Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients

Abstract

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Keywords: African American prostate cancer; UGT2B28; androgen signaling; glucuronidation; metabolic regulation; prostate cancer; tumorigenesis.

Figure 1

Increased UGT2B28 protein expression during prostate cancer progression and in African American prostate cancer patients. (A). Percentage of overall staining of UGT2B28 in benign adjacent tissue, localized tumors, and metastatic tumors. (B). Percentage of staining of nuclear UGT2B28 in African American vs. European American prostate tumors. * p < 0.05. (C). Percent staining of cytoplasmic UGT2B28 in African American vs. European American prostate tumors. * p < 0.05. (D). Percentage of staining of nuclear and cytoplasmic UGT2B28 in African American benign adjacent prostate tissue and prostate tumors. * p < 0.05, **** p < 0.0001. The data are represented as the mean +/− the standard error of the mean (SEM).

Figure 2

(A–C). qPCR verification of UGT2B28 transcript levels in (A) LNCaP, (B) LAPC-4 and (C) VCaP cells upon shRNA-mediated UGT2B28 knockdown (UGT2B28 KD) or re-expression rescue (UGT2B28 R), compared to scrambled controls (UGT2B28 NT, ** p < 0.001, n = 3/group/cell line). The data are represented as the mean +/− the standard deviation (SD). (D). Immunofluorescence images showing UGT2B28 and AR expression and their localization in LNCaP and LAPC-4 cells containing control (UGT2B28 NT), UGT2B28 KD, and re-expression rescue (UGT2B28 R) grown in charcoal-stripped serum (CSS). Scale bar = 10 μm. (E). Same as in (D), but for LNCaP and LAPC-4 cells grown in 1 nM R1881. Scale bar = 10 μm. (F). UGT2B28 transcript expression levels in LNCaP cells treated with 1 nM 5α-dihydrotestosterone (DHT), 10 µM MDV-3100, 1 nM DHT+10 µM MDV-3100, and 300 nM ARCC4 for 24 h. The data are represented as the mean +/− the standard deviation (SD). * p < 0.01, n = 3. (G). ChIP-Exo of the UGT2B28 promoter region in LNCaP cells treated with 1 nM R1881 (full length AR binding, (top)) and LNCaP cells with doxycycline-induced AR-v7 (AR-v7 binding, (bottom)) reveal binding of AR and AR-v7, respectively.

Figure 3

UGT2B28 deficiency in PCa cells impairs organoid formation in vitro and prolongs tumor take and growth in vivo. (A). 10× magnified Green Fluorescence Protein (GFP) images of 3D organoids formed from stably transduced LNCaP cells containing control (NT), UGT2B28 KD, UGT2B28 re-expression (Control + OE), and UGT2B28 re-expression rescue (UGT2B28 R) in the KD setting (UGT2B28 rescue). The 3D organoid formation was conducted in triplicates/group/cell line in Millicell inserts for 24 h. Scale bar = 500 μm. (B). Same as in (A), but for LAPC-4 cells. (C). Tumor growth curve of xenografts generated using LNCaP cells containing NT, UGT2B28 KD, and UGT2B28 R in athymic nude mice supplemented with testosterone. (D). Same as in (C), but for LAPC-4. (E). Same as in (C), but for VCaP. For panels (C–E), please refer to legends for number of replicates in each group. Inset shows verification of UGT2B28 transcript expression in xenograft tumors obtained from each group. The data are represented as the mean +/− the standard deviation (SD). * p < 0.01, ** p < 0.001, ns: not significant.

Figure 4

Prolonged tumor take and delayed tumor growth in response to UGT2B28 downregulation is independent of ligand dependent AR signaling. (A). 10× GFP images of LNCaP organoids formed from non-targeting (NT), UGT2B28 KD, and UGT2B28 overexpression in KD4 background (UGT2B28 R) cells in response to treatment with charcoal-stripped serum (CSS), 1 nM 5α-dihydrotestosterone (DHT), 10 μM enzalutamide (MDV-3100), 300 nM ARCC4, 1 nM DHT + 10 μM MDV-3100, and fetal bovine serum (FBS) for 24 h. n = 3. Scale bar = 500 μm. (B). Tumor growth curve for NT, UGT2B28 KD, and UGT2B28 R VCaP xenografts in castrated, athymic nude mice. The data are represented as the mean +/− the standard deviation (SD). ** p < 0.001, *** p < 0.0001. NT, n = 6, UGT2B28 KD, n = 7, UGT2B28 R, n = 6.