Secretory leukocyte protease inhibitor suppresses HPV E6-expressing HNSCC progression by mediating NF-κB and Akt pathways

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and human papillomavirus (HPV) has been increasingly recognized as a pathogenic factor for the initiation and development of HNSCC. E6 oncogene, an essential component of the HPV 16 virus, acts as a leading cause of the malignant transformation of cancer cells. Therefore, investigating the biological effect and potential mechanisms of E6 oncogene on HNSCC cells and exploring potential therapeutic methods is of great value.

Methods: MTT assay, cell cycle analysis, and apoptosis assay were implemented to detect the biological effect of E6 oncogene on the growth of HNSCC cells. Wound healing assay and transwell assay were used to evaluate the role of E6 in the migration and invasion of HNSCC cells. Western blot and immunofluorescence assay were adopted to explore the regulatory mechanisms underlying E6-induced HNSCC progression. Then, exogenous secretory leukocyte protease inhibitor (SLPI) was added into the cell culture to investigate whether it could maintain its tumor suppressor effect on E6-expressing HNSCC cells.

Results: HPV E6 oncogene could promote the proliferation, cell cycle period, apoptosis resistance, migration and invasion of HNSCC cells by activating NF-κB and Akt pathways. Immunohistochemical analysis conducted on HNSCC tissues illustrated that SLPI was further downregulated in HPV positive HNSCC compared to HNSCC without HPV infection. Exogenous SLPI significantly inhibited HPV E6-mediated malignant phenotypes in HNSCC cells by inhibiting the activation of NF-κB and Akt and signaling pathways.

Conclusions: This study demonstrated that E6 oncogene led to the malignant transformation of HNSCC cells by regulating multiple pathways. SLPI could reverse the effect of E6 oncogene on HNSCC, implying that the functional inhibition of E6 by SLPI may be exploited as an attractive therapeutic strategy.

Keywords: Akt pathway; Head and neck squamous cell carcinoma; Human papillomavirus; NF-κB; Secretory leukocyte protease inhibitor.

Fig. 1

Overexpression of E6 oncogene in HNSCC cells with a stable lentivirus transfection. a mRNA level of E6 oncogene was elevated in HNSCC cells with lentivirus transfection, as demonstrated by qPCR technique. b Immunofluorescence assay illustrated the elevated protein level of E6 oncogene in HNSCC cells after lentivirus transfection. c Western blot results demonstrated the overexpression of HPV E6 oncogene in HN4 and HN30 cells. ***P < 0.001. ****P < 0.0001 (scale bar: 20 μm)

Fig. 2

E6 oncogene promotes the proliferation, migration, and invasion of HNSCC cells. a, b MTT experiment illustrated that E6 oncogene facilitated the proliferation of HNSCC cells. c, d Cell cycle assay demonstrated that E6 promoted cell growth by increasing the number of cells in S phase and decreasing cells in G1 phase. e Apoptosis assay results showed that E6 oncogene significantly resisted the apoptosis activities induced by DMSO in HNSCC cells. f Transwell migration assay revealed E6 oncogene facilitated the migration of HNSCC cells. g Transwell invasion assay illustrated E6 oncogene significantly elevated the invasive abilities of HNSCC cells. h, i E6 oncogene enhanced the migrating capacities of HNSCC cells, suggested by wound healing assay. The fields of migrated and invasive cells on the membrane were captured (magnification × 100). Statistical analysis was performed using the t-tests. The data represented the mean values of three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001

Fig. 3

E6 oncogene activates NF-κB and Akt pathways in HNSCC. a NF-κB luciferase reporter assay demonstrated an increase of NF-κB activities in E6-expressing HNSCC cells, suggesting the activation of NF-κB pathway by E6 oncogene. b Western blot results illustrated that E6 oncogene activated NF-κB pathway and regulated signaling-related proteins expression. c Western blot analysis revealed that p65 was localized mostly in the cytoplasm in E6 negative HNSCC cells, while p65 was translocated from the cytoplasm to the nucleus in E6-expressing cells. d Confocal microscopy analysis confirmed the nuclear accumulation of p65 in E6-expressing HNSCC cells. e, f mRNA levels of several key pro-inflammatory NF-κB-dependent cytokines and genes TNF-α, IL-1β, IL-6, IL-8 and c-myc were elevated in E6 positive HNSCC cells. g E6 oncogene activated Akt signaling pathway in HNSCC, demonstrated by promoting the phosphorylation of Akt protein. h, i Specific inhibitors of NF-κB (PDTC, Beyotime) and Akt pathways (MK-2206, Selleck) were utilized to demonstrate the effect of E6 oncogene on HNSCC was really dependent on these two pathways. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001 (scale bar: 10 μm)

