Poly(C)-binding protein 1 mediates drug resistance in colorectal cancer

Abstract

Oxaliplatin (L-OHP) is standard treatment for colorectal cancer. However, resistance to L-OHP often leads to treatment failure or cancer relapse. Understanding of the mechanism underlying L-OHP resistance is important to overcome the resistance and improve colorectal cancer treatment. This study aimed to identify new proteins that mediates L-OHP resistance in colorectal cancer and elucidate their mode of function. HT-29 cells were exposed to gradually increased concentration of L-OHP to select L-OHP resistant HT-29/L-OHP cell line. Proteomic analysis of HT-29 and HT-29/L-OHP cells were performed to identify differentially expressed proteins, including Poly(C)-binding protein 1 (PCBP1). PCBP1 expression level in 20 cases of L-OHP sensitive patients and 20 cases of L-OHP refractory patients was analyzed by immunohistochemistry. Chemoresistance and Akt activation in HT-29 and HT-29/L-OHP cells were analyzed by MTT assay and Western blot analysis. We identified 37 proteins showing differential expression in HT-29/L-OHP and HT-29 cells. In particular, PCBP1 protein level increased 15.6 fold in HT-29/L-OHP cells compared to HT-29 cells. Knockdown of PCBP1 sensitized HT-29/L-OHP and HT-29 cells to L-OHP, while overexpression of PCBP1 increased L-OHP resistance in HT-29 cells. In addition, PCBP1 expression was significantly higher in tumor samples from L-OHP refractory patients than in those from L-OHP responsive patients. Furthermore, we found that knockdown of PCBP1 inhibited the activation of Akt in HT-29/L-OHP and HT-29 cells. In conclusion, our findings suggest that PCBP1 is a molecular marker of L-OHP resistance in colorectal cancer and a promising target for colorectal cancer therapy.

Keywords: chemoresistance; colorectal cancer; oxaliplatin; poly(C)-binding protein 1.

Figure 1. Establishment of L-OHP resistant HT-29 cell line

(A) IC50 of HT-29 parental cells and HT-29/L-OHP cells. (B) Western blot analysis of MRP and P-gp expression in HT-29 parental cells and HT-29/L-OHP cells. β-actin was loading control.

Figure 2. Proteomic analysis of proteins involved in L-OHP resistance in HT-29/L-OHP cells

(A) 2D gel electrophoresis of HT-29 parental cells and HT-29/L-OHP cells. Arrows indicated protein spots with more than two fold expression difference between HT-29 parental cells and HT-29/L-OHP cells. (B) Enlarged view of protein spots of PCBP1, TUBB2A, ANXA3 and STIP1. (C) MALDI-TOF/TOF analysis of 37 identified proteins. (D) Western blot analysis of PCBP1, ANXA3, and STIP1 expression in HT-29/L-OHP cells. β-actin was loading control.

Figure 3. PCBP1 increased L-OHP resistance in HT-29 cells

(A) Western blot analysis of PCBP1 levels in HT-29, HT-29+PCBP1, HT-29-PCBP1, HT-29/L-OHP and HT-29/L-OHP-PCBP1 cells. β-actin was loading control. *P < 0.05, **P < 0.01 vs. corresponding control. (B) Cell survival curve of HT-29/L-OHP cells with or without PCBP1 knockdown, and cell survival curve of HT-29 cells with or without PCBP1 knockdown and with exogenous PCBP1 expression. (C) IC50 of HT-29/L-OHP cells with or without PCBP1 knockdown, and IC50 of HT-29 cells with or without PCBP1 knockdown and with exogenous PCBP1 expression. *P < 0.05 vs. corresponding control.

Figure 4. Higher PCBP1 expression in samples from L-OHP resistant patients

(A) Representative strong staining of PCBP1 in tumor tissue from L-OHP resistant patient. (B) Representative weak staining of PCBP1 in peri-cancerous tissue from L-OHP resistant patient. (C) Representative weak staining of PCBP1 in tumor tissue from L-OHP sensitive patient. (D) Representative weak staining of PCBP1 in peri-cancerous tissue from L-OHP sensitive patient. Scale bar: 50 μm. (E) PCBP1 level was significantly higher in L-OHP resistant tumor tissue than that in L-OHP sensitive tumor tissue or peri-cancerous tissue (P < 0.05).

Figure 5. Knockdown of PCBP1 led to decreased Akt Ser473 phosphorylation in HT-29 and HT-29/L-OHP cells

Western blot analysis of PCBP1, p-Ser473 Akt and total Akt levels in HT-29 and HT-29/L-OHP cells transfected with scramble siRNA (C) or PCBP1 siRNA (shPCBP1). Β-actin was loading control.