Role of the MUC1-C oncoprotein in the acquisition of cisplatin resistance by urothelial carcinoma

Abstract

Mucin 1 C-terminal subunit (MUC1-C) has been introduced as a key regulator for acquiring drug resistance in various cancers, but the functional role of MUC1-C in urothelial carcinoma (UC) cells remains unknown. We aimed to elucidate the molecular mechanisms underlying the acquisition of cisplatin (CDDP) resistance through MUC1-C oncoprotein in UC cells. MUC1-C expression was examined immunohistochemically in tumor specimens of 159 UC patients who received CDDP-based perioperative chemotherapy. As a result, moderate to high MUC1-C expression was independently associated with poor survival in UC patients. Using human bladder cancer cell lines and CDDP-resistant (CR) cell lines, we compared the expression levels of MUC1-C, multiple drug resistance 1 (MDR1), the PI3K-AKT-mTOR pathway, and x-cystine/glutamate transporter (xCT) to elucidate the biological mechanisms contributing to the acquisition of chemoresistance. MUC1-C was strongly expressed in CR cell lines, followed with MDR1 expression via activation of the PI3K-AKT-mTOR pathway. MUC1-C also stabilized the expression of xCT, which enhanced antioxidant defenses by increasing intracellular glutathione (GSH) levels. MUC1 down-regulation showed MDR1 inhibition along with PI3K-AKT-mTOR pathway suppression. Moreover, it inhibited xCT stabilization and resulted in significant decreases in intracellular GSH levels and increased reactive oxygen species (ROS) generation. The MUC1-C inhibitor restored sensitivity to CDDP in CR cells and UC murine xenograft models. In conclusion, we found that MUC1-C plays a pivotal role in the acquisition of CDDP resistance in UC cells, and therefore the combined treatment of CDDP with a MUC1-C inhibitor may become a novel therapeutic option in CR UC patients.

Keywords: GO-203; MDR1; MUC1-C; PI3K-AKT-mTOR; urothelial carcinoma; xCT.

FIGURE 1

Immunostaining of mucin 1 C‐terminal subunit (MUC1‐C) in patients with urothelial carcinoma (UC). Representative immunostaining of low, moderate, or high expression of MUC1‐C in surgical specimens from (A) patients with upper tract urothelial carcinoma (UTUC) treated with cisplatin (CDDP)‐based adjuvant chemotherapy, and (B) muscle‐invasive bladder cancer (MIBC) patients treated with CDDP‐based neoadjuvant chemotherapy are shown. The histoscore was calculated by applying the following formula: mean percentage × intensity (range, 0‐300). Cases with less than 30 were defined as low, 30‐90 as moderate, and 90 or higher as high MUC1‐C expression. Low power field scale bar, 200 µm and high power field scale bar, 50 µm. A Kaplan‐Meier curve of the cancer‐specific survival in (C) patients with UTUC treated with radical nephroureterectomy and who underwent adjuvant chemotherapy (D) MIBC patients treated with radical cystectomy and who underwent neoadjuvant chemotherapy according to MUC1‐C expression

FIGURE 2

Generation of cisplatin (CDDP)‐resistant cancer cells from 2 urothelial cancer cell lines and comparisons of the expression of mucin 1 C‐terminal subunit (MUC1‐C). A, Acquired CDDP resistance in T24 and UMUC3 urothelial cancer cells. T24CR and UMUC3CR cells were generated by exposing the corresponding wild‐type (T24WT and UMUC3WT) cells to an increasing concentration of 3 μM CDDP over 12 mo. Brief schema showing the generation of CDDP‐resistant (CR) cell lines (upper panel). Graphs show changes in cytotoxicity between WT and CR cells of T24 (middle panel) and UMUC3 cells (lower panel) exposed to various concentrations of CDDP for 48 h. ** P < .01, *** P < .001. B, mRNA levels of MUC1‐C in WT and CR cells measured by RT‐PCR (upper panel, T24WT vs T24CR; lower panel, UMUC3WT vs UMUC3CR). * P < .05. C, Protein levels of MUC1‐C in WT and CR cells measured by a western blot analysis (upper panel, T24WT vs T24CR; lower panel, UMUC3WT vs UMUC3CR). D, mRNA level of ABCB1 (responsive gene of MDR1) in WT and CR cells (upper panel, T24WT vs T24CR; lower panel, UMUC3WT vs UMUC3CR). ** P < .01. E, Protein expression levels of total‐AKT, p‐AKT, total‐mTOR, p‐mTOR, total‐S6K1, p‐S6K1, MDR1, and β‐actin in WT and CR cells (upper panel, T24WT vs T24CR; lower panel, UMUC3WT vs UMUC3CR). F, Immunofluorescence staining of MDR1 and MUC1‐C in T24WT, T24CR, UMUC3WT, and UMUC3CR cells. The nucleus was stained by DAPI, MUC1‐C was stained by Alexa Fluor 488, and MDR1 was stained by an Alexa Fluor 555 antibody

