FAM83A promotes proliferation and metastasis via Wnt/β-catenin signaling in head neck squamous cell carcinoma

Abstract

This research aimed to investigate the expression and function of FAM83A in the proliferation and metastasis in head and neck squamous cell carcinoma (HNSCC). FAM83A mRNA and protein expressions in HNSCC were detected in primary HNSCC samples and cell lines. The associations between FAM83A expression and clinicopathologic variables were evaluated through tissue microarrays. Besides, FAM83A knockdown and overexpression cell lines were constructed to assess cell growth and metastasis in vitro and the relationship between FAM83A and epithelial-mesenchymal transition (EMT). Furthermore, two models of xenograft tumors in nude mice were used to assess the tumorigenicity and metastasis ability of FAM83A in vivo. In the present study, overexpression of FAM83A in HNSCC samples was significantly associated with tumor size, lymph node status and clinical tumor stages. Mechanically, FAM83A could promote HNSCC cell growth and metastasis by inducing EMT via activating Wnt/β-catenin signaling pathway. Rescue experiment demonstrated the inhibition of β-catenin could counteract the function of FAM83A. Also, the FAM83A knockdown could suppress tumor growth and distant metastasis in the xenograft animal models of HNSCC. In conclusion, this study identifies FAM83A as an oncogene of HNSCC. This study provides new insights into the molecular pathways that contribute to EMT in HNSCC. We revealed a previously unknown FAM83A-Wnt-β-catenin signaling axis involved in the EMT of HNSCC. There may be a potential bi-directional signaling loop between FAM83A and Wnt/β-catenin signaling pathway in HNSCC.

Keywords: FAM83A; Head Neck Squamous Cell Carcinoma; Metastasis; Proliferation; Wnt/β-catenin Signaling.

Fig. 1

FAM83A is overexpressed and related to tumor size, lymph node metastasis and clinical tumor stages in HNSCC. A Negative FAM83A expression in HNSCC adjacent normal tissues (A1, 100 × ; A2, 200 ×). B Low FAM83A expression in HNSCC tumor tissues (B1, 100 × ; B2, 200 ×). C High FAM83A expression in HNSCC tumor tissues (C1, 100 × ; C2, 200 ×). D Quantification analysis of FAM83A staining in HNSCC with different tumor size in microarrays. E Quantification analysis of FAM83A staining in HNSCC with different lymph node metastasis in microarrays. F Quantification analysis of FAM83A staining in HNSCC with different clinical tumor stages in microarrays. G Quantification analysis of FAM83A staining in HNSCC with different pathological classifications in microarrays. H The relative expression of FAM83A mRNA was measured by qRT-PCR in 48 freshly collected HNSCC samples and paired adjacent non-tumor tissues. The height of column represents the fold change (log2-transformed) in FAM83A expression in these 48 patients (right panel). I Differential expressions of FAM83A in HNSCC (n = 519) and normal tissues (n = 44) from the TCGA database. J The expressions of FAM83A protein in eight HNSCC tissue and matched adjacent normal tissues. K The expressions of FAM83A protein in HOK and HNSCC cell lines. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

FAM83A knockdown in CAL27 and FADU cells inhibits cell growth and metastasis. A, B FAM83A expression based on real-time PCR and western blotting in CAL27 and FADU cell lines after shFAM83A transfection. C Morphological changes of Cal27 and FADU cells after shFAM83A transfection (100 ×). D CCK8 assays of the proliferation in CAL27 and FADU cells after shFAM83A transfection. E, F The wound-healing assays in CAL27 and FADU cells after shFAM83A transfection (100 ×). G, H Transwell assays of migration and invasion in CAL27 and FADU cells after shFAM83A transfection (100 ×). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

FAM83A overexpression in HN4 and HN6 cells promotes cell growth and metastasis. A, B FAM83A expressions based on real-time PCR and western blotting in HN4 and HN6 cell lines after treated with LV-FAM83A. C Morphological changes of HN4 and HN6 cells after LV-FAM83A transfection (100 ×). D CCK8 assays of the proliferation in HN4 and HN6 cells after LV-FAM83A transfection. E, F A wound-healing assay in HN4 and HN6 cells after LV-FAM83A transfection (100 ×). G, H Transwell assays of migration and invasion in HN4 and HN6 cells after LV-FAM83A transfection (100 ×). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

FAM83A promotes EMT and activates Wnt/β-catenin signaling pathwayin HNSCC cells. A Protein levels of FAM83A, E-cadherin, N-cadherin, Vimentin, Snail and Wnt-responsive genes including β-catenin, phosphor-β-catenin, c-myc and Cyclin D1 were determined by western blotting in CAL27 and FADU cells after shFAM83A transfection. B, C Gene expressions of FAM83A, E-cadherin, N-cadherin, Vimentin, Snail, c-myc and Cyclin D1 were measured by RT-PCR in CAL27 and FAUD cells after shFAM83A transfection. D Protein levels of FAM83A, E-cadherin, N-cadherin, Vimentin, Snail and Wnt-responsive gene including β-catenin, phosphor-β-catenin, c-myc and Cyclin D1 were determined by western blotting in HN4 and HN6 cells after LV-FAM83A transfection. E, F Gene expressions of FAM83A, E-cadherin, N-cadherin, Vimentin, Snail and c-myc and Cyclin D1 were measured by RT-PCR in HN4 and HN6 cells after LV-FAM83A transfection. G Immunofluorescence analysis of the EMT marker E-cadherin and Vimentin in CAL27 cells after shFAM83A transfection (200 ×). H Immunofluorescence analysis of the EMT marker E-cadherin and Vimentin in HN6 cells after LV-FAM83A transfection (200 ×). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

FAM83A promotes EMT through Wnt/β-catenin signaling pathway and β-catenin regulated FAM83A expression in HNSCC cells. A Expression of nuclear and cytoplasmic FAM83A and β-catenin protein in CAL27 and HN6 cells after FAM83A silencing. B The expressions of nuclear and cytoplasmic FAM83A and β-catenin protein in CAL27 and HN6 cells after FAM83A overexpressed. C Immunofluorescence assays of β-catenin in CAL27 and HN6 cells after FAM83A silencing(400 ×). D Immunofluorescence assays of β-catenin in CAL27 and HN6 cells after FAM83A overexpressed (400 ×). E Protein levels of β-catenin, FAM83A, E-cadherin, N-cadherin, Vimentin, and Snail were determined by western blotting in CAL27 and HN6 cells after being treated with XAV-939. F Protein levels of β-catenin and FAM83A were determined by western blotting in CAL27 and HN6 cells after si-β-catenin transfection and plasmid transfection respectively

Fig. 6

FAM83A knockdown inhibits HNSCC growth and metastasis in nude mice. A The shFAM83A cells and control cells were subcutaneously injected into nude mice and general observation of tumor-bearing nude mice injected with cells. B Volumes of xenograft tumors in shNC and shFAM83A groups. C Tumor tissues in shNC and shFAM83A groups were weighed after 24 days. D Representative showings of tumors formed in the nude mice were diagnosed as HNSCC by HE staining (100 ×). E Representative photomicrographs of positive staining of FAM83A in shNC groups and negative staining of FAM83A in shFAM83A groups (100 ×). F, G Representative photographs of lung tissues in mice injected with shFAM83A cells and shNC cells and a summary of the number of lung metastatic nodules in shFAM83A and shNC groups. H HE staining of lung tissues in shNC and shFAM83A groups (H1, H3, 100 × ; H2, H4, 200 ×). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001