MUC1 Drives the Progression and Chemoresistance of Clear Cell Renal Carcinomas

Abstract

While the transmembrane glycoprotein mucin 1 (MUC1) is clustered at the apical borders of normal epithelial cells, with transformation and loss of polarity, MUC1 is found at high levels in the cytosol and is uniformly distributed over the entire surface of carcinoma cells, where it can promote tumor progression and adversely affects the response to therapy. Clear cell renal cell carcinoma (ccRCC), the main histotype of kidney cancer, is typically highly resistant to conventional and targeted therapies for reasons that remain largely unknown. In this context, we investigated whether MUC1 also plays a pivotal role in the cellular and molecular events driving ccRCC progression and chemoresistance. We showed, using loss- and gain-of-function approaches in ccRCC-derived cell lines, that MUC1 not only influences tumor progression but also induces a multi-drug-resistant profile reminiscent of the activation of ABC drug efflux transporters. Overall, our results suggest that targeting MUC1 may represent a novel therapeutic approach to limit ccRCC progression and improve drug sensitivity.

Keywords: MUC1; chemoresistance; kidney cancer.

Figure 1

Distinct MUC1 protein abundance in genetically engineered ccRCC cell line models. Whole-cell protein extracts from either MUC1-overexpressing ACHN clones or MUC1-depleted RCC4 clones were analyzed through Western blotting using antibodies against MUC1-C and β-actin.

Figure 2

Modulation of MUC1 expression influences the proliferation, migration, and invasiveness of ccRCC cells. Proliferation (A,B) and migration (C,D) were assessed using Incucyte technology, whereas cell invasion (E,F) was evaluated using 24-well Boyden chambers coated with Matrigel^® with 10% fetal bovine serum as a chemoattractant. MUC1 overexpression promotes the proliferation, migration, and invasiveness of ACHN cells (A,C,E), whereas MUC1 depletion in RCC4 cells (B,D,E) has the opposite effect. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 3

Expression of MUC1 in ccRCC cells is associated with a multi-drug-resistant phenotype. MUC1 ACHN-overexpressing cells (MUC1FL) (A), MUC1 RCC4-depeleted cells (MUC1KO) (B), and their respective controls were exposed to increasing concentrations of cisplatin, oxaliplatin, etoposide, SN38, paclitaxel, or vinblastine. IC50 (half-maximal inhibitory concentration), a valid marker of drug sensitivity, was determined in each condition after 72 h of drug treatment using the MTS assay. The overexpression of MUC1 confers the ACHN cells a multi-drug-resistant profile (A), whereas the depletion of MUC1 in the RCC4 cells (B) has the opposite effect. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 4

The expression of MUC1 in ccRCC cells is associated with their resistance to targeted therapies. MUC1-overexpressing ACHN cells (MUC1FL) (A), MUC1 RCC4-depeleted cells (MUC1KO) (B), and their respective controls were exposed to increasing concentrations of cabozantinib, crizotinib, dovitinib, or sunitinib. IC50 (half-maximal inhibitory concentration), a valid marker of drug sensitivity, was determined in each condition after 72 h of drug treatment using the MTS assay. While the modulation of MUC1 has no effect on sunitinib sensitivity (A,B), its overexpression impaired the responses of ACHN cells to the other tested targeted agents (A), whereas its depletion in RCC4 cells (B) has the opposite effect. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05 and ** p < 0.01).

Figure 5

MUC1 increases the expression of numerous efflux pumps. Relative expression of ABCC1–6, ABCB1, and ABCG2 was determined using qPCR in ACHN (A) and RCC4 cells (B). Overexpression of MUC1 in ACHN cells increases the expression of many members of the ABC transporter family (A), whereas its depletion in RCC4 cells (B) has the opposite effect. PPIA was used as an internal control. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05 and ** p < 0.01).

Figure 6

Modulation of MUC1 expression in ccRCC cells influences drug efflux pump activity. The intracellular fluorescence of the eFluxx-ID^TM gold detection reagent in ACHN (A) and RCC4 (B) cells is shown. The multi-drug resistance activity factor (MRAF) represented the P-gP, MDR1, and BCRP activities in ACHN (C) and RCC4 (D) cells and was determined using the eFluxx-ID^TM gold detection reagent in the presence (or not) of verapamil (an ABCB1/P-gP inhibitor), MK-571 (an ABCC1/MRP1 inhibitor), and novobiocin (an ABCG2/BCRP inhibitor). Overexpression of MUC1 in ACHN cells promotes overall drug efflux pump activity (A) and ABCB1-, ABCC1-, and ABCG2-specific activity (C), whereas its depletion in RCC4 cells decreases the overall drug efflux pump activity (B) and ABCB1-specific activity (D). Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05 and ** p < 0.01).

Figure 7

MUC1 increases the expression of cancer stem cell markers. Relative expression of Nanog and Sox2 was determined using qPCR in ACHN (A) and RCC4 cells (B). Overexpression of MUC1 in ACHN cells increases the expression of the stem cell markers Nanog and Sox2 (A), whereas its depletion in RCC4 cells (B) has the opposite effect. PPIA was used as an internal control. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).