A Chemical Biology Approach Identifies AMPK As a Modulator Of

ORIGINAL ARTICLE A chemical biology approach identiﬁes AMPK as a modulator of melanoma oncogene MITF

V Borgdorff1,2, U Rix2,3, GE Winter2, M Gridling2,ACMu¨ ller2, FP Breitwieser2, C Wagner1, J Colinge2, KL Bennett2, G Superti-Furga2 and SN Wagner1,2

The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-speciﬁc expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identiﬁed as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.

Oncogene (2014) 33, 2531–2539; doi:10.1038/onc.2013.185; published online 3 June 2013 Keywords: MITF; melanoma; AMPK; compound C; midostaurin; sunitinib

INTRODUCTION Thus, the activation of MITF is tightly coupled to subsequent The basic helix–loop–helix leucine zipper transcription factor, degradation. Additional post-translational modifications of 11 microphthalmia-associated transcription factor (MITF), has been MITF include phosphorylation by GSK3b at serine 298 and 12,13 shown to be a melanoma-specific oncogene in addition to a sumoylation, both of which have been shown to modulate master regulator of melanocytes.1 Most melanomas express MITF activity. MITF and a significant proportion of melanomas harbour an The importance of a tight regulation of MITF protein levels is amplification of this gene.1 In addition, two groups recently reflected by emerging evidence showing that the protein level of indentified a germline mutation in MITF that predisposes to MITF dictates function, a concept formulated in the rheostat 5,14,15 melanoma.2,3 One of the transcriptional targets of MITF is the anti- model of MITF function. This model reconciles the, at first apoptotic protein Bcl-2,4 thus explaining the dependence of both sight, paradoxical observation that MITF is of crucial importance melanocytes and melanoma cells on MITF for survival. Recently, it in both differentiated melanocytes and melanoma cells. The was shown that MITF induces transcription of multiple genes model does so by postulating that high levels of MITF result in a involved in DNA replication, DNA damage repair and mitosis differentiated phenotype, intermediate levels in a proliferative in melanoma cells.5,6 All these processes are a prerequisite for phenotype, low levels in a stem cell/invasive phenotype and 6,16 proper cell proliferation. Therefore, targeting MITF as a means complete absence in a senescent phenotype. Each state on of combatting melanoma is attractive. Therapeutic efforts to the MITF rheostat is characterised by the expression of distinct target this protein, however, are hindered by the fact that molecular markers, for example, low MITF levels are associated 17 transcription factors lack a catalytic domain that can be targeted. with the expression of stem cell marker Oct-4; intermediate Given that the expression and activity of MITF are tightly levels of MITF with high expression of proliferation markers 18 regulated, it should be possible to indirectly target MITF by such as CDK2 and CDK4, and cells highly expressing MITF are interfering with its upstream modulators. Key established pigmented and express tyrosinase. modulators of MITF are ERK and RSK,7 members of the mitogen- We hypothesised that drug-mediated inhibition of MITF- activated protein kinase pathway that is hyperactivated in the activating molecules could impair the viability of melanocytic majority of melanomas because of the presence of an oncogenic cells and assessed a panel of different classes of drugs for their mutation in BRAF.8 Activation of this pathway results in phospho- capacity to (indirectly) downregulate MITF. We observed that the ERK- and phospho-RSK1-mediated phosphorylation of MITF at multi-kinase inhibitors midostaurin and sunitinib, compounds with serine 73 and 409, respectively, inducing its transcriptional an overlapping target-spectrum,19,20 downregulated MITF protein activity.7,9,10 Phosphorylation of MITF at these sites is followed levels. Subsequent chemical proteomic analysis of the cell-specific by its ubiquitination and proteasome-mediated degradation. target molecules shared by both drugs led to the identification of

1Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria. Correspondence: Dr SN Wagner or Dr V Borgdorff, Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: [email protected] or [email protected] 3Current address: Drug Discovery Department, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA. Received 6 April 2012; revised 29 January 2013; accepted 12 April 2013; published online 3 June 2013 AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2532 AMP-activated kinase (AMPK) as a kinase important not only reduced cell growth and induction of apoptosis (Supplementary for the modulation of post-translational phosphorylation of Figures S2a–c), comparable to what we observed with BRAFV600E MITF, but also for the maintenance of MITF protein levels in melanocytes. melanocytic cells. We assessed a panel of 14 compounds that are either already approved by the US Food and Drug Administration or under clinical evaluation. Together, these compounds target a wide spectrum of signalling pathways. Most of the drugs used were RESULTS kinase inhibitors (Supplementary Table S1). Initially, we screened Kinase inhibitors midostaurin and sunitinib downregulate MITF for reversion of the phenotype induced by MITF in BRAFV600E/MITF protein levels melanocytes, that is, rescue of contact inhibition as indicated by We hypothesised that by identifying drugs that downregulate the absence of foci formation. Next, we assessed whether the thus MITF protein expression, we could highlight modulators of MITF selected drug(s) also downregulated MITF protein levels. From through which its expression or activity could be indirectly this two-step procedure, the multi-kinase inhibitor midostaurin targeted. As a model, we used p’Mel/hTERT/CDK4(R24C)/p53DD was identified as a drug that downregulates MITF protein levels. cells, a human telomerase (hTERT)-immortalised primary human In support of this finding, sunitinib (a kinase inhibitor with an melanocyte line (hereafter indicated as (parental) immortalised overlapping target spectrum19,20) also downregulated MITF melanocytes) and derivatives thereof. These latter cells have protein levels (Figures 2a and b). been genetically engineered to express either BRAFV600E alone (hereafter indicated as BRAFV600E melanocytes) or a combination of BRAFV600E and haemagglutinin (HA)-tagged MITF (hereafter Midostaurin and sunitinib share kinase targets in melanocytic cells indicated as BRAFV600E/MITF melanocytes).1 Ectopic expression of To ascertain which of the shared kinase(s) targeted by midostaurin BRAFV600E in parental immortalised melanocytes resulted in a and sunitinib is/are responsible for the maintenance of MITF strong reduction of MITF protein expression1,18 (Figure 1a) and its protein levels in melanocytic cells, the target profile of each drug target gene Bcl-2 (Figure 1b),4 while co-expression of MITF in BRAFV600E/MITF melanocytes was determined using a chemical induced Bcl-2 (Figure 1b). Importantly, the remaining low MITF proteomic approach. Analogues of both drugs were prepared, protein levels in BRAFV600E expressing human melanocytes which allowed for immobilisation followed by affinity purification (Supplementary Figure S1a) are still of functional importance, of drug-interacting proteins (Supplementary Figure S3). In vitro evidenced by the fact that RNA interference with MITF expression kinase assays with the respective cognate targets confirmed in BRAFV600E melanocytes results in reduction of cell growth retention of inhibitory activity for each analogue (Supplementary (Supplementary Figure S1b). In contrast to BRAFV600E melanocytes, Table S2). Drug pull-downs with midostaurin and sunitinib were BRAFV600E/MITF melanocytes have been shown to form colonies performed with lysates of BRAFV600E/MITF melanocytes and in soft agar.1 Consistently, BRAFV600E/MITF melanocytes showed protein eluates analysed by gel-free one-dimensional liquid contact-independent growth when cultured on plastic (Figure 1c). chromatography mass spectrometry (1D-LCMS). Of 42 kinases RNA interference with MITF or Bcl-2 expression in BRAFV600E/MITF detected, in total, 12 and 11 kinase targets of midostaurin and melanocytes using two different siRNAs for each gene resulted in sunitinib, respectively, were identified as the most prominent

Figure 1. Ectopic expression of BRAFV600E and MITF in immortalised human melanocytes induces molecular and phenotypic changes. (a) MITF expression is strongly reduced upon ectopic BRAFV600E expression and can be regained by concomitant ectopic expression of MITF. MITF western blot of immortalised human melanocytes, BRAFV600E melanocytes and BRAFV600E/MITF melanocytes. (b) Bcl-2 expression is induced by MITF. Bcl-2 western blot of immortalised melanocytes with and without ectopically expressed BRAFV600E and MITF. (c) MITF overexpression in BRAFV600Emelanocytes confers the ability of contact-independent growth. Cropped, representative images are shown.

Oncogene (2014) 2531 – 2539 & 2014 Macmillan Publishers Limited AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2533

Figure 2. Multi-kinase inhibitors midostaurin and sunitinib down-regulate MITF protein levels in BRAFV600E/MITF melanocytes. (a) HA western V600E blot of BRAF /MITF melanocytes treated with 10 mM midostaurin, 10 mM sunitinib or DMSO control. Cells were treated 1 day after seeding and whole-cell extracts prepared 24 h after start of treatment. (b) Fluorescence images of BRAFV600E/MITF melanocytes stained with MITF antibody. One day after seeding, cells were treated with 3 mM midostaurin or 3 mM sunitinib and 24 h later cells were ﬁxed and immunoﬂuorescent staining performed. Cropped, representative images are shown.

Table 1. Shortlist of candidate kinases and signalling pathways modulating MITF as identiﬁed by gel-free one-dimensional liquid chromatography mass spectrometry after drug pull-downs with midostaurin and sunitinib in BRAFV600E/MITF melanocytes

