12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

Masahiro Oka,Naoko Sumita,Masanobu Sakaguchi,Tetsushi Iwasaki,Toshinori Bito,Toshiro Kageshita,Ken-Ichi Sato,Yasuo Fukami,Chikako Nishigori

doi:10.1074/jbc.m109.001073

Abstract

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

Highlights

The transcription factor signal transducer and activator of transcription 3 (STAT3) was originally identified as a DNAbinding protein that responds to stimulation by epidermal growth factor and interleukin-6 (IL-6) and has an important role in their signaling [10, 11]
2 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; Dimethyl sulfoxide (DMSO), dimethyl sulfoxide; DN, dominant negative; EMEM, Eagle’s minimal essential medium; FCS, fetal calf serum; IL-6, interleukin-6; JAK, Janus kinase; PKC, protein kinase C; aPKC, atypical PKC; cPKC, classical/conventional PKC; nPKC, new/novel PKC; pS-STAT3, STAT3 phosphorylated on Ser727; pY-STAT3, STAT3 phosphorylated on Tyr705; PTP, protein-tyrosine phosphatase; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3
TPA significantly inhibited the in vitro growth as well as DNA synthesis of WM98-1, WM115, WM164, WM239A, and WM1205Lu cells, whereas it did not affect those of WM35 and WM39 cells (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Antibodies against STAT3 phosphorylated on Tyr705 (pY-STAT3) and STAT3 phosphorylated on Ser727 (pS-STAT3) were obtained from Cell Signaling Technology (Beverly, MA). Immunoblot Analysis—Immunoblot analysis for the determination of total STAT3, pY-STAT3, pS-STAT3, actin, PKC␣, PKC␤II, PKC␦, and PKC⑀ was carried out essentially as described previously [26]. The cells were washed with phosphate-buffered saline and lysed in 20 mM Tris-HCl (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM ␤-mercaptoethanol, 10 mM NaF, 1 mM sodium orthovanadate, and 50 ␮g/ml phenylmethylsulfonyl fluoride and centrifuged at 17,000 ϫ g for 10 min. Immunoblot analysis for the determination of total c-Src and phospho-c-Src was carried out essentially as described previously [27]. Equal amounts of protein extracts from the supernatants of samples for total STAT3, pYSTAT3, pS-STAT3, actin, PKC␣, PKC␤II, PKC␦, and PKC⑀, and the immunoprecipitates from samples for total c-Src and phospho-c-Src were separated by SDS-PAGE and transferred onto an Immobilon P membrane (Millipore, Bedford, MA). Statistics—Differences between results were assessed for significance using Student’s t test. p Ͻ 0.05 was considered to be statistically significant

RESULTS

All seven melanoma cell lines expressed similar levels of total

DISCUSSION