FIGURE 3

Mucin 1 C‐terminal subunit (MUC1) knockdown downregulates multiple drug resistance 1 (MDR1) via the inhibition of the PI3K‐AKT‐mTOR pathway and increases cisplatin (CDDP) sensitivity in CDDP‐resistant (CR) cells. A, mRNA expression of MUC1‐C was down‐regulated with siRNA for MUC1 (siMUC1#1 and siMUC1#2), but not with siRNA for a non‐targeting control (NTC) (upper panel, T24CR; lower panel, UMUC3CR). *** P < .001. B, Western blot analysis of MUC1‐C, total‐AKT, p‐AKT, total‐mTOR, p‐mTOR, total‐S6K1, p‐S6K1, MDR1, and β‐actin after transfection with siRNA for NTC and MUC1 (siMUC1#1 and siMUC1#2). (upper panel, T24CR; lower panel, UMUC3CR). C, The graph shows the viability of cells exposed to various concentrations of CDDP for 48 h after transfection with siRNA for NTC, transfection agent only, and MUC1 (siMUC1#1, and #2) (upper panel, T24CR cells; lower panel, UMUC3CR cells). ** P < .01. *** P < .001

FIGURE 4

Mucin 1 C‐terminal subunit (MUC1‐C) stabilizes x‐cystine/glutamate transporter (xCT) expression and decreases reactive oxygen species (ROS) generation by increasing intracellular glutathione (GSH) levels in T24CR cells. A, Protein expression level of xCT in T24WT and T24CR cells. B, Intracellular GSH levels in T24WT and T24CR cells after a 24‐h exposure to the vehicle control, cisplatin (CDDP) (1 and 10 µM), and 100 µM l‐buthionine‐sulfoximine (BSO). *** P < .001. C, Intracellular ROS production in T24WT and T24CR cells after a 24‐h exposure to CDDP (10 µM) measured by flow cytometry. The graph shows ROS levels in cells treated with the vehicle control and 10 µM CDDP in T24WT and T24CR cells. *** P < .001. D, Protein expression of xCT in T24CR cells transfected with NTC and siMUC1. E, Intracellular GSH levels in T24CR cells transfected with NTC and siMUC1 after a 24‐h exposure to the vehicle control, CDDP (1 and 10 µM), and 100 µM BSO. *** P < .001. F, Intracellular ROS production in T24CR cells transfected with NTC and siMUC1 after exposure to CDDP (10 µM) measured by flow cytometry. The graph shows ROS levels in T24CR cells treated with the vehicle control and 10 µM CDDP after the transfection with NTC and siMUC1. *** P < .001

FIGURE 5

The MUC1‐C inhibitor GO‐203 restores sensitivity to cisplatin (CDDP) in chemoresistant tumor cells. A, mRNA expression of ABCB1 in T24CR and UMUC3CR cells treated with 5 µM control peptide (CP‐2) and 5 µM GO‐203. *** P < .001. B, Protein expression of MUC1‐C, total‐AKT, p‐AKT, MDR1, xCT, and β‐actin after a 48‐h exposure to CP‐2 and after 24‐h and 48‐h exposures to GO‐203. C, The cell viabilities of T24CR cells and UMUC3CR cells exposed to various concentrations of CDDP with a combination of 5 µM CP‐2 or 5 µM GO‐203 48 h after exposure (upper panel, T24CR cells; lower panel, UMUC3CR cells). * P < .05, ** P < .01, and *** P < .001. D, Treatment in vivo. UMUC3CR cells (2 × 10⁶ cells) were implanted subcutaneously into the flank of nude mice. When a palpable tumor had developed, each group of 8 mice were treated with vehicle control of CP‐2 (daily ip injection of 18 mg/kg), CDDP alone (ip injection of 5 mg/kg on day 1 and 15), GO‐203 alone (daily ip injection of 18 mg/kg), or a combination of CDDP and GO‐203. (Upper panel) Representative subcutaneous tumors extracted from mice treated with CP‐2 (vehicle), CDDP alone, GO‐203 alone, and a combination of CDDP and GO‐203. (Lower panel) Growth of tumor volume in each group. Mean tumor volumes are shown in mm³. * P = .012, vehicle control vs GO‐203 alone; * P = .044, GO‐203 alone vs the combination of CDDP and GO‐203; *** P < .001, CDDP alone vs the combination of CDDP and GO‐203. E, Schema of the functional role of MUC1‐C for the acquisition of CDDP resistance by UC cells. MUC1‐C promotes ABCB1/MDR1 expression via PI3K/AKT pathway activation. MDR1 functions as an energy‐dependent efflux pump to discharge CDDP from tumor cells. MUC1‐C also contributes to the stabilization of xCT protein expression. xCT stabilization interacts to increase intracellular GSH levels, which results in a decrease in ROS production. The 2 crucial mechanisms induce CDDP resistance upon UC cells