Target kinases Signalling pathways

Midostaurin Sunitinib Midostaurin Midostaurin and Sunitinib and Sunitinib

TBK1 TBK1 TBK1 NFKB (TNF) signalling AAK1 CAMK2D AAK1 Clathrin-mediated endocytosis AMPKa1 CAMK2G AMPKa1 AMPK signalling AURKA AMPKa1 CAMK2D Calcium signalling CAMK2D AAK1 IKBKE NFKB (TNF) signalling BMP2K IKBKE CAMK2G Calcium signalling GSK3B ULK3 CAMK2B Calcium signalling IKBKE CAMK2B GSK3A YES1 CAMK2G MAP2K1 CAMK2B MAP2K2 MAP3K11 Kinases are listed in descending order of peptide count. Cutoff criterion was a minimal protein sequence coverage of 10%. interactors, when target selection was based on a minimum protein sequence coverage of 10%. Seven of the targets were shared by both the drugs (Table 1, Supplementary Table S3). In addition to the gel-free one-dimensional liquid chromatography mass spectrometry experiments, a quantitative proteomic analysis of midostaurin- and sunitinib-enriched proteins was Figure 3. Kinome-wide target profiles of midostaurin and sunitinib performed using isobaric tag for relative and absolute quantitation in BRAFV600E/MITF melanoma cells as determined by a quantitative (iTRAQ) labelling combined with gel-free two-dimensional liquid chemical proteomic approach (iTRAQ-labelling plus gel-free chromatography mass spectrometry. With this approach, kinases two-dimensional liquid chromatography mass spectrometry). equally enriched by both drugs and proteins preferentially The Human Kinome Map was adapted with permission from Cell targeted by either of the compounds (cutoff value: X2-fold Signaling Technology (www.cellsignal.com). Kinases that were enrichment) were detected. In total, 47 kinases were identified, of enriched more strongly with midostaurin than with sunitinib which 11 kinases were equally enriched by both the drugs (by at least 2-fold) are depicted in brown, kinases equally enriched by both the drugs (between 0.5- and 2-fold) are represented by tan (Figure 3 and Supplementary Table S4). Comparison of the seven circles and kinases more strongly enriched with sunitinib than with candidate kinases obtained from the one-dimensional liquid midostaurin (by at least 2-fold) are shown in grey. Kinases that could chromatography mass spectrometry analysis with the 11 candi- not be quantitated by iTRAQ are displayed in white. The actual date kinases obtained by iTRAQ combined with two-dimensional relative quantitative values are given in Supplementary Table S4. liquid chromatography mass spectrometry revealed two kinases that were identified by both approaches: AMPKa1 and AAK1. Given the identified role of AMPK in cancer biology,21 this protein V600E V600E was selected for further validation experiments. immortalised melanocytes, BRAF and BRAF /MITF melanocytes, as well as several melanoma cell lines using an antibody targeting acetyl coenzyme A carboxylase (ACC) phosphorylated at AMPK inhibition downregulates MITF protein levels serine 79, a site uniquely targeted by AMPK.22,23 Parental First, we asked whether AMPK signalling is active in human immortalised human melanocytes showed low levels of pACC, melanocytic cells and performed western blot analysis of parental whereas BRAFV600E melanocytes contained rather high levels of

& 2014 Macmillan Publishers Limited Oncogene (2014) 2531 – 2539 AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2534

Figure 4. Inhibition of AMPK is associated with downregulation of MITF in BRAFV600E/MITF melanocytes. (a) pACC western blot analysis of parental immortalised melanocytes and derived cell lines overexpressing BRAFV600E and MITF. (b) pACC and MITF western blot analysis of BRAFV600E/MITF melanocytes after treatment with the indicated doses of compound C for 24 h. (c) Images of MITF immunoﬂuorescence V600E performed with BRAF /MITF melanocytes after 20 mM compound C treatment. Cells were treated 1 day after seeding and ﬁxed 24 h later. Green: MITF; red: DAPI-stained nuclei. Cropped, representative images are shown. (d) BRAFV600E/MITF melanocytes were reverse transfected with 30 nM of the indicated siRNAs/ siRNA sets and 4 days later cell lysates were prepared for MITF western blotting.

pACC (Figure 4a), comparable to the levels observed in and BRAFV600E melanocytes as shown by annexin V/propidium human melanoma cells WM983A, SK-Mel-5 and MALME-3M iodide (PI) staining (Figure 5). Here, BRAFV600E melanocytes (Supplementary Figure S4a). BRAFV600E/MITF melanocytes showed overexpressing MITF showed lower percentages of annexin lower levels of pACC (Figure 4a, Supplementary Figure S4a). These V/PI-positive cells as compared to BRAFV600E melanocytes, data show the AMPK pathway to be active in human melanocytic suggesting that higher levels of MITF and lower levels of pACC cells, both melanocytes and melanoma cells. To investigate the (and thus of active AMPK, Figure 4a) are associated with decreased effect of AMPK inhibition on MITF protein expression, we sensitivity to compound C. Interestingly, pACC expression could performed western blot analysis and immunofluorescence also be detected in SK-Mel-5 melanoma cells carrying with BRAFV600E/MITF melanocytes treated with compound C, a an inactivating mutation in AMPK’s upstream activator LKB1 compound identified in a chemical library screen as a potent (also named serine/threonine kinase 11 (STK11)) (Supplementary inhibitor of AMPK.24 Compound C treatment resulted in an Figure S4a), consistent with LKB1-independent activation of AMPK inhibition of AMPK activity (reflected by a decrease of pACC in these cells. Compound C-mediated AMPK inhibition of SK-Mel-5 protein levels) along with a downregulation of MITF protein levels cells resulted in reduced viability (Supplementary Figures S4b and (Figures 4b and c). c), indicating that AMPK activity is required for the viability of AMPK is a heterotrimeric protein consisting of an a-catalytic these cells as well. In addition, compound C treatment of several subunit (either AMPKa1 or AMPKa2) and a b- and g-regulatory other melanoma cell lines showed a similar reduction of cell subunit.25 To further validate the results obtained by chemical viability as observed in SK-Mel-5 melanoma cells (Supplementary inhibition, we performed RNA interference with the expression of Figure S4d). AMPKa1 and AMPKa2 in BRAFV600E/MITF melanocytes. As shown in Figure 4d and Supplementary Figure S5, siRNA-mediated knockdown of AMPKa1 alone and in combination with AMPKa2 AMPK inhibition induces phosphorylation of MITF followed resulted in a clear reduction of MITF protein levels. These data by its degradation further substantiate the role of AMPK in maintaining cellular MITF To dissect the mechanism underlying MITF downregulation upon protein levels. AMPK inhibition, a time-course experiment was performed with compound C. MITF phosphorylation9,10 was induced by compound C within the first 4 h, followed by an almost complete AMPK inhibition impairs cell viability loss of detectability after 24 h of treatment (Figure 6a). At the latter Downregulation of both pACC and MITF upon AMPK inhibition timepoint we also observed a downregulation of MITF target with compound C (at a dose of 10 mM and higher) was associated gene Bcl-2. Downregulation of MITF could be prevented by with reduced cell viability of both BRAFV600E/MITF melanocytes pre-treatment with proteasome inhibitor MG132 (Figure 6b).

