Low disabled-2 expression promotes tumor progression and determines poor survival and high recurrence of esophageal squamous cell carcinoma

Abstract

Patients with esophageal squamous cell carcinomas (ESCCs) have poor survival and high recurrence rate, but lack a prognostic biomarker. Disabled-2 (DAB2) is a crucial tumor suppressor, but its roles in ESCCs are uncertain. We investigated whether low DAB2 expression in ESCCs could lead into tumor progression and poor prognosis. Our results found patients with low-DAB2 expression ESCCs had significantly larger tumor size, deeper tumor invasion depth, lymph node metastasis, worse survival, and higher recurrence rate (P<0.05). The Cox-regression model revealed low-DAB2 expression was an independent factor of poor survival (P<0.05), and also of tumor recurrence with the predictive performance superior to clinical TNM stage (P<0.05). Low-DAB2 cancer cells, validated by DAB2 knockdown or over-expression, had higher phosphorylated ERK and migration abilities, which could be suppressed by ERK inhibitor treatment. TGF-β-induced epithelial-to-mesenchymal transition (EMT) only existed in the high-DAB2 cells, and related to worse prognosis of high-DAB2 ESCCs (P<0.05). In conclusion, DAB2 can suppress the ERK signaling, but correlate to have TGF-β-induced EMT in ESCCs. DAB2 expression could be a biomarker to identify patients with worse survival and high recurrence. Our data suggest DAB2 expression can stratify patients in need of aggressive surveillance and with possible benefit from anti-ERK or anti-TGF-β therapies.

Keywords: disabled-2; esophageal cancer; recurrence; survival.

Figure 1. The DAB2 protein of ESCC can be correlated to survival and tumor recurrence rate

A. High-DAB2 ESCC with cytoplasmic brownish staining of DAB2 protein; B. Low-DAB2 ESCC with loss of DAB2 protein (original magnification x 200); C. The survival rate by Kaplan-Meier survival curve was lower in patients with low-DAB2 ESCC than in those with high-DAB2 ESCC (P=0.016). D. The tumor recurrence rate was higher in patients with low-DAB2 ESCC than in those with high-DAB2 ESCC (P=0.007).

Figure 2. The low-DAB2 esophageal cancer cells had a higher phosphorylated ERK (p-ERK) expression and migration abilities

A. The western blot analysis of DAB2 protein in five ESCC cell lines (KYSE) and human normal esophageal squamous epithelial cell line (Het-1A). B. The wound healing assay (upper panel) and transwell migration assay (lower panel) to evaluate the horizontal and vertical migration abilities in ESCC cell lines. C. TheERK, p-ERK and β-catenin levels in ESCC cell lines. D. The quantitative representation of migration abilities in ESCC cell lines.

Figure 3. The associations between DAB2 and p-ERK in clinical tumor samples and cell lines

A. The high-DAB2 cancer (upper panel) had lower p-ERK (upper-right panel) expression, evaluated by immunohistochemistry in clinical tumor samples. The low-DAB2 cancer had higher p-ERK level (lower panel). B. The western blot confirmed the suppression of Erk phosphorylation by Erk inhibitor; C. The wound healing test (upper panel) and transwell migration assay (lower panel) showed the treatment with ERK inhibitor suppress cancer cell motility of low-DAB2 cell lines; D. The quantitative representation of migration abilities in low-DAB2 ESCC cell lines before and after ERK inhibitor treatment.

Figure 4. The down-regulation of DAB2 by siRNA in high-DAB2 cell lines promoted ERK phosphorylation and cell motility

A. Western blot confirmed down-regulation of DAB2 by siRNA in KYSE-50 and KYSE-70 cell lines. B. DAB2 down-regulation promoted ERK phosphorylation. The wound healing C. and transwell migration assay D. showed DAB2 down-regulation promoted cancer cell migration (Abbreviation: s#1: siRNA#1;s#2: siRNA#1; NT, Non-target).

Figure 5. The over-expression of DAB2 can suppress ERK phosphorylation and cancer cell motility

A. Reverse transcription PCR confirmed the over-expression of DAB2 in Low-DAB2 (KYSE-150) cell line. B. DAB2 over-expression inhibited ERK phosphorylation. C, D. The wound healing and transwell migration assay showed DAB2 over-expression suppressed cell migration.

Figure 6. The high-DAB2 esophageal cancer cell had the presence of epithelial-mesenchymal transition (EMT)

A. Western blot showed EMT phenotype appeared only in high-DAB2 cell lines, KYSE-70 & -170. B. Treatment with TGF-β (5 ng/ml) for high-DAB2 (KYSE-50) and low-DAB2 (KYSE-150) ESCC cells, respectively. TGF-β can induce N-cadherin and β-catenin expression in KYSE-50, but not in KYSE-150. C. EMT phenotype with reduced E-cadherin and increased N-cadherin expression (arrow) in clinical tumor tissues by immunohistochemistry. The survival analysis by Kaplan-Meier survival curves showed EMT had survival impact on the patients with high-DAB2 cancers (p=0.04) D. but not in low-DAB2 cancers E.

Figure 7. The methylation status of DAB2 promoter and the schematic algorithm demonstrated the three clinical ESCC phenotypes

A. The analysis of DAB2 promoter methylation by MSP in the ESCC cell lines (upper panel) and representative primary tumors (lower panel). U and M refer to the un-methylated and the methylated PCR reactions, respectively. B. The schematic algorithm demonstrated the three clinical ESCC phenotypes, defined by DAB2 and EMT expressions, and their corresponding survival and tumor recurrence rates.