ECM1 regulates the resistance of colorectal cancer to 5-FU treatment by modulating apoptotic cell death and epithelial-mesenchymal transition induction

Abstract

5-Fluorouracil (5-FU) chemoresistance is a persistent impediment to the efficient treatment of many types of cancer, yet the molecular mechanisms underlying such resistance remain incompletely understood. Here we found CRC patients resistant to 5-FU treatment exhibited increased extracellular matrix protein 1 (ECM1) expression compared to CRC patients sensitive to this chemotherapeutic agent, and higher levels of ECM1 expression were correlated significantly with shorter overall survival and disease-free survival. 5-FU resistant HCT15 (HCT15/FU) cells expressed significantly higher levels of ECM1 relative to parental HCT15 cells. Changes in ECM1 expression altered the ability of both parental and HCT15/FU cells to tolerate the medication in vitro and in vivo via processes associated with apoptosis and EMT induction. From a mechanistic perspective, knocking down and overexpressing ECM1 in HCT15/FU and HCT15 cell lines inhibited and activated PI3K/AKT/GSK3β signaling, respectively. Accordingly, 5-FU-induced apoptotic activity and EMT phenotype changes were affected by treatment with PI3K/AKT agonists and inhibitors. Together, these data support a model wherein ECM1 regulates CRC resistance to 5-FU via PI3K/AKT/GSK3β pathway-mediated modulation of apoptotic resistance and EMT induction, highlighting ECM1 as a promising target for therapeutic intervention for efforts aimed at overcoming chemoresistance in CRC patients.

Keywords: ECM1; PI3K/AKT/GSK3βsignaling pathway; cell apoptosis; colorectal cancer; drug resistance; epithelial-mesenchymal transition.

FIGURE 1

ECM1 is associated with 5-FU resistance and poor prognosis in CRC patients. (A) Photographs illustrating the presence of ECM1 protein in tumor tissue samples from CRC patients. (B) Kaplan-Meier analyses of the OS of 5-FU-sensitive and -resistant patients. (C) The ECM1 expression of 5-FU-resistant and -sensitive patient samples was assessed. (D,E) The OS (D) and DFS (E) of patients expressing low and high levels of ECM1 were compared. Data are means ± SD (***p < 0.001, ****p < 0.0001).

FIGURE 2

ECM1 is involved in 5-FU resistance in CRC cells. (A) After treatment for 24 h with a range of 5-FU doses (0, 1, 2.5, 5, or 10 µg/ml), ECM1 levels in HCT15 cells were detected via Western immunoblotting. (B) After treatment for 24, 48, 72, or 96 h with 5 µg/ml of 5-FU, ECM1 protein levels in HCT15 cells were detected via Western immunoblotting. (C,D) qPCR and Western immunoblotting approaches detected ECM1 levels within HCT15 and HCT15/FU cells. (E–H) Western immunoblotting and qPCR were used to evaluate the efficacy of ECM1 overexpression or knockdown. Through CCK-8 and colony formation tests, the susceptibility of these cells to 5-FU was further examined. (I,J) The effects of ECM1 knockdown on 5-FU resistance in HCT15/FU cells. (K,L) The effect of excessive ECM1 expression on 5-FU sensitivity of HCT15 cells. Data are means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 3

5-FU-resistant CRC cells exhibit EMT induction and apoptosis-resistant phenotypes. (A) CCK-8 assays were used to compute IC50 and resistance index (RI) values for HCT15 and HCT15/FU cells. (B) HCT15 and HCT15/FU cell morphological characteristics were visualized. (C,D) Transwell and wound healing experiments investigated these cells’ relative migratory and invasive activity. (E) Both HCT15 and HCT15/FU cells have EMT-related protein levels. (F,G) TUNEL staining and flow cytometry were employed to examine the apoptotic death of HCT15 and HCT15/FU cells. (H) Apoptosis-associated protein expression was detected in HCT15/FU cells. Data are represented as means ± SD (**p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 4

Knocking down ECM1 alters CRC cell EMT induction. The morphology of the respective HCT15 cell types was examined when ECM1 was either knocked down (A) or overexpressed (D). (B,C) A high dose of 5-FU (150 µg/ml) was used to treat HCT15/FU-NC and shECM1 cells, after which their migration/invasion was assessed through wound healing and Transwell assays. (E,F) A 5 µg/ml dose of 5-FU was used to treat HCT15-NC and HCT15-ECM1 cells, after which their migration/invasion was detected through Transwell and wound healing assays. (G,H) EMT-associated protein expression was detected following ECM1 knocked down or overexpressed. Data are means ± SD (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 5

ECM1 regulates cellular resistance to 5-FU-induced apoptosis. The apoptotic death of 5-FU-treated cells was examined through TUNEL staining and flow cytometry. (A–H) The impact of altered ECM1 expression on the apoptosis of 5-FU-treated cells. (I,J) Apoptosis-related protein expression was detected via Western immunoblotting. Data are means ± SD (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 6

ECM1 enhances CRC cell resistance to 5-FU via the PI3K/AKT/GSK3β pathway. (A,B) The indicated PI3K/AKT pathway proteins and phosphoproteins were detected in HCT15/FU or HCT15 cells in which ECM1 was silenced or overexpressed. (C) PI3K/AKT-related protein expression was detected following 740 Y-P treatment for 24 h to confirm treatment efficacy. (D–G) After treatment with 740 Y-P for 24 h, the invasive and migratory activities of HCT15/FU-shECM1 cells were examined via Transwell and wound healing analyses (D,E). In contrast, their apoptotic death was detected via TUNEL staining and flow cytometric analyses (F,G). (H,I) Western immunoblotting was used to identify proteins involved in apoptosis and EMT. The data are shown as means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 7

Knocking down ECM1 enhances the in vivo chemosensitivity of HCT15/FU cells. (A–E) Knocking down ECM1 enhanced the 5-FU sensitivity of tumor cells, as evidenced by decreased tumor volume and weight on day 28 post-treatment with 5-FU. (F–I) Western immunoblotting and IHC staining were used to detect apoptosis-, EMT- and pathway-related proteins. Data are means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (J) Western immunoblotting was used to detect PI3K/AKT-related protein expression.