Fig. 4

Relative activation of NF-κB and Akt pathways and SLPI downregulation in HPV positive HNSCC. a, b The proportion of p65 expressed in the cell nucleus in HPV positive tissues was significantly higher than that in HPV negative tissues. c, d The expression level of p-Akt in HPV positive HNSCC samples was much higher when compared to that in HNSCC samples without HPV infection (scale bar: main = 50 μm; insert = 15 μm). e mRNA expression level of SLPI was significantly decreased in HPV positive HNSCC samples (diagnosis both by FISH testing and p16 testing) according to the analysis results of mRNA data from the Cancer Genome Atlas (TCGA, http://xenabrowser.net). f Immunohistochemistry assay showed that HPV positive HNSCC tissues displayed a widespread expression of E6 oncogene, both in nuclear and cytoplasm while HPV negative tissues presented no E6 expression. Meanwhile, the staining intensity of SLPI was obviously lower in HPV positive HNSCC when compared to HPV negative HNSCC. g Statistical analysis of immunohistochemistry conducted on 24 HPV positive HNSCC tissues and 28 HPV negative tissues illustrated that SLPI protein level in HPV positive HNSCC was statistically lower than that in HPV negative ones

Fig. 5

Exogenous SLPI could get internalized into HNSCC cells. a, b The images of HN4 cells incubated without exogenous SLPI protein. c, d The images of HN4 cells incubated with 40 μg/mL exogenous SLPI protein for 1 h. Cell nucleus was stained with DAPI (blue). Cytoskeleton was stained with phalloidine (red). SLPI was stained with FITC secondary antibody (green) (scale bar: 50 μm)

Fig. 6

Exogenous SLPI inhibits cell growth and induces apoptosis activities in E6 positive HNSCC cells. a, b MTT assay revealed that exogenous SLPI significantly suppressed HPV E6 positive or E6 negative HNSCC cells proliferation. c, d Exogenous SLPI affected the cell distribution in HNSCC cells with or without E6 expression, mainly manifested by decreasing cells in S phase and increasing cells in G1 phase. e, f Apoptosis assay results suggested that apoptosis resistance of E6-expressing HNSCC cells was functionally reversed by SLPI treatment *P < 0.05. **P < 0.01. ***P < 0.001

Fig. 7

Exogenous SLPI remarkably inhibited the migration and invasion of HPV E6-expressing HNSCC cells. a, b Transwell migration and invasion assay demonstrated that SLPI could inhibit the migrating and invasive abilities of HPV E6 positive and E6 negative HNSCC cells. c, d Wound healing assay revealed the inhibitory role of SLPI in HNSCC cells migration. *P < 0.05. **P < 0.01

Fig. 8

Exogenous SLPI reverses E6-mediated activation of NF-κB and Akt pathways in E6-expressing HNSCC cells. a NF-κB luciferase reporter assay demonstrated that administration of SLPI abolished the increase of NF-κB activities caused by E6 oncoprotein in HNSCC cells. b Western blot analysis showed that SLPI presented a suppressive effect on the phosphorylation of p65 and IκBα, and prevented the degradation of IκBα. c Nuclear and cytoplasmic proteins were extracted from cells and it was demonstrated that SLPI inhibited the translocation of p65 from the cytoplasm to the nucleus caused by E6 oncogene in HNSCC cells. d SLPI could reverse E6-mediated activation of Akt signaling pathway. e, f Specific inhibitors of NF-κB (PDTC, Beyotime) and Akt pathways (MK-2206, Selleck) were utilized to demonstrate that the effect of SLPI on HPV E6-expressing HNSCC cells may be obtained by mediating NF-κB and Akt pathways. *P < 0.05. **P < 0.01. ****P < 0.0001

Fig. 9

Schematic representation of the working model. The modal indicates that exogenous SLPI may affect HPV-E6 expressing HNSCC progression by inhibiting the activation of NF-κB and Akt pathways