Human prostatic acid phosphatase, an authentic tyrosine phosphatase, dephosphorylates ErbB-2 and regulates prostate cancer cell growth

Abstract

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.

FIGURE 1.

Analyses of tyrosine phosphorylation of ErbB-2 in different LNCaP and MDA PCa2b cells. Upper panels, A, LNCaP C-33 and C-81 cells were seeded in regular culture medium at a density of 5 × 10⁵/T25 flask for 3 days. p indicates phosphorylation. B, MDA PCa2b cells (passages 38 and 89) were seeded with a density of 1 × 10⁶/T25 flask in regular culture medium. Cells were fed with fresh medium and cultured for another 2 days. Immunoblottings were performed with Abs to different tyrosine phosphorylation sites of ErbB-2. After stripping, the membrane was hybridized with Ab to detect ErbB-2 protein. Similar results were obtained from over three sets of independent experiments. Lower panels, growth rates of different LNCaP and MDA PCa2b cells. A, LNCaP cells were seeded at a density of 3 × 10⁴ cells/well in 6-well plates in the regular culture medium. 3 days after plating, one set of attached cells was harvested and counted as day 0. The remaining cells were refreshed with the regular medium, and the total cell numbers were counted on the indicated days. B, for MDA PCa2b cell growth kinetics, cells were seeded in regular medium at a density of 1 × 10⁵ cells/well in 6-well plates and counted on days 3, 5, and 7. Data shown are the ratios of cell numbers normalized to the corresponding number on day 0. Bars = the range of duplicates. Similar results were obtained from at least three sets of independent experiments. Y, tyrosine.

FIGURE 2.

Co-immunoprecipitation of cPAcP and ErbB-2. A, confluent C-33 cells were maintained in a steroid-reduced medium for 48 h. Cells were then harvested, lysed, and immunoprecipitated by PAcP Ab (ATM-3) (36). The immunoprecipitate was electrophoresed and blotted (IB) with anti-ErbB-2 Ab (C-terminal (C-Ter) Ab) and anti-PAcP Ab (Sigma catalog number P-9808). B, C-33 cells in a steroid-reduced medium for 48 h were harvested and lysed for immunoprecipitation by ErbB-2 Ab (N-terminal (N-Ter) Ab). The immunoprecipitate was immunoblotted with anti-PAcP Ab (ATM-3) and anti-ErbB-2 Ab (C-terminal Ab). Similar results were obtained from three sets of independent experiments. C, LNCaP C-33 cells were seeded at a density of 0.6 × 10⁶/T75 (Subconfluent) and 4.5 × 10⁶/T75 (Confluent) for 3 days and then maintained in steroid-reduced medium for 2 days. Cells were harvested and separated into membrane (Mem), cytoplasmic (Cyto), and nuclear (Nu) protein subfractions using the subcellular protein fractionation kit. Immunoblottings were performed with respective Abs to detect ErbB-2, cPAcP, Shc proteins, EGFR, PYK2, and HNF3α. The intensity of the hybridization band was semiquantified and calculated for a total of 100% for each group. EGFR, PYK2, and HNF3α served as markers for the respective subcellular fractionation. D and E, confluent LNCaP C-33 cells were harvested after being maintaining in a steroid-reduced medium for 2 days. The total cell lysates, membrane, and cytoplasmic fractions, separated by the subcellular protein fractionation kit, were immunoprecipitated by cPAcP Ab (ATM-3) or ErbB-2 Ab (N-terminal Ab). The immunoprecipitate was immunoblotted with anti-PAcP Ab (Santa Cruz Biotechnology (Santa), sc-80908) and anti-ErbB-2 Ab (C-terminal Ab). Similar results were obtained from three sets of independent experiments.

FIGURE 3.

