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. 2021 May 17:2021:5510753.
doi: 10.1155/2021/5510753. eCollection 2021.

Clinicopathological Significance of FOXO4 Expression and Correlation with Prx1 in Head and Neck Squamous Cell Carcinoma

Yunping Lu  1 , Yajun Shen  1 , Lingyu Li  1 , Min Zhang  1 , Min Wang  1 , Lihua Ge  1 , Jing Yang  1 , Xiaofei Tang  1
Affiliations

Clinicopathological Significance of FOXO4 Expression and Correlation with Prx1 in Head and Neck Squamous Cell Carcinoma

Yunping Lu et al. Anal Cell Pathol (Amst). .

Abstract

Objective: Forkhead box O 4 (FOXO4), a key albumen in the forkhead box O (FOXOs) family, plays crucial roles as a tumor suppressor in the cancer development. In our previous study, Peroxiredoxin1 (Prx1) promoted the development of oral cancer and was predicted to bind to FOXO4. The aim of this study was to investigate the clinicopathological significance of FOXO4 expression and its potential mechanism in head and neck squamous cell carcinomas (HNSCC).

Methods: The function of FOXO4 correlation with HNSCC prognosis was analyzed via ONCOMINE, UALCAN, Human Protein Atlas, and cBioPortal. The expression of FOXO4 was detected in Prx1 silenced CaL27 and SCC9 cell lines by Western blot. FOXO4 protein expression was observed via immunohistochemistry (IHC) and the binding of Prx1 to FOXO4 measured by Duolink analysis in a 4-nitro-quinoline-1-oxide- (4NQO-) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/- mice.

Results: By the analysis of Bioinformation Databases, there was a significant interaction of FOXO4 down expression to clinical tumor stages and pathological grades in the patients with HNSCC. Reduced mRNA and protein expression of FOXO4 were found to be significantly correlated with the poor overall survival (OS) of HNSCC patients. FOXO4 expression is negatively related to Prx1 significantly in HNSCC tissues. By employing a 4NQO-induced oral carcinogenesis mouse model, we confirmed that FOXO4 expression was reduced in 4NQO-induced squamous cell carcinoma (SCC) tongue tissues compared with those in normal tissues. Prx1 knockdown resulted in the upregulation of FOXO4 expression in the SCC tissues and CaL27 and SCC9 cell lines. Furthermore, the interaction of Prx1 with FOXO4 was observed in mouse tongue tissues by Duolink analysis.

Conclusion: FOXO4 plays an important role in the development of HNSCC. The lower expression of FOXO4 is significantly correlated with the shorter OS in patients with HNSCC. FOXO4 is negatively regulated via interaction with Prx1. FOXO4 could be a potential molecular target for the treatment and prognosis of HNSCC.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
mRNA expression of FOXO4 from ONCOMINE data. (a) mRNA expression of FOXO4 in HNSCC samples. (b–d) Validation of mRNA expression level of FOXO4 in three datasets. Cut-off p = 0.01, fold change > 1.5, Gene Rank = 10%; ∗∗p < 0.01, data type: mRNA.
Figure 2
Figure 2
Relationship between mRNA expression of FOXO4 and cancer stages of patients with HNSCC (UALCAN). (a) Expression of FOXO4 in normal and HNSCC samples. (b) Expression of FOXO4 in individuals of gender. (c) Expression of FOXO4 in normal individuals or any age in HNSCC patients aged 21-40, 41-60, 61-80, or 81-100 yr. (d) Expression of FOXO4 in normal individuals or in HNSCC patients in stage 1, 2, 3, or 4. (e) Expression of FOXO4 in normal individuals or HNSCC patients with grade 1, 2, 3, or 4 tumors. Grade 1: well differentiated (low grade); grade 2: moderately differentiated (intermediate grade); grade 3: poorly differentiated (high grade); grade 4: undifferentiated (high grade). (f) Prognostic value of mRNA expression of FOXO4 in HNSCC patients (Kaplan-Meier Plotter). ∗p < 0.05; ∗∗p < 0.01.
Figure 3
Figure 3
Immunohistochemistry images of FOXO4 in normal tissues or HNSCC tissues (Human Protein Atlas) and genetic alteration of FOXO4 in HNSCC patients (cBioPortal). (a) Low protein expressions of FOXO4 in normal tissues and FOXO4 proteins were not detected in HNSCC tissues. (b) Survival analysis of protein expression of FOXO4 in HNSCC patients (Kaplan-Meier Plotter, ∗∗p < 0.01). (d) Low FOXO4 mutation rate (6%) in HNSCC patients. (e) Associated OS and (f) DFS were no significantly difference between the altered group and unaltered group of HNSCC patients (p > 0.05).
Figure 4
Figure 4
FOXO4 coexpression genes in HNSCC (LinkedOmics). (a) Genes positively and negatively correlated with FOXO4 in HNSCC were shown as volcano plot. Red indicates positively correlated genes, and green indicates negatively correlated genes. (b) The Pearson test was used to analyze correlation between FOXO4 and Prx1 in HNSCC. (c, d) The expression of FOXO4 in shPrx1 Cal27 and shPrx1 SCC9 cells. The relative levels of protein were normalized to VCL used as an internal control. Values presented as the means ± SD (∗p < 0.05; ∗∗p < 0.01). (e) FOXO4 protein levels measured by IHC in tongue tissues from Prx1+/+ and Prx1+/− mice. (f) FOXO4 MOD value in mouse tongue tissues. (magnification: 200x, the value represents the mean ± SD, and ∗p < 0.05).
Figure 5
Figure 5
Interaction between Prx1 and FOXO4 (ZDOCK and PLA of Duolink analysis). (a) Structure of Prx1 bound to FOXO4 and FOXO4-Prx1 interaction plot. Prx1(A) is shown in green, Prx1(B) is shown in red, and FOXO4 is shown in cyan. Hydrogen bonds are shown as yellow dashed lines. The images were constructed using Discovery Studio (DS) 2016. (b) Interactions of Prx1 with FOXO4 were detected in tongue tissues from Prx1+/+ and Prx1+/− mice. PLA signal is red, and the nuclei is blue (magnification: 1000x).
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