Androgen Receptor Signaling Induces Cisplatin Resistance via Down-Regulating GULP1 Expression in Bladder Cancer

Abstract

The underlying molecular mechanisms of resistance to cisplatin-based systemic chemotherapy in bladder cancer patients remain to be elucidated, while the link between androgen receptor (AR) activity and chemosensitivity in urothelial cancer has been implicated. Our DNA microarray analysis in control vs. AR knockdown bladder cancer lines identified GULP1 as a potential target of AR signaling. We herein determined the relationship between AR activity and GULP1 expression in bladder cancer cells and then assessed the functional role of GULP1 in cisplatin sensitivity. Androgen treatment in AR-positive cells or AR overexpression in AR-negative cells considerably reduced the levels of GULP1 expression. Chromatin immunoprecipitation further showed direct interaction of AR with the promoter region of GULP1. Meanwhile, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment compared with respective controls. GULP1 knockdown also resulted in a significant decrease in apoptosis, as well as a significant increase in G2/M phases, when treated with cisplatin. In addition, GULP1 was immunoreactive in 74% of muscle-invasive bladder cancers from patients who had subsequently undergone neoadjuvant chemotherapy, including 53% of responders showing moderate (2+)/strong (3+) expression vs. 23% of non-responders showing 2+/3+ expression (P = 0.044). These findings indicate that GULP1 represents a key downstream effector of AR signaling in enhancing sensitivity to cisplatin treatment.

Keywords: androgen receptor; chemoresistance; cisplatin; immunohistochemistry; urothelial cancer.

Figure 1

Relationship between AR and GULP1 expression in bladder cancer cells. (a) Western blotting of AR and GULP1 in 5637, 647V, UMUC3, and TCCSUP. (b) Western blotting of AR and GULP1 in 5637-vector vs. 5637-AR, 647V-vector vs. 647V-AR, and UMUC3-AR-shRNA vs. UMUC3-control-shRNA. (c) Western blotting of AR and GULP1 in 647V/647V-AR or UMUC3-AR-shRNA/UMUC3, cultured for 24 h with ethanol (mock), 10 nM DHT, or 5 µM HF. GAPDH served as a loading control.

Figure 2

Binding of AR to the GULP1 promoter in bladder cancer cells. There are three putative binding sites in the GULP1 promoter region. A ChIP assay using UMUC3 cell lysates immunoprecipitated with an anti-AR antibody or mouse IgG as a negative control. The DNA fragments were PCR amplified with sets of GULP1 promoter-specific primers, and the PCR products (i.e., 338 bp for binding site 1, 173 bp for binding site 2, 131 bp for binding site 3) were electrophoresed on 1% agarose gel. A fraction of the mixture of protein-DNA complex (i.e., 1% of total cross-linked, reserved chromatin prior to immunoprecipitation) was used as “input” DNA.

Figure 3

Effects of GULP1 knockdown on CDDP cytotoxicity in bladder cancer cells. Western blotting of GULP1 in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines (a) or UMUC3-control-shRNA vs. UMUC3-GULP1-shRNA sublines (b). GAPDH served as a loading control. An MTT assay in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines (c) or UMUC3-control-shRNA vs. UMUC3-GULP1-shRNA sublines (d) cultured for 72 h in the presence of various concentrations (0–20 µM) of CDDP. Cell viability is presented relative to that of each subline without CDDP treatment. Each value represents the mean (±SD) from a total of 6 determinants. * p < 0.05 (vs. control-shRNA).

Figure 4

Effects of GULP1 knockdown on the growth of bladder cancer cells. (a) MTT assay in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines cultured for 1–4 days. Cell viability is presented relative to that of the control subline at day 1. Each value represents the mean (±SD) from a total of 6 determinants. (b) TUNEL assay in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines in the absence or presence of 5 µM CDDP cultured for 72 h. Apoptosis counted as a percentage of at least 500 cells is presented relative to that of the control subline. Each value represents the median (±SE) from a total of 16 determinants. (c) Cell cycle phase analysis in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines in the absence or presence of 5 µM CDDP cultured for 72 h. Color changes are associated with cells in G1 (yellow), S (light green), G2 (dark green), and M (intense blue) phases (original magnification: 100×). Proportion of G2/M counted as a percentage represents the mean (+SD). (d) Wound-healing assay in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines gently scratched and cultured for 24 h. Cell migration determined by the rate of cells filling the wound area is presented relative to that of the control subline (original magnification: 40×). Each value represents the mean (+SD) from a total of 10 determinants. (e) Transwell invasion assay in 647V-control-shRNA vs. 647V-GULP1-shRNA sublines. Cell invasion determined by counting the number of invaded cells in the lower chamber under a microscope is presented relative to that of the control subline (original magnification: 40×). Each value represents the mean (+SD) from a total of 10 determinants. * p < 0.001 (vs. control-shRNA).

Figure 5

Immunohistochemistry of GULP1 in surgical specimens. (a) Expression of GULP1 in non-neoplastic urothelium and urothelial tumor (original magnification: 200×). Immunoreactivity is seen mainly in the cytoplasm of urothelial cells. (b) Kaplan–Meier curves for progression-free survival in patients with GULP1-negative (n = 13) vs. GULP1-positive (n = 65) non-muscle-invasive tumors. (c) Kaplan–Meier curves for progression-free survival in patients with GULP1-negative (n = 20) vs. GULP1-positive (n = 31) muscle-invasive tumors.