Hypoxia-induced MFAP5 Promotes Tumor Migration and Invasion via AKT Pathway in Head and Neck Squamous Cell Carcinoma

Abstract

Objective: Microfibrillar-associated protein 5 (MFAP5) is highly expressed in many types of cancers. Our previous study has observed that overexpression of MFAP5 was correlated with lymph nodes metastasis and poor prognosis in head and neck squamous cell carcinoma (HNSCC), but the underlying mechanism is poorly understood. Materials and methods: The MFAP5 expression is detected under hypoxia condition. HNSCC cell lines are transfected with MFAP5-expressing lentivirus vector to establish stable overexpression model. Wound-healing, migration and invasion assay are used to determine the effect of MFAP5 on HNSCC and metastasis-related proteins are examined by Western blot. In vivo lung metastasis assays are conducted by the tail vein injection. In addition, immunohistochemistry is applied to analyze the correlation of MFAP5, hypoxia-induced factor-1 α (HIF-1α), and vimentin in 84 HNSCC patients' tissue samples. Results: Firstly, MFAP5 expression can be markedly induced under hypoxia condition in HNSCC cell lines. Cell lines with MFAP5 overexpression has a significant higher ability of migration and invasion. In addition, in vivo assay observes that overexpression of MFAP5 can promote tumor lung metastasis. Furthermore, MFAP5 facilitates this process by activating epithelial-mesenchymal transition (EMT) program via AKT pathway in HNSCC cell lines. The pro-metastatic effect of MFAP5 can be reversed by MK2206, an AKT phosphorylation inhibitor. Lastly, the positive correlation among HIF-1α, MFAP5 and vimentin from tissue samples and TCGA dataset are also observed in HNSCC. Conclusion: Our study demonstrates MFAP5 plays a critical role in hypoxia-induced EMT program via AKT pathway in HNSCC, which would be a very promising therapeutic target.

Keywords: AKT; Head and neck squamous cell carcinoma; Invasion; Microfibrillar-associated protein 5; Migration.

Figure 1

Hypoxia induces MFAP5 expression in HNSCC. (A) The protein level of HIF-1α, snail and MFAP5 in hypoxia environment for 0h, 1h and 10h. β-actin was used as an internal control. The proteins level is significant higher in hypoxia environment (**P<0.01). (B) The expression level of MFAP5 and HIF-1α is related in same tumor. (C) The correlation analysis of immunohistochemistry intensity for HIF-1α and MFAP5 in 84 patients' tissue sample (r=0.493, **P<0.01).

Figure 2

Overexpressed MFAP5 promotes migration and invasion on HNSCC cell lines. (A) Relative mRNA expression of MFAP5 in Cal27 and HN30 transfected with MFAP5 and vector (**P<0.01). (B) The migration assay was performed to testify the migration ability of Cal27 and HN30 transfected with MFAP5 or negative control lentivirus (**P<0.01). (C) The wound healing assay was performed to testify the migration ability of Cal27 and HN30 transfected with MFAP5 or negative control lentivirus (**P<0.01). (D) The invasion assay was performed to testify the invasion ability of Cal27 and HN30 transfected with MFAP5 or negative control lentivirus (**P<0.01).

Figure 3

Overexpressed MFAP5 facilitates lung metastasis in vivo. (A) Bioluminescence imaging showed MFAP5 overexpression significantly increased lung metastasis burden (**P<0.01). (B) Gross specimen and H&E staining are shown (×100).

Figure 4

Western blot was performed to detect the protein level of Cal27 and HN30 transfected with MFAP5 or negative control lentivirus. The expression of MMP9, MMP2, vimentin, snail and p-AKT/AKT are significant higher along with MFAP5 (**P<0.01).

Figure 5

The EMT program enhanced by MFAP5 could be reversed by MK2206. (A) The protein level of MMP9, snail, vimentin and p-AKT/AKT was reduced after MK2206 was applied to Cal27-MFAP5 and HN30-MFAP5 (**P<0.01) (B) Wound healing assay was performed to Cal27-MFAP5 and HN30-MFAP5 with or without MK2206 (**P<0.01). (C) Migration assay was performed to Cal27-MFAP5 and HN30-MFAP5 with or without MK2206 (**P<0.01). (D) Invasion assay was performed to Cal27-MFAP5 and HN30-MFAP5 with or without MK2206 (**P<0.01).

Figure 6

Overexpression of MFAP5 can be observed in HNSCC and is associated with aggressive clinical features, poor outcome and EMT-related proteins. (A) Overexpression of MFAP5 can be observed in HNSCC. (B) MFAP5 expression is related with advanced stage (P<0.01) and cervical lymph node metastasis (P=0.04). (C) TCGA data shows high level of MFAP5 lead to poor prognosis (P=0.013). (D) Correlation analysis of relative mRNA expression shows MFAP5 is associated with EMT-related proteins. (E)The correlation analysis of immunohistochemistry intensity for vimentin and MFAP5 in 84 patients' tissue sample (r=0.399, P<0.01).