Oncogene (2014) 2531 – 2539 & 2014 Macmillan Publishers Limited AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2535

Figure 5. AMPK inhibition induces apoptosis in BRAFV600E and BRAFV600E/MITF melanocytes. Cells were treated with the indicated doses of compound C and 48 h later annexin V/PI staining was performed.

Figure 6. Compound C induces ERK-dependent phosphorylation of MITF, which is followed by its degradation. (a) BRAFV600E/MITF melanocytes were treated with 20 mM compound C 1 day after seeding. After 1, 4, 8 and 24 h, cell extracts were prepared and western blotting V600E performed with the indicated antibodies. (b) One day after seeding, BRAF /MITFmelanocytes were pre-treated with 25 mM proteasome inhibitor MG132 or DMSO for 1 h and subsequently treated with 20 mM compound C in the presence or absence of MG132. After 6.5 h, cell extracts were prepared and western blotting performed with the indicated antibodies. (c) One day after seeding, BRAFV600E/MITF melanocytes were pre-treated for 4 h with 100 nM or 1 mM MEK inhibitor U0126, 20 mM PD98059 or DMSO control and subsequently treated with 20 mM compound C in the presence or absence of U0126 or PD98059. After 2 h, cell extracts were prepared and western blotting performed with the V600E indicated antibodies. (d) BRAF /MITF melanocytes were reverse transfected with 30 nM of the indicated siRNA sets and 4 days later cell lysates were prepared for pERK western blotting.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2531 – 2539 AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2536 U0126 These data show that downregulation of MITF via AMPK inhibition MEK LKB1 CAMKK2 is a consequence of proteasomal degradation and that, in PD98059 agreement with a previous report,10 degradation of MITF was preceded by increased phosphorylation (Figures 6a and b). PP2A? ERK AMPK Compound C DUSP? MITF phosphorylation upon AMPK inhibition is dependent on p90RSK active ERK Key kinases responsible for MITF phosphorylation are the MITF ACC mTORC1 mitogen-activated protein kinase pathway members ERK1/ ERK2.7,9,10 To determine whether these molecules contribute to the initial induction of MITF phosphorylation upon AMPK inhibition with compound C, we performed a western blot analysis Bcl-2 p70S6K of phospho-ERK levels upon treatment of BRAFV600E/MITF melanocytes with compound C. As shown in Figure 6c, compound C induced phospho-ERK together with phosphorylated MITF. In eIF4B addition, RNA interference with expression of AMPK using two Figure 7. A schematic depiction of AMPK signalling in melanocytic independent siRNA sets targeting AMPKa1 and AMPKa2 resulted cells. AMPK is activated by LKB1 and calmodulin-dependent in an upregulation of pERK (Figure 6d), further advocating a role protein kinase 2. AMPK activates ERK phosphatases, either protein for pERK in the regulation of MITF via ERK. Conversely, pre- and co- phosphatase 2A or dual specificity phosphatase, or both. Inhibition treatment with MEK1/MEK2 inhibitors U0126 or PD98059 inter- of AMPK by compound C results in ERK activation because of fered with the induction of phospho-ERK and (at least partially) reduced activity of the ERK phosphatases. Activated ERK subse- prevented the induction of MITF phosphorylation by AMPK quently phosphorylates MITF, either directly or indirectly through activation of p90RSK. This can be rescued by inhibition of MEK by inhibition (Figure 6c), substantiating the contribution of phos- U0126 and PD98059. pho-ERK in the phosphorylation of MITF upon AMPK inhibition. AMPK has been shown to activate protein phosphatase 2A26 and 27 to induce dual specificity phosphatases. Both classes of 34,35 28 LKB1. Our results show that, even in the context of an phosphatases can dephosphorylate phospho-ERK. Consistently, V600E V600E activating BRAF mutation and an inactivating mutation in we observed an induction of pERK levels in BRAF /MITF LKB1, AMPK still plays an important role in melanocytic cells as it melanocytes upon treatment with okadaic acid (Supplementary may profoundly regulate MITF protein levels and activity. Figure S6), an established inhibitor of protein phosphatase 2A, 29 A possible explanation may be that AMPK can be activated which can also inhibit dual specificity phosphatases. not only by LKB1, but also by