High expression of cytoplasmic FOXO3 protein associated with poor prognosis of rectal cancer patients: A study from Swedish clinical trial of preoperative radiotherapy to big database analysis

Abstract

Introduction: Accumulating evidence has implicated a pivotal role for FOXO3, FOXM1 and SIRT6 in cancer progression. The majority of researches focused on the functions of these proteins in drug resistance, but their relationships with radiotherapy (RT) response remain unclear. In this study, we examined protein expression of FOXO3, FOXM1 and SIRT6 and their clinical significance in a Swedish rectal cancer trial of preoperative RT.

Methods: Expression of FOXO3, FOXM1 and SIRT6 protein was examined by immunohistochemistry in patient samples. Genetic analysis of FOXO3, FOXM1 and SIRT6 were performed by cBioportal and MEXPRESS database. Gene-gene network analysis was conducted using GeneMANIA. Functional enrichment analysis was performed based on LinkedOmics and Metascape online software.

Results: FOXO3 and FOXM1were mainly expressed in the cytoplasm in both normal and tumour tissues, and SIRT6 in both the cytoplasm and nucleus in normal and tumour tissues. FOXO3 and FOXM1 expression increased from normal mucosa to primary cancer (P < 0.001), while SIRT6 expression decreased from normal mucosa to primary cancer (P < 0.001). High FOXO3 expression correlated with late TNM stage (P = 0.040), distant metastasis (P = 0.032) and independently with disease free survival (DFS) in the RT patients (HR = 7.948; P = 0.049; 95% CI = 1.002-63.032) but not in non-RT patients (P > 0.05). Genetic analysis indicated that DNA methylation status contributed to FOXO3 overexpression. Functional enrichment analysis demonstrated that FOXO3 was closely related to metabolism-related signalling pathway which in turn associated with cancer radioresistance. Moreover, there were strong gene-gene interactions between FOXO3 and metabolism-related signalling.

Conclusions: Our findings suggest that FOXO3 may be a prognostic factor in rectal cancer patients with RT.

Keywords: FOXM1; FOXO3; Prognosis; Radiotherapy; Rectal cancer patients; SIRT6.

Fig. 1

Expression of FOXO3/FOXM1/SIRT6 protein in various tissues from the rectal cancer patients. Immunohistochemical staining for the expression of FOXO3 (A), FOXM1 (C) and SIRT6 (E) in the normal mucosa (left panel), primary tumour (middle panel) and lymph node metastasis (right panel) in both low and high power, respectively. In the top rows, the scale bars indicate 200 μm, whereas in the bottom rows, the scale bars correspond to 50 μm. Magnification 20x and 40x. Percentages of the cases with high expression levels of FOXO3 (B), FOXM1 (D) and SIRT6 (F) in the distant normal mucosa, adjacent normal mucosa, primary cancer and lymph node metastasis in non-radiotherapy (non-RT) and radiotherapy (RT) rectal cancer patients.

Fig. 2

The relationship between FOXO3 expression and survival in radiotherapy (RT) and non-radiotherapy (non-RT) rectal cancer patients. Cancer-specific survival analysis showed that the cancer-specific survival probability was significantly different in the rectal cancer patients between high and low FOXO3 expression in non-RT (A) and RT (B) groups. Disease free survival analysis revealed that rectal cancer patients with high and low FOXO3 expression was statistically different in non-RT (2C) and RT (2D) rectal cancer patients.

Fig. 3

Gene-gene interaction networks among FOXO3, FOXM1, SIRT6 and genes in NF-κB signalling in colorectal cancer. Each node represents an individual gene. The inter-node connection lines represent the types of gene-gene interactions and colour of the lines represents the types of interactions. The colour nodes represent the possible functions of respective genes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Genetic alterations of FOXO3, FOXM1 and SIRT6 in colorectal cancer. The OncoPrint analysis summaries genetic variations of FOXO3, FOXM1 and SIRT6 in TCGA dataset (A). Alteration frequencies of FOXO3 in various subtypes of the cancers. COAD: Colon Adenocarcinoma; READ: Rectal Adenocarcinoma; MAC: Mucinous Adenocarcinoma of the Colon and Rectum; COADREAD: Colorectal Adenocarcinoma (B). Detail mutation information of FOXO3 and FOXM1 in colorectal cancer (C).

Fig. 5

Correlation analysis of FOXO3-related genes in colorectal cancer. Top 20 GO enrichment terms are listed in the bar graph (A). A volcano plot of FOXO3-related genes (B). Heat map showing the genes positively associated with FOXO3 (C). heat map revealing the genes negatively associated with FOXO3 (D).

Fig. 6

Functional enrichment analysis of FOXO3-associated genes in colorectal cancer. We select the term between upregulated and downregulated groups with the best p-value within each cluster as its representative term and display them in a dendrogram. The heatmap cells are coloured by their p-values, white cells indicate the lack of enrichment for that term in the corresponding gene list. Top 20 GO enrichment terms are listed in the dendrogram (A). Network of the top 20 GO enriched terms coloured by clusters (B). Network of the top GO 20 enriched terms coloured by P-value (C). Top 20 KEGG enrichment terms are listed in the dendrogram (D). Gene-gene interaction networks between FOXO3 and the genes in metabolism-related pathways in colorectal cancer. Each node represents an individual gene. The inter-node connection lines represent the types of gene-gene interactions and colours of the lines represent the types of interactions. The colour nodes represent the possible functions of respective genes (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Schematic diagram of research flow.