Stage IV colon cancer patients without DENND2D expression benefit more from neoadjuvant chemotherapy

Abstract

According to the EPOC study, chemotherapy could improve 5-year disease-free survival of stage IV colon cancer patients by 8.1%. However, more molecular biomarkers are required to identify patients who need neoadjuvant chemotherapy. DENND2D expression was evaluated by immunohistochemistry in 181 stage IV colon cancer patients. The prognosis was better for patients with DENND2D expression than patients without DENND2D expression (5-year overall survival [OS]: 42% vs. 12%, p = 0.038; 5-year disease-free survival: 20% vs. 10%, p = 0.001). Subgroup analysis of the DENND2D-negative group showed that patients treated with neoadjuvant chemotherapy achieved longer OS than patients without neoadjuvant chemotherapy (RR = 0.179; 95% CI = 0.054-0.598; p = 0.003). DENND2D suppressed CRC proliferation in vitro and in vivo. Downregulation of DENND2D also promoted metastasis to distant organs in vivo. Mechanistically, DENND2D suppressed the MAPK pathway in CRC. Colon cancer patients who were DENND2D negative always showed a worse prognosis and were more likely to benefit from neoadjuvant chemotherapy. DENND2D may be a new prognostic factor and a predictor of the need for neoadjuvant chemotherapy in stage IV colon cancer.

Fig. 1. DENND2D is more highly expressed in adjacent normal tissues than colorectal cancer tissues, confirmed by online database and our clinical samples.

A Expression level of DENND2D across 31 TCGA cancer types, in comparison with their normal controls if available. The middle line in the box is the median, the bottom and top of the box are the first and third quartiles, the whiskers extend to 1.5 IQR of the lower quartile and the upper quartile respectively, and the black solid circles represent outliers. P values were derived from two-sided Wilcoxon’s rank-sum test. B DENND2D gene expression among normal, tumor, and metastatic tissues in colorectal cancer patients obtained from the TNMplot.com web tool. C Protein expression of DENND2D in colon cancer tissues from 181 stage IV CRC patients. The level of DENND2D was classified as negative, weak, moderate, and strong. Protein expression of DENND2D in normal tissue (×40 and ×200). D Protein expression of DENND2D in normal tissues. E, F Western blot analysis of six pairs of tumor tissues and normal tissues from six colon cancer patients.

Fig. 2. DENND2D-negative patients with R0 resction benefit more from neoadjuvant chemotherapy.

A Overall survival of 181 stage IV colon cancer patients with 95% confidence intervals. B Disease-free survival of 181 stage IV colon cancer patients with 95% confidence intervals. C Comparison of the survival rate between patients who had received R0 resection with or without neoadjuvant chemotherapy in the DENND2D-negative group. D Comparison of the survival rate between patients who had received R0 resection with or without neoadjuvant chemotherapy in the DENND2D-positive group.

Fig. 3. DENND2D suppressed tumor proliferation, migration and colony formation in vitro.

A, B DENND2D was knockdown by siRNA in HCT116, HT29 and SW620 confirmed by qPCR and western blot. C, D CRC cell proliferation was promoted by DENND2D knockdown. E, F CRC cell migration was promoted by DENND2D knockdown. G–I CRC cell proliferation was promoted by DENND2D knockdown. J, K DENND2D was knocked down by shRNA in HCT116 confirmed by qPCR, western blot, and IHC. L, M CRC cell proliferation was promoted by DENND2D knockdown. N CRC cell migration was promoted by DENND2D knockdown. O, P DENND2D was overexpressed in CRC cells by shRNA confirmed by qPCR, western blot, and IHC. Q, R CRC cell proliferation was suppressed by DENND2D overexpression. S CRC cell migration was suppressed by DENND2D overexpression. T, U CRC cells are more sensitive to 5FU after DENND2D knockdown.

Fig. 4. DENND2D suppressed CRC cell proliferation and metastasis in vivo.

A–C Volumes of xenografts generated from HCT116-sh1/sh2 and HCT-shNC cells. D–F The volumes of xenografts generated from HCT116 and HCT overexpressing cells. G, H DENND2D and Ki67 expression in xenografts generated from HCT116-sh1/sh2, HCT-shNC, HCT116, and DENND2D-overexpressing cells. I–K Liver metastasis model generated by spleen injection of HCT116-sh2, HCT-shNC, HCT116, and DENND2D-overexpressing cells.

Fig. 5. DENND2D suppressed CRC cell proliferation and metastasis in vitro by MAPK pathway.

A Differential pathway-related protein expression between shNC and sh2 DENND2D cell lines. B The relationship between six key molecules using Pearson correlation analysis of the data from the TCGA database. C DENND2D overexpression or knockdown changed the levels of p-MET1/2 (Ser221) and p-ERK1/2 (Thr202/Tyr204) in HCT116 cells, as determined by WB assay. D, E DENND2D and pERK were expressed in xenografts generated from HCT116-sh1/sh2, HCT-shNC, HCT116, and DENND2D-overexpressing cells. F–H Restoring DENND2D expression in DENND2D-silenced CRC cells rescued the activity of the MAPK pathway.