BPTF Transduces MITF-Driven Prosurvival Signals in Melanoma Cells
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BPTF transduces MITF-driven prosurvival signals in melanoma cells Altaf A. Dara, Shahana Majidb, Vladimir Bezrookovea, Binh Phana, Sarah Ursua, Mehdi Nosratia, David De Semira, Richard W. Sagebiela, James R. Miller IIIa, Robert Debsa, James E. Cleaverc,1, and Mohammed Kashani-Sabeta,1 aCenter for Melanoma Research and Treatment, California Pacific Medical Center Research Institute, San Francisco, CA 94107; bDepartment of Urology, Veterans Affairs Medical Center, University of California, San Francisco, CA 94121; and cDepartment of Dermatology and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Contributed by James E. Cleaver, April 19, 2016 (sent for review December 10, 2015; reviewed by Peter Hersey and Hasan Mukhtar) Microphthalmia-associated transcription factor (MITF) plays a critical of the proliferative and metastatic capacity of melanoma cells, and complex role in melanocyte transformation. Although several along with induction of the apoptotic phenotype. BPTF exerted its downstream targets of MITF action have been identified, the precise oncogenic effects in melanoma, at least in part, by activation of mechanisms by which MITF promotes melanocytic tumor progression several genes, including BCL2, BCL-XL,andCCND2.Herewe are incompletely understood. Recent studies identified an oncogenic explore the role of BPTF in the MITF signal transduction pathway, role for the bromodomain plant homeodomain finger transcription and demonstrate activation of BPTF by MITF in melanoma cells. factor (BPTF) gene in melanoma progression, in part through activa- tion of BCL2, a canonical target of MITF signaling. Analysis of the BPTF Results promoter identified a putative MITF-binding site, suggesting that MITF Is Not Regulated by BPTF. Given that BCL2 is regulated by MITF may regulate BPTF expression. Overexpression of MITF resulted both MITF and BPTF, we explored whether BPTF regulates MITF in up-regulation of BPTF in a panel of melanoma and melanocyte cell expression. Stable, shRNA-mediated suppression of BPTF ex- lines. shRNA-mediated down-regulation of MITF in melanoma cells pression in C8161.9 and 1205-Lu melanoma cells had no effect on was accompanied by down-regulation of BPTF and BPTF-regulated MITF expression at the RNA (Fig. 1A) and protein levels (Fig. 1 B genes (including BCL2) and resulted in reduced proliferative capacity and C), respectively. Furthermore, overexpression of BPTF did not of melanoma cells. The suppression of cell growth mediated by MITF affect MITF expression in 1205-Lu cells (Fig. 1 D and E). These silencing was rescued by overexpression of BPTF cDNA. Binding of results indicate that BPTF does not regulate expression of MITF. MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of Effects of Regulation of MITF Expression on BPTF Levels. We then BPTF, as evidenced by increased luciferase activity driven by the BPTF assessed whether MITF could regulate BPTF expression. We promoter. These results indicate that BPTF transduces key prosurvival initially examined the consequences of overexpression of MITF signals driven by MITF, further supporting its important role in pro- cDNA to determine its effects on expression of BPTF and of moting melanoma cell survival and progression. BPTF-regulated genes. Overexpression of MITF in a panel of melanoma and melanocyte cell lines (Fig. 2 and Fig. S1) signifi- melanoma | signaling cascade | oncogenes cantly up-regulated the expression of BPTF and downstream icrophthalmia-associated transcription factor (MITF) is Significance Mknown to play a key role in melanocyte biology and pro- gression. MITF performs these functions by activating tran- Microphthalmia-associated transcription factor (MITF) is a master scription of many genes through interaction with the consensus regulator of survival of the melanocyte lineage, exerting its ef- DNA-binding sequence (CACGTG, termed E box) present on fects through a cascade of transcriptional activation by inter- the promoters of target genes. MITF controls expression of acting with a consensus DNA-binding sequence on the several proteins required for melanin synthesis, including ty- promoters of target genes. This study found that MITF reg- rosinase (TYR), tyrosinase-related protein 1 (TYRP1), and ulates expression of bromodomain plant homeodomain dopachrome tautomerose (DCT) (1–3). In addition, MITF plays a finger transcription factor (BPTF), which has been recently critical role in melanocyte survival and serves as a lineage- shown to promote melanoma cell growth and metastasis, specific oncogene in melanoma, as evidenced by its amplifica- and serves as a prognostic marker of survival associated tion in a subset of melanomas (4–6). MITF expression is reg- with melanoma. These results suggest that MITF directs its ulated by several factors, including SOX-10, CREB, PAX3, prosurvival program in melanoma cells, at least in part by LEF1, and ATF2 (7, 8). MITF in turn regulates a plethora of activating BPTF expression, providing additional insight into genes, with its complex role in melanoma biology, as its over- the MITF signaling cascade. The participation of BPTF in the expression has been shown to result in both proproliferative MITF pathway suggests a critical role for BPTF in maintain- and antiproliferative stimuli (4, 5, 9, 10). Accordingly, verified targets ing melanoma cell survival, further supporting its role in of MITF include both genes that promote cell survival and block melanoma progression. apoptosis, (e.g., CDK2, BCL2, DIAPH1, and TBX2), and genes that block cell cycle progression (e.g., CDKN2A and p21Cip1). Thus, Author contributions: A.A.D. and M.K.