Upregulation of NETO2 expression correlates with tumor progression and poor prognosis in colorectal carcinoma

Abstract

Background: Neuropilin and tolloid-like 2 (NETO2) has been found to be overexpressed in different human cancers, but its expression pattern and clinical relevance in colorectal carcinoma (CRC) remains unknown.

Methods: Real-time quantitative PCR, western blot and immunohistochemistry analyses were used to analyze the expression of NETO2 in CRC clinical samples. The correlation of NETO2 expression with clinicopathologic features was estimated in a cohort containing 292 patients with primary CRC. Kaplan-Meier and Cox proportional hazards regression analyses were used to assess the prognostic value of NETO2 expression in CRC.

Results: The expression of NETO2 was frequently upregulated in CRC clinical samples at both the mRNA and protein levels, and its upregulation was significantly correlated with poor tumor differentiation (p = 0.013), advanced local invasion (p = 0.049), increased lymph node metastasis (p = 0.009), advanced TNM stage (p = 0.041) and increased patient death (p = 0.001). Kaplan-Meier analysis of the complete study cohort revealed that patients with high-NETO2 tumors had a significantly shorter disease-specific survival (DSS) than those with low-NETO2 tumors (p < 0.001). Importantly, high levels of NETO2 protein predicted poor DSS for patients with early stage tumors (p = 0.027) and for those with advanced stage tumors (p = 0.020). Furthermore, multivariate analyses indicated that increased NETO2 expression was an independent unfavorable prognostic factor for patients with early stage tumors (hazard ratio [HR] = 1.937, 95% CI = 1.107-3.390, p = 0.021) as well as patients with advanced stage tumors (HR = 2.241, 95% CI = 1.245-4.035, p = 0.007).

Conclusions: Our findings suggest that NETO2 upregulation could serve as a potential biomarker for the prediction of advanced tumor progression and unfavorable prognosis in patients with CRC.

Fig. 1

Expression of NETO2 in primary CRC tissues. a NETO2 mRNA expression in 57 paired human CRC and corresponding adjacent normal mucosa specimens were determined by real-time qPCR method. Gene expression results were normalized by internal control β-actin. (T, tumor tissues; N, adjacent normal tissues). b Protein expression levels of NETO2 in an independent set of 24 paired CRC and corresponding adjacent normal specimens were determined by Western blot analysis. β-actin was used as a loading control. The relative protein expression of NETO2 was quantified and normalized to β-actin. Each N was arbitrarily designated 1.0. (T: Tumor tissues; N: adjacent normal tissues, T vs N, p < 0.001)

Fig. 2

Immunostaining of NETO2 protein in human CRC and adjacent normal tissues. a Representative immunohistochemical expression patterns of NETO2 in cancerous and adjacent normal mucosa specimens were shown. (Magnification, upper panel, ×100; lower panel, ×400) Right panel: human CRC cell line HCT116 cells were used as a negative control and HCT15 cells were used as a positive control for immunostaining of NETO2 protein. (Magnification × 100) (b) Percentage of cases with different staining intensity of NETO2 in the tumor or adjacent normal tissues in the study cohort. (p < 0.001)

Fig. 3

Kaplan-Meier survival analysis for CRC patients. a Kaplan-Meier curves for disease-specific survival of all CRC patients in the study cohort according to NEOT2 expression status. b Kaplan-Meier curves for disease-specific survival of all CRC patients according to TNM stage. c-d Kaplan-Meier curves for disease-specific survival of patients with early stage tumors (c) or advanced stage tumors (d) according to NETO2 expression status. The p-value was determined using the log-rank test. The absolute number of patients at risk in each subgroup is listed below