calmodulin-dependent protein kinase 2.36–38 By sequence homology, calmodulin-dependent protein kinase 2 is the mammalian kinase closest to LKB1.39 DISCUSSION Although tissue-specific deletion has shown that LKB1 is the major The transcription factor MITF plays a crucial role in maintaining the activator of AMPK in several tissue types, calmodulin-dependent viability of melanocytic cells,1,4,6 and its essential function is protein kinase 2 seems to be a key activator of AMPK in neurons restricted to cells of the melanocyte cell lineage, mast cells and and T cells.39 Given that neurons and melanocytes are derived osteoclasts.30 Moreover, MITF is an oncogene in at least a subset from the same progenitor cell population (both cell types stem of melanoma tumours.1–3 Direct chemical targeting of MITF, from the neural crest), it is reasonable to assume that calmodulin- however, remains a challenge because of the lack of a catalytic dependent protein kinase 2 could also be an important activator domain. An elegant indirect targeting approach in which MITF of AMPK in melanocytic cells. Furthermore, our data point towards was targeted by interfering with mechanisms required for its a regulation of MITF protein levels by AMPK inhibition via the expression has been shown to be successful.31 Multiple histone induction of ERK signalling, consistent with the recently deacetylase inhibitors reduced MITF expression in human demonstrated inhibition of ERK signalling by AMPK in cardiac melanoma cells both in vitro and in vivo when xenografted onto fibroblasts.40 Figure 7 schematically depicts how AMPK signalling mice, thus suppressing melanoma cell proliferation both in vitro may impact on MITF in melanocytic cells. and in vivo. However, histone deacetylase inhibitors affect The link between AMPK and MITF may also be interesting in the numerous additional proteins, thereby potentially inducing light of the previously demonstrated relationship between LKB1 multiple unexpected and adverse effects when applied (an upstream activator of AMPK) and pigmentation. Patients with therapeutically.31,32 We aimed at identifying a drug with a Peutz-Jeghers syndrome, a dominantly inherited disorder often narrower target spectrum that could still decrease MITF activity caused by inactivating germline mutations in LKB1, present with by targeting an upstream modulator of MITF. We found that melanocytic macules at the skin and the mucosa.41 These data both midostaurin and sunitinib (two multi-kinase inhibitors with a suggest that, in contrast to our observations, inactivation of the highly overlapping target spectrum) mediated downregulation of AMPK signalling pathway may rather result in the permanent MITF in melanocytic cells. Using a chemical proteomic approach to activation of MITF. A likely explanation for this difference is the deconvolute the targets of these drugs in melanocytes, we could fact that salt-inducible kinase 2, an AMPK family member and show that AMPK is essential to maintain MITF protein levels in also a target of LKB1, represses CREB-specific coactivator TORC1 these cells. Consistently, AMPK has recently been identified as one (also called CRTC1), thereby interfering with CREB-induced of the targets of sunitinib in intact PC3 human prostate cancer MITF transcription.42 In patients with Peutz-Jeghers syndrome, cells and in cell-free kinase assays with AMPKa1-containing the failure to activate salt-inducible kinase 2 because of inactive complexes immunoprecipitated from PC3 cells.33 LKB1 is expected to result in increased CREB-mediated MITF AMPK is an energy sensor of the cell that maintains cellular transcription, rather than in downregulation of MITF protein levels energy homeostasis by stimulating catabolic pathways upon an as described in our study. Together these data suggest that the increase of the cellular AMP/ATP ratio.25 Recent studies have AMPK family of proteins may have a dual impact on MITF shown that in melanomas harbouring a BRAFV600E mutation, AMPK expression. AMPK (family members) may inhibit MITF transcription activation is impaired because of the mitogen-activated protein via the repression of CREB-specific co-activator TORC1 on one kinase--dependent inactivation of AMPK’s upstream activator hand, but maintain MITF protein expression on the other.

