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.
Highlights
Because testicular androgens are involved in prostate carcinogenesis, systematic androgen ablation therapies are usually performed in advanced stages of prostate cancer (PCa)3 [1, 2]
ERK1/2 are activated by phosphorylation in prostate carcinomas and PCa cells in which cPAcP is decreased, suggesting that decreased cPAcP resulted in activating ErbB-2 and ERK1/2 signaling [11, 32]
CPAcP plays a regulatory role of controlling the activation of ErbB-2, and in cPAcP-null PCa cells, activated ErbB-2-induced signaling can be involved in ligand-independent AR activation, leading to HR progression of these cells
Summary
Materials—RPMI 1640 medium, gentamicin, FBS, trypsin/ EDTA, and Lipofectamine PLUSTM reagents were purchased from Invitrogen. Anti-phospho-Erk1/2 (Thr202/Tyr204) Ab was from United States Biological (Swampscott, MA). Anti-PCNA Ab, anti-ErbB-2/Neu Ab (C-18) (9G6), anti-cyclin D1Ab, anti-Erk1/2 Ab, anti-SHP1 (SH-PTP1) Ab, anti-SHP-2 (SH-PTP2) Ab, anti-Akt Ab, anti-PAcP Ab (045), and horseradish peroxidase-conjugated polyclonal secondary. Anti-PAcP antiserum (ATM-3) was obtained as described previously [22]. LNCaP cells were routinely maintained in the regular medium, i.e. phenol red-positive RPMI 1640 medium supplemented with 5% FBS, 2 mM glutamine, and 50 g/ml gentamicin. The LNCaP cell model was described originally by Lin et al [30] and further characterized by Igawa et al. 126 consistently showed the high efficiency of PAcP knockdown, stable subclones were established in LNCaP C-33 cells by transfecting with siPAcP-126 pSUPER plasmid
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