eIF4E binding protein 1 expression is associated with clinical survival outcomes in colorectal cancer

Abstract

eIF4E binding protein 1 (4E-BP1), is critical for cap-dependent and cap-independent translation. This study is the first to demonstrate that 4E-BP1 expression correlates with colorectal cancer (CRC) progression. Compared to its expression in normal colon epithelial cells, 4E-BP1 was upregulated in CRC cell lines and was detected in patient tumor tissues. Furthermore, high 4E-BP1 expression was statistically associated with poor prognosis. Hypoxia has been considered as an obstacle for cancer therapeutics. Our previous data showed that YXM110, a cryptopleurine derivative, exhibited anticancer activity via 4E-BP1 depletion. Here, we investigated whether YXM110 could inhibit protein synthesis under hypoxia. 4E-BP1 expression was notably decreased by YXM110 under hypoxic conditions, implying that cap-independent translation could be suppressed by YXM110. Moreover, YXM110 repressed hypoxia-inducible factor 1α (HIF-1α) expression, which resulted in decreased downstream vascular endothelial growth factor (VEGF) expression. These observations highlight 4E-BP1 as a useful biomarker and therapeutic target, indicating that YXM110 could be a potent CRC therapeutic drug.

Keywords: YXM110; colorectal cancer (CRC); eIF4E binding protein 1; hypoxia; prognosis.

Figure 1. Compare 4E-BP1 expression in normal and colorectal cancer cells under normoxia and hypoxia

The detection of 4E-BP1 expression in normal colon cell line (CCD-18co) and colorectal cancer cells (SW480, SW620, Colo205, Caco-2, HCT116 and HT29) under A. normoxia and B. hypoxia. C. Also campare the difference of 4E-BP1 between normoxia and hypoxia on each one cell line.

Figure 2. Immnuohistochemical staining of 4E-BP1 in a tissue microarray

A. Representive 4E-BP1 staining of CRC specimens and corresponding non-cancerous tissues. B. The statisitc result of 4E-BP1 expression in CRC and paired normal specimens by Image J. ***P < 0.001.

Figure 3. Evaluation of the clinincal importance of 4E-BP1 expression in CRC patients

The correlation between 4E-BP1 and A. survival time and B. recurrence time were analyzed by Kaplan-Meier analysis. C. The representative of immunohistochemical staining of 4E-BP1 on different stages of sections. Left lane shows the negative staining and right lane are the positive results.

Figure 4. YXM110 suppresses cap-independent translation through 4E-BP1 deletion

A. HCT116 cells were treated with vehicle (0.1% DMSO) or YXM110 (0.1 or 0.3 μM) under normoxia or hypoxia conditions for 24 h. The cells were then harvested for detection of p-mTOR (Ser2448), eIF4E, 4E-BP1, p-4E-BP1 (Thr37/46), p-S6 (Ser240/244) and actin by western blot analysis. B. Bicistronic luciferase vector (pFR-Luc) contains a cap-dependent firefly (Fir) and cap-independent Renilla (Ren) luciferase. C. HCT116 cells were cotransfected bicistronic vector and wt 4E-BP1 for testing the effect of YXM110 on cap-dependent and cap-independent translation by using a dual Luciferase reporter system. *P < 0.05; ***P < 0.001; ^###P < 0.001.

Figure 5. YXM110 inhibits HIF-1α translation

A. HIF-1α mRNA was analyzed with quantitative real-time RT-PCR after 6 h treatment of YXM110 (0.1 or 0.3 μM) under normoxia or hypoxia conditions. B. HCT116 cells were treated with vehicle (0.1% DMSO) or YXM110 (0.1 or 0.3 μM) under normoxia or hypoxia conditions for 24 h or C. pretreated with vehicle or 10 μM MG132 and then incubated with vehicle or YXM110 (0.1 or 0.3 μM) for 24 h under hypoxia. The cells were then harvested for detection of HIF-1α and actin by western blot analysis. *P < 0.05; ^## P < 0.01.

Figure 6. YXM110 inhibits HRE-Luciferases activity and VEGF transcription and translation

A. HCT116 cells transiently transfected with 5-HRE-Luciferase reporter were treated with YXM110 under normoxia or hypoxia conditions for 24 h. Data have shown represented luciferase activity relative to the control. B. VEGFA mRNA was analyzed with quantitative real-time RT-PCR after 6 h treatment of YXM110 (0.1 or 0.3 μM) under normoxia or hypoxia conditions. C. VEGF levels in culture medium were determined using Human VEGF Quantikine ELISA Kit. ***P < 0.001; ^###P < 0.001.