Dihydrotestosterone upregulates the expression of epidermal growth factor receptor and ERBB2 in androgen receptor-positive bladder cancer cells

Abstract

Androgen receptor (AR) signals play important roles in bladder carcinogenesis and tumor progression. Activation of the epidermal growth factor receptor (EGFR) family, including EGFR and ERBB2, leads to bladder cancer cell growth and correlates with poor patients' prognosis. However, cross talk between AR and EGFR/ERBB2 pathways in bladder cancer remains poorly understood. In AR-positive bladder cancer UMUC3 and TCC-SUP cells, dihydrotestosterone (DHT) increased the expression of EGFR and ERBB2 both in mRNA and in protein levels, and an anti-androgen hydroxyflutamide antagonized the effect of DHT. The necessity of AR was confirmed by silencing the receptor, using short hairpin RNA (shRNA), in UMUC3 cells, as well as by expressing the receptor in AR-negative 5637 cells. Of note were much higher basal levels of EGFR and ERBB2 in UMUC3-control-shRNA than in UMUC3-AR-shRNA and those of EGFR in 5637-AR than in 5637-V. DHT additionally upregulated the levels of phosphorylation of EGFR (pEGFR) and its downstream proteins AKT (pAKT) and ERK1/2 (pERK), induced by EGF treatment, in AR-positive cells. Immunohistochemistry on cystectomy specimens showed strong associations between expressions of AR and EGFR (PZ0.0136), pEGFR (PZ0.0041), ERBB2 (PZ0.0331), or pERK (PZ0.0274), but not of pAKT (PZ0.5555). The Kaplan-Meier and log-rank tests further revealed that positivity of AR (PZ0.0005), EGFR (PZ0.2425), pEGFR (PZ0.1579), ERBB2 (PZ0.2997), or pERK (PZ0.1270) and negativity of pAKT (PZ0.0483) were associated with tumor progression. Our results indicate that AR activation upregulates the expression of EGFR and ERBB2 in bladder cancer cells. AR signals may thus contribute to the progression of bladder cancer via regulation of the EGFR/ERBB2 pathways.

Loss of GATA3 in bladder cancer promotes cell migration and invasion

“…Simply, in bladder cancer tissues GATA3 expression correlates with ERβ loss, an indicator of better prognosis, but with AR overexpression, an indicator of worse prognosis. 2,[25][26][27][28]31 Therefore, the clinical consequence of GATA3 loss as to tumor progression may be dependent on not only the predominance or functional activity of sex hormone receptors but also the levels of circulating sex hormones and the systemic hormonal milieu.…”

“…2,31 Briefly, cells (3 × 10 3 ) seeded in 96-well tissue culture plates were incubated with medium for 24-96 h. Before measuring, we added 10 μl of MTT (Sigma) stock solution (5 mg/ml) to each well with 0.1 ml of medium for 4 h at 37 °C. Then, we replaced the medium with 100 μl of DMSO, incubated for 5 min at room temperature, and measured the absorbance at a wavelength of 570 nm with background subtraction at 630 nm.…”

“…Western blot Protein extraction and western blot were performed, as described previously, 31,37 with minor modifications. Equal amounts of protein obtained from cell extracts were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore) by electroblotting, using a standard protocol.…”

“…Real-time PCR was then performed in 15 μl system by using SYBR GreenER qPCR SuperMix for iCycler (Invitrogen), as described previously. 31,38 The primer sequences are given as below: GATA3 (forward, 5′-CAGCCTTCGC TTGGGCTTAA T-3′; reverse, 5′-GATGGCAGGC TCAGTGATGT C-3′), VEGF (forward, 5′-CTGTACCTCC ACCATGCCAA G-3′; reverse, 5′-GGTACTCCTG GAAGATGTCC ACC-3′), MMP-2 (forward, 5′-TACAGGATCA TTGGCTACAC ACC-3′; reverse, 5′-GGTCACATCG CTCCAGACT-3′), and MMP-9 (forward, 5′-TGTACCGCTA TGGTTACACT CG-3′; reverse, 5′-GGCAGGGACA GTTGCTTCT-3′). GAPDH (forward, 5′-AAGGTGAAGG TCGGAGTCAA C-3′; reverse, 5′-GGGGTCATTG ATGGCAACAA TA-3′) was used as an internal control.…”

Androgen activates β-catenin signaling in bladder cancer cells

“…AR signals have been implicated in bladder carcinogenesis and tumor progression. Specially, promising evidence further documents a critical role of AR in bladder cancer cell proliferation (Miyamoto et al 2007, Johnson et al 2008, Boorjian et al 2009, Wu et al 2010, Zheng et al 2011. Nonetheless, the mechanism by which AR signaling modulates bladder cancer progression remains poorly understood.…”