Oncogene (2014) 2531 – 2539 & 2014 Macmillan Publishers Limited AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2537 The role of AMPK in tumorigenesis seems to be context- melanoma cell lines A375, MALME-3M and SK-Mel-5 were obtained from dependent.21 It has been shown to inhibit mammalian target the American Type Culture Collection (ATCC, Manassas, VA, USA). WM983A of rapamycin complex 1 (mTORC1), which is activated by the cells were obtained from Meenhard Herlyn (The Wistar Institute, AKT signalling pathway.25 Consistently, tumour growth of Philadelphia, PA, USA). All melanoma cell lines were cultured in RPMI 1 xenotransplanted human breast cancer cells in mice was (Gibco, Invitrogen) containing 10% FBS at 37 C/5% CO2. suppressed by AMPK activator OSU-5343 and a retrospective analysis of diabetes patients taking the anti-diabetic drug Drug screen metformin, which activates AMPK, showed a significantly All compounds used in the screen were dissolved in DMSO at a 44 reduced risk for breast cancer. In contrast, AMPK was shown concentration of 10 mM. to aid in the adaptation of tumour cells to a hypoxic environment Cells were plated at a density of 3500 cells per well of a 96-well plate in and H-Ras-transformed AMPKa-null mouse embryonic fibroblasts 100 ml media. Twenty-four hours after plating, all cells were adherent and formed much smaller tumours than wild-type mouse embryonic drugs, prediluted in media, were added. Final cellular DMSO concentration was 0.1% in every well. Each compound was assayed at nine different fibroblasts.45 Furthermore, AMPK was found to be activated in concentrations ranging from 20 mM to 0.3 nM in triplicate. At 24, 48 and 72 h human prostate cancer specimens and cell lines as indicated by after drug addition, cell growth was monitored for the presence/absence increased expression of pACC compared to normal prostate tissue, of contact inhibition and foci formation. and inhibition of AMPK in human prostate cancer cells decreased cellular proliferation.46 In melanoma, only a limited amount of Chemical proteomics data are available on AMPK and tumour biology. Recently, Tomic et al.47 showed that metformin has an anti-proliferative effect on Immobilisation and affinity purification. c-Sunitinib was synthesised by Gateway Pharma (Freeland, UK). c-Midostaurin was synthesised internally human melanoma cells by stimulating autophagy and apoptosis 48 by amidification of staurosporine with N-Boc-protected m-aminomethyl- and Janjetovic et al. observed similar effects in B16 mouse benzoic acid in the presence of HATU and diisopropylethylamine and melanoma cells. In both the experimental settings, however, subsequent deprotection with trifluoroacetic acid (all from Sigma-Aldrich). metformin-induced anti-tumour effects were mostly independent The coupleable drug analogues were immobilised on NHS-activated of AMPK. In agreement with studies showing that MITF is crucial Sepharose 4 Fast Flow resin (GE Healthcare Bio-Sciences AB, Uppsala, for the viability of melanocytic cells,1,4,6 we found that MITF Sweden) as described previously.49 Affinity chromatography and elution downregulation through inhibition of AMPK signalling decreased were performed in duplicate for each drug and cell line with a minimum of 50 cell viability in a dose-dependent manner in BRAFV600E/MITF 10 mg total protein, as reported previously. Competition experiments immortalised melanocytes. To our knowledge, this is the first were performed by incubation of a 10-mg aliquot of each cell lysate with the c-midostaurin drug affinity matrix in the presence of 20 mM unmodified study showing that AMPK inhibition interferes with MITF protein midostaurin. expression and melanocytic cell viability. Although we cannot formally exclude activation of the mTOR pathway upon AMPK Kinase inhibition analysis. In vitro kinase inhibition assays were performed inhibition in our system, our results show that these effects are not on the Millipore KinaseProfiler platform. c-Midostaurin was compared to sufficient to prevent the reduction of viability in human melanoma midostaurin for inhibition of FLT3 (at 0.01, 0.1 and 1 mM), c-sunitinib was cells. These data suggest that AMPK inhibition as a potential compared to sunitinib