-S. designed research; A.A.D., S.M., V.B., B.P., and S.U. performed research; M.N., D.D.S., and J.R.M. contributed new reagents/analytic tools; although several downstream targets of MITF action have been A.A.D., S.M., V.B., M.N., D.D.S., R.W.S., J.R.M., R.D., J.E.C., and M.K.-S. analyzed data; and identified, the precise mechanisms by which MITF promotes A.A.D. and M.K.-S. wrote the paper. melanocytic tumor progression are still poorly understood. Reviewers: P.H., Melanoma Institute Australia; and H.M., University of Wisconsin. Recently, we identified a role for the bromodomain plant The authors declare no conflict of interest. homeodomain finger transcription factor (BPTF) gene in mela- Freely available online through the PNAS open access option. noma progression (11). BPTF is the largest subunit of the 1To whom correspondence may be addressed. Email: [email protected] or kashani@ nucleosome-remodeling factor, a key member of the imitation cpmcri.org. switch (ISWI) family of ATP-dependent chromatin-remodeling This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. factors. shRNA-mediated silencing of BPTF resulted in suppression 1073/pnas.1606027113/-/DCSupplemental. 6254–6258 | PNAS | May 31, 2016 | vol. 113 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1606027113 Downloaded by guest on September 26, 2021 ABMITF Binds to the BPTF Promoter. To understand the mechanism by C8161.9 1205-Lu which MITF regulates BPTF, we analyzed the BPTF promoter 120 120 C8161.9 and identified a putative consensus MITF-binding site (CACGTG) 100 100 BPTF 265 bp from the transcriptional initiation site (Fig. 6A). To deter- 80 80 1 0.27 mine if MITF activates the BPTF promoter, cotransfection of 60 60 MITF cDNA along with a vector encoding the luciferase gene MITF BPTF BPTF 40 40 * 1 0.78 driven by the promoter significantly up-regulated * 20 GAPDH transcriptional activity, as evidenced by increased reporter gene 20 expression compared with the control promoter vector (Fig. 6B). 0 Neg- BPTF- Relative mRNA expression (%) Relative mRNA 0 BPTF MITF expression (%) Relative mRNA BPTF MITF shRNA shRNA Finally, to determine whether the transcriptional activation of Neg shRNA Neg shRNA BPTF by MITF occurs through a direct interaction of MITF with BPTF-shRNA BPTF-shRNA the BPTF promoter, ChIP analysis was performed in two mela- CDE noma cell lines (C8161.9 and 1205-Lu) using the anti-MITF anti- body. A specific PCR band was obtained when primers specific for 1205-Lu * 140 BPTF the BPTF promoter were used, confirming the binding of MITF to 1 4.64 C BPTF the BPTF promoter (Fig. 6 ). These results indicate that MITF 2.5 1 0.34 MITF binds directly to the BPTF promoter to activate its transcription 2.0 1 1.14 and to transduce several of its prosurvival signals. MITF 1.5 GAPDH 1 0.98 Association Between Expression Levels of MITF and BPTF in Melanoma 1.0 pCMV6 pCMV-BPTF GAPDH Tissues. Finally, we assessed the potential association between 0.5 Neg- BPTF- expression levels of MITF and BPTF in human melanoma shRNA shRNA expression (Fold) Relative mRNA 0 specimens using The Cancer Genome Atlas (TCGA) analysis BPTF MITF of cutaneous melanoma (cancergenome.nih.gov). Normalized pCMV6 pCMV-BPTF RNA expression levels for MITF and BPTF were available for Fig. 1. Effects of suppression of BPTF by a specific anti-BPTF shRNA on 473 cases. Initially, a significant association was identified be- MITF expression at the RNA (A) and protein (B and C) levels in C8161.9 and tween expression of MITF and BPTF. Specifically, BPTF ex- 1205–Lu melanoma cells. (D and E) Effects of overexpression of BPTF cDNA pression was elevated in samples with high MITF expression on MITF RNA or protein expression in 1205-Lu melanoma cells. In A and D, (i.e., above the median) and reduced in samples with low MITF ± < data presented reflect mean SEM of three replicates. *P 0.05. expression (i.e., below the median, P < 0.0001). In addition, prosurvival genes—including BCL2, BCL-XL,andCCND2—at the RNA level. Overexpression of MITF cDNA (Fig. 3) signifi- A B cantly up-regulated expression of BPTF protein in C8161.9 cells, * C8161.9 + pCMV6 * 1205-pCMV6 along with that of BCL2, BCL-XL, and CCND2, compared with 250 C8161.9 + pCMV-MITF 300 1205-pCMV-MITF * B C * overexpression of control pCMV6 vector (Fig.