, FCGBP level was lower in tumor tissues and HPV⁻ tumor tissues in HNSCC. b FCGBP protein expression in HNSCC samples and normal tissues based on Ualcan database. c Differential expression of FCGBP mRNA between tumor and normal tissues of HNSCC based on 3 GEO datasets (GSE37991, GSE29330 and GSE30784). d FCGBP mRNA level was negatively correlated with clinical stage in HNSCC, although there was no statistical significance. e the detailed information of Kaplan–Meier analysis, for OS, the cut-off value was set as median of FCGBP mRNA expression, while for DFS, the cut-off was set as quartile. d and e were analyzed by GEPIA 2. * means P < 0.05, ** means P < 0.01, *** means P < 0.001, **** means P < 0.0001

Fig. 2

Low FCGBP mRNA level was related with a suppressive immune infiltration pattern. Immune infiltration analysis showed that FCGBP expression was positively correlated with immune cells which play a dominant role in anti-tumor immune response and negatively correlated with suppressive immune cells MDSCs, a–f showed B cells, CD8⁺T cells, dendritic cells, monocytes, neutrophils and MDSCs respectively

Fig. 3

Pathways affected by FCGBP in HNSCC by GO/KEGG analysis and GSEA. GO and KEGG analysis were conducted for genes co-expressed with FCGBP by Omicshare website (https://www.omicshare.com/tools/). a GO-Cellular compartment analysis showed these genes were mainly located in plasma membrane and cell periphery, b GO-Molecular function showed these genes were enriched in signaling receptor activity, molecular transducer activity, transmembrane signaling receptor activity and cytokine receptor activity, c GO-Biological process showed these genes were enriched in immune-related biological process, such as lymphocyte activation, leukocyte activation, immune response, immune system process, d KEGG analysis showed these genes were enriched in cytokine-cytokine receptor interaction, hematopoietic cell lineage, Th17 cell differentiation and so on. e GSEA showed allograft rejection, apoptosis and complement were activated in the group with high FCGBP expression, while DNA repair, glycolysis and angiogenesis were activated in the group with low FCGBP expression

Fig. 4

FCGBP was an independent prognostic marker for HNSCC. Prognostic value of FCGBP were investigated by using GSE41613 dataset. a FCGBP was a significant protective factor for HNSCC prognosis, while stage was a risk factor by univariate cox analysis. b FCGBP was an independent prognostic marker for HNSCC by multivariate cox analysis. c Survival analysis validation by GSE41613 dataset. d FCGBP mRNA level were significantly down-regulated in HNSCC cell lines compared with HOK by qRT-PCR. *** means P < 0.001, **** means P < 0.0001

Fig. 5

Knockdown of FCGBP enhanced migration and invasion of SCC9 and CAL27 cell lines. a Validation of knockdown efficiency in SCC9 by qRT-PCR, si-1 and si-3 were selected for the following experiments. b Proliferation of SCC9 and CAL27 after knockdown of FCGBP were detected by CCK8. c Migration and invasion of SCC9 and CAL27 after knockdown of FCGBP were detected by transwell assay, all scale lables represented 50 μm. d Statistic analysis of migration. e Statistic analysis of invasion. ** means P < 0.01, *** means P < 0.001, **** means P < 0.0001

Fig. 6

Knockdown of FCGBP promoted an immunosuppressive phenotype formation of PBMCs. The immune phenotype of PBMCs after cultured by conditioned media from SCC9 and CAL27 which FCGBP had been knockdown were detected by flow cytometry. a The proportion of CTL/T cells in PBMCs. b The proportion of MDSCs/live cells in PBMCs. c The proportion of B cells/live cells in PBMCs. * means P < 0.05, ** means P < 0.01