for inhibition of PDGFRa (at 0.1, 1 and 10 mM). therapeutic strategy for melanoma treatment deserves further clinical exploration. Solution tryptic digestion and peptide purification. Using the c-midostaurin and c-sunitinib affinity matrices, the enriched protein eluates obtained from BRAFV600E and BRAFV600E/MITF melanocytes, respectively, were MATERIALS AND METHODS reduced with dithiothreitol, cysteine residues alkylated by incubation Antibodies and chemical reagents with iodoacetamide and the samples digested with modified porcine trypsin. Three percent (and multiples thereof) of the digested eluates Antibodies raised against the following antigens were used: MITF (C5) were purified and concentrated by C18 reversed-phase material51 for (sc-56725), HA (sc-7392), Bcl-2 (sc-509), GAPDH (sc-25778) (Santa Cruz subsequent duplicate analysis by one-dimensional gel-free liquid Biotechnology, Santa Cruz, CA, USA); HA (2367); AMPKa (2532), AMPKa1 chromatography mass spectrometry. (2795) (Cell Signaling Technology, Danvers, MA, USA); MITF (C5) (Ab-1, MS-771) (Labvision/Neomarkers, Fremont, CA, USA); MITF (C5) (ab12039) iTRAQ-labelling and reversed-phase peptide fractionation. The remaining (Abcam, Cambridge, UK); a-tubulin (CP06) (Calbiochem, Gibbstown, NJ, peptides were individually derivatised with 4-plex iTRAQ52 (ABI, USA); pACC (Ser79) (Millipore, Billerica, MA, USA); HRP-conjugated Framingham, MA, USA) and labelled according to the instructions secondary antibody raised against mouse and rabbit IgG (Bio-Rad, provided by the manufacturer. All four samples were pooled, peptides Hercules, CA, USA). Midostaurin and staurosporin were purchased separated by reversed-phase liquid chromatography, pH 10, and fractions from LC Laboratories (Woburn, MA, USA); sunitinib was purchased from collected. Details of the procedure are essentially as previously LC Laboratories and Selleck Chemicals (Houston, TX, USA); compound C described.53,54 was purchased from Tocris Bioscience (Bristol, UK); U0126 was purchased from Selleck Chemicals; PD98059 and okadaic acid were purchased from Liquid chromatography mass spectrometry. Mass spectrometry was Calbiochem; iodoacetamide, dithiothreitol, 1 M triethylammonium bicarbo- nate and formic acid were purchased from Sigma-Aldrich (St Louis, MO, performed on a hybrid LTQ-Orbitrap XL mass spectrometer (ThermoFisher USA); modified porcine trypsin was purchased from Promega Corp. Scientific, Waltham, MA, USA) coupled to an Agilent 1200 HPLC nanoflow (Madison, WI, USA). system (Agilent Biotechnologies, Palo Alto, CA, USA) via a nanoelectrospray ion source using a liquid junction (Proxeon, Odense, Denmark). Analyses were performed in a data-dependent acquisition mode using a top Cell lines 6 collision-induced dissociation method for peptide identification alone; or Primary human melanocytes immortalised by transduction with hTERT, a top 3 high-energy collision-induced dissociation method for peptide p53DD, CDK(R24C) (primary melanocytes/hTERT/CDK4(R24C)/p53DD) with identification plus relative quantitation of iTRAQ reporter ions. Details of and without ectopically expressed BRAFV600E and HA-MITF have been the LC-MS configuration and mass spectrometer instrument settings are 54 described earlier.1 The parental cells (without overexpression of BRAFV600E given elsewhere. and/or HA-MITF) were cultured in Ham’s F10 plus glutamine (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 7% FBS, penicillin/ Data extraction, database search and iTRAQ relative quantitation. Peak list streptomycin (Gibco, Invitrogen), 0.1 mM IBMX (Sigma-Aldrich), 50 ng/ml information was extracted from the acquired MS data and searched TPA (Sigma-Aldrich), 1 mM Na3VO4 and 1 mM dbcAMP (Sigma-Aldrich) as against the human SwissProt database version v2010.09_20100812 (35 149 1 V600E described earlier and cultured at 37 1C/5% CO2. Immortalised BRAF sequences, including isoforms as obtained from varsplic.pl) with the search melanocytes with and without ectopically expressed MITF were cultured in engines MASCOT (v2.2.03, MatrixScience, London, UK) and Phenyx (v2.5.14, 55 RPMI (Gibco, Invitrogen) containing 10% FBS at 37 1C/5% CO2. Human GeneBio, Geneva, Switzerland). Details of the protein database search

