Sialylation Inhibition Can Partially Revert Acquired Resistance to Enzalutamide in Prostate Cancer Cells

Abstract

Prostate cancer is a lethal solid malignancy and a leading cause of cancer-related deaths in males worldwide. Treatments, including radical prostatectomy, radiotherapy, and hormone therapy, are available and have improved patient survival; however, recurrence remains a huge clinical challenge. Enzalutamide is a second-generation androgen receptor antagonist that is used to treat castrate-resistant prostate cancer. Among patients who initially respond to enzalutamide, virtually all acquire secondary resistance, and an improved understanding of the mechanisms involved is urgently needed. Aberrant glycosylation, and, in particular, alterations to sialylated glycans, have been reported as mediators of therapy resistance in cancer, but a link between tumour-associated glycans and resistance to therapy in prostate cancer has not yet been investigated. Here, using cell line models, we show that prostate cancer cells with acquired resistance to enzalutamide therapy have an upregulation of the sialyltransferase ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) and increased levels of α2,6-sialylated N-glycans. Furthermore, using the sialyltransferase inhibitor P-SiaFNEtoc, we discover that acquired resistance to enzalutamide can be partially reversed by combining enzalutamide therapy with sialic acid blockade. Our findings identify a potential role for ST6GAL1-mediated aberrant sialylation in acquired resistance to enzalutamide therapy for prostate cancer and suggest that sialic acid blockade in combination with enzalutamide may represent a novel therapeutic approach in patients with advanced disease. Our study also highlights the potential to bridge the fields of cancer biology and glycobiology to develop novel combination therapies for prostate cancer.

Keywords: castrate resistance; combination therapies; enzalutamide; prostate cancer; sialic acid; sialyltransferase inhibitor.

Figure 1

Enzalutamide-resistant prostate cancer cells have upregulation of ST6GAL1. (A) Western blot analysis of ST6GAL1 in the VCaP control and enzalutamide-resistant VCaP (VCaP^EnzR) cells. ST6GAL1 levels are increased in both cell pellet and conditioned media samples from the VCaP^EnzR cells. GAPDH is included as a loading control. (B) Analysis of ST6GAL1 levels in conditioned media samples from the VCaP control and VCaP^EnzR cells using pre-validated sandwich ELISA assays [77]. The levels of ST6GAL1 are significantly higher in conditioned media from VCaP^EnzR cells (n = 3, unpaired t-test, *** p = 0.0002). (C) Western blot analysis of ST6GAL1 in the LNCaP control and enzalutamide-resistant LNCaP (LNCaP^EnzR) cells. GAPDH is used as a loading control. The levels of ST6GAL1 are increased in both cell pellet and conditioned media samples (D) Analysis of ST6GAL1 levels in conditioned media samples from the LNCaP control and LNCaP^EnzR cells using pre-validated sandwich ELISA assays [77]. The levels of ST6GAL1 are significantly higher in conditioned media from the LNCaP^EnzR cells compared to the control LNCaP cells (n = 6, unpaired t-test, ** p = 0.0014). Results are representative of three biological repeats and are presented as the mean ± standard error. Original western blots are presented in File S1.

Figure 2

Enzalutamide-resistant prostate cancer cells have upregulation of ST6GAL1 and increased levels of α2,6-sialylated glycans. (A) SNA lectin immunofluorescence shows VCaP^EnzR cells have increased levels of ST6GAL1 and α2-6 sialylation (SNA, the lectin from Sambucus nigra, recognises α2-6-linked sialylated N-glycans [91]). (B) SNA lectin immunofluorescence shows LNCaP^EnzR cells have increased levels of ST6GAL1 and α2-6 sialylation compared to control LNCaP cells. DNA is stained with Hoechst. Scale bar = 200 µM.

Figure 3

The sialyltransferase inhibitor P-SiaFNEtoc blocks α2,6 sialylation in the VCaP^EnzR and LNCaP^EnzR prostate cancer cells. (A) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N-glycans in the VCaP control and VCaP^EnzR cells treated with 20 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 6 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N-glycans (detected using SNA lectin). Control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats. (B) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N-glycans in LNCaP control and LNCaP^EnzR cells treated with 2 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 3 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N-glycans (detected using SNA lectin). The control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats.

Figure 3

The sialyltransferase inhibitor P-SiaFNEtoc blocks α2,6 sialylation in the VCaP^EnzR and LNCaP^EnzR prostate cancer cells. (A) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N-glycans in the VCaP control and VCaP^EnzR cells treated with 20 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 6 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N-glycans (detected using SNA lectin). Control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats. (B) Detection of immunofluorescent staining of ST6GAL1 and α2,6-sialylation of N-glycans in LNCaP control and LNCaP^EnzR cells treated with 2 µM of the sialyltransferase inhibitor P-SiaFNEtoc for 3 days. Treatment of both cell lines with P-SiaFNEtoc inhibits α2,6-sialylation of N-glycans (detected using SNA lectin). The control cells were treated with DMSO. Scale bar = 50 µm. The images are representative of three biological repeats.

Figure 4

Sialic acid blockade using P-SiaFNEtoc partially re-sensitises prostate cancer cells to the second-generation androgen receptor antagonist enzalutamide. (A) The VCaP^EnzR cells have increased resistance to enzalutamide compared to the control VCaP cells. The control VCaP and VCaP^EnzR cells were treated with a range of enzalutamide concentrations (0–500 µM). Cell viability was measured after 6 days using a CellTitrer-Glo^® luminescence assay. The IC₅₀ values (the concentration of enzalutamide which reduced cellular viability by 50% relative to the DMSO control) was 3.46-fold greater in the VCaP^EnzR cells (8.95 µM for VCaP control cells and 30.97 µM for the VCaP^EnzR cells). (B) Treatment of the VCaP^EnzR cells with P-SiaFNEtoc partially reverts resistance to enzalutamide. The control VCaP and VCaP^EnzR cells were treated with 20 µM P-SiaFNEtoc and a range of concentrations of enzalutamide (0–500 µM) for 6 days. Inhibiting sialylation in the VCaP^EnzR cells reduced the IC₅₀ value from 30.97 µM to 19.10 µM, indicating a partial reversion of their resistance to enzalutamide. (C) The LNCaP^EnzR cells have increased resistance to enzalutamide compared to the control LNCaP cells. The control LNCaP and LNCaP^EnzR cells were treated with a range of enzalutamide concentrations (0–500 µM). Cell viability was measured after 3 days using a CellTitre-Glo^® luminescence assay. The IC₅₀ value was 2.27-fold higher in the LNCaP^EnzR cells (53.33 µM for LNCaP control cells and 121.06 µM for the LNCaP^EnzR cells). (D) Treatment of the LNCaP^EnzR cells with P-SiaFNEtoc partially reverts their resistance to enzalutamide. The control LNCaP and LNCaP^EnzR cells were treated with 2 µM P-SiaFNEtoc and a range of concentrations of enzalutamide (0–500 µM) for 3 days. A DMSO-only control arm was included for each cell line. Inhibiting sialylation in the LNCaP^EnzR cells reduced the IC₅₀ value from 121.06 µM to 74.30 µM, indicating a partial reversion of their resistance to enzalutamide. A line of best fit was utilised for interpolating the IC₅₀ value. Results are presented as the cell viability (luminescence) relative to the respective DMSO control against the log of the enzalutamide concentration (nM) used for each cell line. Results are presented as the mean ± standard error and are representative of three biological repeats.