Effect of cPAcP expression on tyrosine phosphorylation of ErbB-2 in LNCaP C-81 cells. A, LNCaP C-81 cells were transiently transfected with WT PAcP, mutant PAcP H12A cDNAs, or vector alone. Cells were harvested after 48 and 72 h, respectively, and an equal amount of cellular lysates was separated by SDS-PAGE for Western blot analyses. cPAcP, the total and the specific tyrosine phosphorylation (p) level of ErbB-2, and phosphorylation of p52^Shc and PCNA were detected by the corresponding Ab. The protein levels of ErbB-2, Shc, and β-actin were examined with the respective Ab after the membranes were stripped. B, cPAcP, ErbB-2, and total tyrosine phosphorylation of ErbB-2 protein in WT PAcP cDNA-transfected LNCaP-28 and LNCaP-40 stable sublines were detected with anti-PAcP, anti-ErbB-2, and 4G10 anti-Tyr-phosphorylation Abs, respectively. For cell growth kinetics, cPAcP-28, cPAcP-40, and LNCaP C-81 cells were seeded at a density of 5 × 10⁴ cells/well in 6-well plates in regular medium for 3 days. One set of attached cells was harvested and counted as day 0. The remaining cells were fed with regular medium. Cells were harvested on days 2, 4, and 7 for counting. Similar results were obtained in two sets of independent experiments in duplicate wells. *, p < 0.05 versus LNCaP C-81 cells; **, p < 0.01 versus LNCaP C-81 cells.

FIGURE 4.

Effect of transient knockdown of cPAcP by siRNA in LNCaP C-33 cells on tyrosine phosphorylation of ErbB-2. A, lysate proteins of transiently transfected cells with different amounts of PAcP siRNA-126 plasmids for 48 h were analyzed for cPAcP, cyclin D1, PCNA, and the phosphorylation (p) levels of ErbB-2, p52^Shc, ERK1/2, Akt, Src, STAT3, and STAT5 proteins. After stripping the membrane, the protein levels of ErbB-2, Shc, ERK1/2, Akt, Src, STAT3, STAT5, and α-tubulin were detected, respectively. Similar results were obtained from five sets of independent experiments. B, LNCaP C-33 cells were plated at a cell density of 3 × 10⁵/well in 6-well plates for 48 h and then transfected with different amounts of PAcP siRNA-126 plasmids. Control cells were transfected with the vector containing scramble oligonucleotides. Cell numbers were counted 3 days after transfection. The ratio of cell growth was calculated by normalizing the cell number to that of the control. The result shown is the average from three sets of independent experiments in duplicates (n = 2 × 3). Bars = S.E.

FIGURE 5.

Kinetic effects of knockdown of cPAcP on tyrosine phosphorylation of ErbB-2. LNCaP C-33 cells were transiently transfected with siPAcP-126 plasmid or vector alone and harvested at 30, 36, and 48 h after transfection. An equal amount of cellular lysates was separated by SDS-PAGE for Western blot analyses. cPAcP, the specific tyrosine phosphorylation (p) level of ErbB-2, and phosphorylation of p52^Shc were detected by the corresponding Ab. The protein levels of ErbB-2, Shc, and α-tubulin were examined after the membranes were stripped. Similar results were obtained from three sets of independent experiments.

FIGURE 6.

Tyrosyl phosphorylation of ErbB-2 protein and the growth properties of PAcP knockdown stable subclones. A, siPAcP stable sublines were seeded in regular culture medium with 5 × 10⁵/T25 flask for 3 days. Total lysates from PAcP siRNA-transfected stable subclone cells (C-11, C-3, C-17) and the control subclone cells (V-3) were analyzed by Western blotting. The data shown are representative of three sets of independent experiments. B, for growth analyses, cells were seeded in duplicates at a density of 5 × 10⁴/well in 6-well plates. After 3 days, the numbers of each stable subclone cell were counted at the time points as indicated in the figure. The relative cell growth was represented by the ratio of total cell number normalized to the respective number on day 0. Similar results were obtained from three sets of independent experiments (n = 2 × 3). Bar = S.E. C, Cells were seeded in duplicates in the condition as described above. After 4 days of culturing in steroid-reduced medium, the cell number was counted. The relative cell growth was represented by the ratio of total cell number normalized to that of V-3 control cells. Similar results were obtained from three sets of independent experiments (n = 2 × 3). Bar = S.E. Inset, cPAcP protein was analyzed by Western blotting from one set of experiments. D, xenograft animal model for siPAcP subclone cells. 1 × 10⁶ cells of each subline in 0.1 ml of medium with 0.1 ml of Matrigel were injected into five female nude mice/group. The tumor growth was monitored weekly. *, p < 0.05 versus V-3 control cells.