& 2014 Macmillan Publishers Limited Oncogene (2014) 2531 – 2539 AMPK is a modulator of melanoma oncogene MITF V Borgdorff et al 2538 54 criteria are given elsewhere. iTRAQ relative quantitation analysis was 2 Bertolotto C, Lesueur F, Giuliano S, Strub T, de Lichy M, Bille K et al. A sumoylation- 56 performed with the isobar algorithm. defective MITF germline mutation predisposes to melanoma and renal carcinoma. Nature 2011; 480: 94–98. Immunofluorescence and microscopy. Cells were fixed in 4% paraformal- 3 Yokoyama S, Woods SL, Boyle GM, Aoude LG, MacGregor S, Zismann V et al. dehyde (Merck KGaA, Darmstadt, Germany) for 10 min at room tempera- A novel recurrent mutation in MITF predisposes to familial and sporadic mela- ture followed by permeabilisation with 90% methanol (Fisher Scientific noma. Nature 2011; 480: 99–103. GmbH, Schwerte, Germany) for 10 min at room temperature. Cells were 4 McGill GG, Horstmann M, Widlund HR, Du J, Motyckova G, Nishimura EK et al. Bcl2 stained overnight at 4 1C with primary antibody (4 mg/ml) diluted in regulation by the melanocyte master regulator MITF modulates lineage survival antibody diluent (Dako, Glostrup, Denmark). The next day, cells were and melanoma cell viability. Cell 2002; 109: 707–718. incubated for 2 h at room temperature with AlexaFluor-488-conjugated 5 Goding CR. Commentary. A picture of MITF in melanoma immortality. Oncogene goat anti-mouse antibody diluted in antibody diluent (Dako). 2011; 30: 2304–2306. The last phosphate-buffered solution wash step was combined with 6 Strub T, Giuliano S, Ye T, Bonet C, Keime C, Kobi D et al. Essential role of DAPI (300 nM, Sigma-Aldrich). Cells were imaged on a Zeiss Axiovert 200 M microphthalmia transcription factor for DNA replication, mitosis and genomic inverted microscope and images were acquired using Zen Software stability in melanoma. Oncogene 2011; 30: 2319–2332. (Manchester, UK). 7 Widlund HR, Fisher DE. Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival. Oncogene 2003; 22: 3035–3041. siRNA transfection. Cells were reverse transfected with 30 nM final 8 Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Mutations of the concentration siRNA using HiPerfect (Qiagen, Hilden, Germany), siRNAs BRAF gene in human cancer. Nature 2002; 417: 949–954. (Ambion, Austin, TX, USA) siPRKAA1 (s101, s102), siMITF (110566, s8791) 9 Hemesath TJ, Price ER, Takemoto C, Badalian T, Fisher DE. MAP kinase links the and siBcl-2 (s1916, s194310), and negative control siRNA #1 (4390843). transcription factor microphthalmia to c-Kit signalling in melanocytes. Nature 1998; 391: 298–301. Western blotting. Cells were lysed in Laemmli sample buffer. Cell lysates 10 Wu M, Hemesath TJ, Takemoto CM, Horstmann MA, Wells AG, Price ER et al. c-Kit were sonicated and proteins separated by SDS-PAGE and transferred to triggers dual phosphorylations, which couple activation and degradation of the Immobilon-P polyvinylidenedifluoride membranes (Millipore). Membranes essential melanocyte factor Mi. Genes Dev 2000; 14: 301–312. were blocked with PBST (PBS/0.05% Tween-20 (Sigma-Aldrich)) containing 11 Takeda K, Takemoto C, Kobayashi I, Watanabe A, Nobukuni Y, Fisher DE et al. 5% non-fat dry milk (Bio-Rad). Membranes were incubated overnight at Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phos- 4 1C with primary antibodies diluted in PBST containing 1% non-fat dry phorylation site with functional significance. Hum Mol Genet 2000; 9: 125–132. milk. After washing with PBST, membranes were incubated for 1 h with 12 Miller AJ, Levy C, Davis IJ, Razin E, Fisher DE. Sumoylation of MITF and its related horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit family members TFE3 and TFEB. J Biol Chem 2005; 280: 146–155. antibodies diluted in PBST containing 1% non-fat dry milk. Proteins were 13 Murakami H, Arnheiter H. Sumoylation modulates transcriptional activity of MITF visualised using ECL Western Blotting Detection Reagents (GE Healthcare in a promoter-specific manner. Pigment Cell Res 2005; 18: 265–277. Europe, Munich, Germany). 14 Carreira S, Goodall J, Denat L, Rodriguez M, Nuciforo P, Hoek KS et al. MITF regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Cell viability assay. Cell viability was assayed by incubating the cells for Dev 2006; 20: 3426–3439. 4 h with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 15 Hoek KS, Goding CR. Cancer stem cells versus phenotype-switching in melanoma. (MTT, Sigma-Aldrich) followed by formazan solubilisation with DMSO. Pigment Cell Melanoma Res 2010; 23: 746–759. 16 Giuliano S, Cheli Y, Ohanna M, Bonet C, Beuret L, Bille K et al. Microphthalmia- The OD492 of the supernatants was determined with an ELISA plate reader associated transcription factor controls the DNA damage response and a lineage- (Bender MedSystems GmbH, Vienna, Austria) using OD620 as the reference wavelength. specific senescence program in melanomas. Cancer Res 2010; 70: 3813–3822. 17 Cheli Y, Giuliano S, Botton T, Rocchi S, Hofman V, Hofman P et al. MITF is the key molecular switch between mouse or human melanoma initiating cells and their Annexin V assay. Cells were collected and resuspended in 1 binding Â differentiated progeny. Oncogene 2011; 30: 2307–2318. buffer (eBioscience, San Diego, CA, USA) after one wash in PBS (Invitrogen). 18 Wellbrock C, Rana S, Paterson H, Pickersgill H, Brummelkamp T, Marais R. 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Blood 2009; 114: 2984–2992. 21 Fay JR, Steele V, Crowell JA. Energy homeostasis and cancer prevention: the AMP-activated protein kinase. Cancer Prev Res (Phila) 2009; 2: 301–309. CONFLICT OF INTEREST 22 Carling D, Zammit VA, Hardie DG. A common bicyclic protein kinase cascade The authors declare no conflict of interest. inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 1987; 223: 217–222. 23 Ha J, Daniel S, Broyles SS, Kim KH. Critical phosphorylation sites for acetyl-CoA carboxylase activity. J Biol Chem 1994; 269: 22162–22168. ACKNOWLEDGEMENTS 24 Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J et al. Role of AMP-activated We thank Hans Widlund (Harvard Skin Disease Research Center, Brigham and protein kinase in mechanism of metformin action. J Clin Invest 2001; 108: Women’s Hospital, Boston, MA, USA) for providing us with human immortalised 1167–1174. melanocytes with and without ectopic expression of BRAFV600E and HA-MITF as 25 Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all described in Garraway et al.1 We thank Gaurav Pathria for helpful discussions and aspects of cell function. Genes Dev 2011; 25: 1895–1908. critical reading of the manuscript. This study was funded by FWF-Austrian Science 26 Kim KY, Baek A, Hwang JE, Choi YA, Jeong J, Lee MS et al. Adiponectin-activated Fund (L590-B12) to SNW. The team at CeMM is supported by the Austrian Academy AMPK stimulates dephosphorylation of AKT through protein phosphatase 2A of Sciences, and the GEN-AU initiative of the Austrian Federal Ministry for Science and activation. Cancer Res 2009; 69: 4018–4026. Research (PLACEBO GZ BMWF-70.081/0018-II/1a/2008 and APP-III 820965). 27 Kim MJ, Park IJ, Yun H, Kang I, Choe W, Kim SS et al. 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