Expression and function of ATIP/MTUS1 in human prostate cancer cell lines

Abstract

Background: We have previously demonstrated Ang II type 2 (AT(2)-) receptor-mediated inhibition of EGF-induced prostate cancer cell growth in androgen-dependent (LNCaP) and independent (PC3) prostate cancer cell lines.

Methods: To explore the signaling pathways involved in this inhibitory effect, we examined the interaction of the AT(2)-receptor with its novel regulatory partner ATIP using real time PCR, over-expression, siRNA and [(3)H]thymidine incorporation assays.

Results: The results in human prostate cancer cell lines demonstrate the presence of ATIP in both cell lines examined, and suggest that (i) the AT(2)-receptor through an interaction with ATIP mediates an anti-growth factor effect in both androgen-dependent and androgen-independent cell lines; (ii) ATIP expression decreases as the rate of cell growth and androgen-independence increase; and (iii) EGF may act on cell growth in part by reducing the content of ATIP present in the cells.

Conclusions: The results support our earlier proposal in normal cell lines that ATIP is an important component of the cellular response to AT(2)-receptor activation. The results further suggest that a critical level of ATIP is required to mediate the effect of AT(2)-receptor activation to inhibit EGF mediated increases in cell growth. They also suggest that EGF may in part induce cell growth by suppressing the level of ATIP expression.

Fig. 1.

Effects of EGF and Ang II on (A) ATIP and (B) ATIP1 mRNA expression in LNCaP prostate cancer cells and (C) ATIP and (D) ATIP1 mRNA expression in PC3 cells grown to approximately 70% confluence.Values are expressed as % fold difference relative to untreated (control) expression (100%).^*,**,*** denote a significant decrease in ATIP mRNA expression in treated compared to untreated cells at P < 0.05, P < 0.01, and P < 0.001, respectively. Statistics were calculated using parametric ANOVAs with Tukey’spost-tests on ΔCt values from seven to nine independent determinations.

Fig. 2.

A : Fold-decrease in ATIP and ATIP1 mRNA expression following transfection of ATIP siRNA into LNCaP cells.^***indicatesa significant decrease in ATIP mRNA expression, P < 0.001. B : % Change in DNA synthesis in LNCaP cells transiently transfected with either ATIP siRNA or Alexa Fluor (negative control, reported as 100%) in the presence or absence of 10 ng/ml EGF and 500 nM Ang II or the combination of EGF and Ang II.C : 10 ng/ml EGF and 500 nM CGP42112A or the combination of EGF and CGP42112A for 72 hr.*and^***denote that there are significant decreases in ATIP or ATIP1or increases in DNA synthesis compared to untreated LNCaP cells transfected with the same plasmid (P < 0.05 and 0.001, respectively, repeated-measures ANOVA test with Tukey post-test).+and+++ denote that in cells treated with EGF there is a significant increase in DNA synthesis in cells transfected with siRNA compared to cells transfected with Alexa fluor P < 0.05 and 0.001, respectively.^# denotes that there is significantly less DNA synthesis in Alexa-fluor transfected cells treated with the combination of EGF and CGP42112A than in cells treated with EGF P < 0.05.^@ indicates that in cells treated with EGF and CGP42112A there is significantly higher DNA synthesis in cells transfected with siRNA compared to cells transfected with Alexa-fluor P < 0.05. Each value represents the mean standard error of the mean of four independent experiments. Statistics were calculated using a repeated measures ANOVA, with aTukey-Kramer Multiple Comparisons Test post-test.

Fig. 3.

A : Fold-decreasein ATIP and ATIP1mRNA expression following transfection of ATIP siRNA into PC3 cells.^*** indicates a significant decreasein ATIP mRNA expression, P < 0.001.B,C : % Change in DNA synthesis in PC3 cells transiently transfected with either ATIP siRNA or Alexa Fluor (negative control) in the presence or absence of (B) 10 ng/ml EGF and 500 nM Ang II and the combination of EGF and Ang II; or (C) 10 ng/mlEGF and 500 nM CGP42112A and the combination of EGF and CGP42112A for 72 hr.^***denotes that there is a significant increase in DNA synthesis compared to untreated PC3 cells transfected with the same plasmid(P < 0.001, repeated measures ANOVA test with Tukey post-test).- denotes that there is a significant difference in DNA synthesis between ATIP silenced and untreated control cells.^### denotes that there is significantly less DNA synthesisin Alexa Fluor-transfected cells treated with the combination of EGF and Ang II or EGF and CGP42112A thanin cells treated with EGF P < 0.001.Each value represents the mean + standard error of the mean of four independent experiments.

Fig. 4.

A: ERK1 and ERK2 phosphorylation (phospho-ERK1 and phospho-ERK2, respectively) after 5 min stimulation with the indicated dose of EGF and Ang II in PC3 cells transiently transfected with pcDNA3 or ATIP1 plasmid in a single representative experiment.Unphosphorylated ERK2 (ERK2) in the same nitrocelluloseblot. B : Summary of ERK2 phosphorylation in PC3 cells transfected with either pcDNA3 or pcDNA3-ATIP1 following a 5 min stimulation with EGF alone or in the presence of Ang II. Values are expressed as themean and standard error ofmean ofphospho- ERK2relativeto theamountofunphosphorylatedERK2.^**indicates that there is a significant increase in ERK2 phosphorylation in EGF-treated compared to untreated PC3 cells transfected with the same plasmid at P < 0.01 (Friedman Nonparametric Repeated Measures ANOVA with Dunn’s post-test).^# denotes that in pcDNA3-transfected PC3 cells ERK2 phosphorylation is significantly less in cells co-administered EGF and Ang II than in cells treated with EGF alone, P < 0.05.⁺⁺ indicates that there is significantly less ERK2 phosphorylation in untreated ATIP1-transfected than pcDNA3 transfected PC3 cells, P < 0.01.^@@ indicates that there is significantly less ERK2 phosphorylation in EGF-treated ATIP1-transfected than EGF-treated pcDNA3 transfected PC3 cells, P < 0.01.^% denotes that there is a significant increase in ERK2 phosphorylation in EGF + Ang II-treated compared to untreated PC3 cells transfected with ATIP1 P < 0.05.^&&& indicates that there is a significant decrease in ERK2 phosphorylation in EGF + Ang II treated ATIP1-transfected cells when compared to pcDNA3 transfected PC3 cells treated in the same way, P < 0.001 (Students t-test).