Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics
2014; Elsevier BV; Volume: 13; Issue: 7 Linguagem: Inglês
10.1074/mcp.m113.037424
ISSN1535-9484
AutoresVito W. Rebecca, Elizabeth R. Wood, Inna V. Fedorenko, Kim H.T. Paraiso, H. Eirik Haarberg, Yi Chen, Yun Xiang, Amod A. Sarnaik, Geoffrey T. Gibney, Vernon K. Sondak, John M. Koomen, Keiran S.M. Smalley,
Tópico(s)Heat shock proteins research
ResumoThe evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic. The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic. Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ∼5.3 months) (1.Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. Dummer R. Garbe C. Testori A. Maio M. Hogg D. Lorigan P. Lebbe C. Jouary T. Schadendorf D. Ribas A. O'Day S.J. Sosman J.A. Kirkwood J.M. Eggermont A.M. Dreno B. Nolop K. Li J. Nelson B. Hou J. Lee R.J. Flaherty K.T. McArthur G.A. 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There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK) 1The abbreviations used are: LC-MRM, liquid chromatography-multiple reaction monitoring mass spectrometry; RTK, receptor tyrosine kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HSP, heat shock protein; PDGFR, PDGF receptor; mTOR, mammalian target of rapamycin; APC, adenomatous polyposis coli. 1The abbreviations used are: LC-MRM, liquid chromatography-multiple reaction monitoring mass spectrometry; RTK, receptor tyrosine kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HSP, heat shock protein; PDGFR, PDGF receptor; mTOR, mammalian target of rapamycin; APC, adenomatous polyposis coli. signaling, may be involved in acquired BRAF inhibitor resistance (5.Nazarian R. Shi H. Wang Q. Kong X. Koya R.C. Lee H. Chen Z. Lee M.K. Attar N. Sazegar H. Chodon T. Nelson S.F. McArthur G. Sosman J.A. Ribas A. Lo R.S. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.Nature. 2010; 468: 973-977Crossref PubMed Scopus (1733) Google Scholar, 6.Paraiso K.H. Xiang Y. Rebecca V.W. Abel E.V. Chen Y.A. Munko A.C. Wood E. Fedorenko I.V. Sondak V.K. Anderson A.R. Ribas A. Palma M.D. Nathanson K.L. Koomen J.M. Messina J.L. Smalley K.S. PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression.Cancer Res. 2011; 71: 2750-2760Crossref PubMed Scopus (416) Google Scholar, 7.Whittaker S.R. Theurillat J.P. Van Allen E. Wagle N. Hsiao J. Cowley G.S. Schadendorf D. Root D.E. Garraway L.A. A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition.Cancer Discov. 2013; 3: 350-362Crossref PubMed Scopus (261) Google Scholar). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8.Paraiso K.H. 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Activity of the heat shock protein 90 inhibitor ganetespib in melanoma.PLoS ONE. 2013; 8: e56134Crossref PubMed Scopus (31) Google Scholar). Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15–20% of melanoma patients whose tumors harbor activating NRAS mutations (10.Fedorenko I.V. Gibney G.T. Smalley K.S. NRAS mutant melanoma: biological behavior and future strategies for therapeutic management.Oncogene. 2013; 32: 3009-3018Crossref PubMed Scopus (105) Google Scholar). Although there is some evidence that MEK inhibitors have activity in NRAS mutant melanoma patients, responses tend to be short-lived (mean progression-free survival ∼3 months) and resistance is nearly inevitable (11.Ascierto P.A. Schadendorf D. Berking C. Agarwala S.S. van Herpen C.M. Queirolo P. Blank C.U. Hauschild A. Beck J.T. St-Pierre A. Niazi F. Wandel S. Peters M. Zubel A. Dummer R. 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Here, we have developed a novel multiplexed LC-MRM assay to quantify the expression of >80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions (22.Remily-Wood E.R. Liu R.Z. Xiang Y. Chen Y. Thomas C.E. Rajyaguru N. Kaufman L.M. Ochoa J.E. Hazlehurst L. Pinilla-Ibarz J. Lancet J. Zhang G. Haura E. Shibata D. Yeatman T. Smalley K.S. Dalton W.S. Huang E. Scott E. Bloom G.C. Eschrich S.A. Koomen J.M. A database of reaction monitoring mass spectrometry assays for elucidating therapeutic response in cancer.Proteomics Clin. Appl. 2011; 5: 383-396Crossref PubMed Scopus (44) Google Scholar). In this study, we present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors. Through this method, we identify the degradation of key HSP90 client proteins in vivo and elucidate a novel mechanism of adaptation to MEK inhibition through increased RTK signaling. WM1361A, WM1366, and WM1346 melanoma cell lines were a kind gift from Dr. Meenhard Herlyn (The Wistar Institute, Philadelphia, PA), and M318 and M245 cell lines were a gift from Antoni Ribas (UCLA, Los Angeles, CA). All cell lines were grown in RPMI 1640 medium supplemented with 5% FBS. MTT assays were performed as described previously (15.Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar). HSPs were quantified from digests of whole cell lysates; protein extracts from ∼2,000 cells (200 ng of total protein digest) were analyzed with LC-MRM after denaturation with 8 m urea, reduction, alkylation, and in-solution digestion. The GeLC-MRM approach to quantify lower abundance cancer signaling proteins was developed based on previous implementations of SDS-PAGE fractionation combined with LC-MRM quantification (22.Remily-Wood E.R. Liu R.Z. Xiang Y. Chen Y. Thomas C.E. Rajyaguru N. Kaufman L.M. Ochoa J.E. Hazlehurst L. Pinilla-Ibarz J. Lancet J. Zhang G. Haura E. Shibata D. Yeatman T. Smalley K.S. Dalton W.S. Huang E. Scott E. Bloom G.C. Eschrich S.A. Koomen J.M. 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From each cell lysate or tissue homogenate, an aliquot of protein extract (50 μg) was fractionated by SDS-PAGE into five regions of 4–12% BisTris gels (Criterion XT, Bio-Rad) and visualized with Coomassie Brilliant Blue G-250 (Aldrich), as described in supplemental Fig. 1. The approximate molecular weight ranges are as follows: band 1, >250 kDa; band 2, 120–250 kDa; band 3, 70–120 kDa; band 4, 30–70 kDa; and band 5, <30 kDa. Gel regions were excised and diced (to ∼1 mm3) for processing. After destaining, cysteines were reduced with 2 mm tris(carboxyethyl)phosphine and alkylated with 20 mm iodoacetamide prior to overnight digestion with sequencing grade trypsin (Promega, Madison, WI). The resulting proteolytic peptides were extracted with aqueous 50% acetonitrile, 0.01% trifluoroacetic acid and concentrated vacuum centrifugation (SC210A, Speedvac, Thermo). Peptides were resuspended in 2% acetonitrile with 0.1% formic acid (loading solvent), containing the internal standards. LC-MRM analysis was performed in triplicate on a nanoLC (EasynLC, Proxeon, Thermo, San Jose, CA) interfaced with an electrospray triple quadrupole mass spectrometer (TSQ Quantum Ultra or Vantage, Thermo, San Jose, CA). The following solvent system is used for LC-MRM analysis; solvent A is aqueous 5% acetonitrile with 0.1% formic acid, and solvent B is aqueous 90% acetonitrile with 0.1% formic acid. For each sample, an aliquot of the peptide mixture (5 μl, ∼1/6 of the sample) was loaded onto the trap column at 6 μl/min and washed with loading solvent for 5 min. Then, a gradient of 5% B to 50% B was applied over 35 min prior to washing the column and re-equilibrating over a total of 45 min for the LC experiment. Mass spectrometry instrument parameters include the following: 2400-V spray voltage; 250 °C transfer tube temperature; Q1 resolution 0.4 when transitions were monitored for the entire LC separation (HSPs and band 1) and 0.7 when scheduled methods were used (bands 2–5); 1.5 millitorr collision gas pressure; Q3 resolution 0.7; and 20-ms scan time per transition. The list of proteins, peptides, and transitions is given in supplemental Table 1. Briefly, the number of peptides and transitions for each gel band ranged from band 1 with six peptides and 30 transitions to band 3 with 68 peptides and 252 transitions. Collision energy values were optimized by infusion of the standard peptides. Skyline version 1.3 was used for data evaluation (26.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2964) Google Scholar). Peaks were evaluated by comparison of their elution time and fragment ion signal ratios to their matched internal standards. All transitions above 10% of the base peak were used for quantification. Data were exported to Excel for calculations of protein quantity, standard deviation, and column volumes (%). Melanoma cells were treated with either 300 nm XL-888 for up to 48 h or with 1 μm AZD6244 for 24 h before protein lysate was collected and run on 8–16% Tris/glycine gels. Proteins were subsequently immunoblotted with the antibodies to AKT1, APC, phospho-NFκB (Ser-536), mTOR, phospho-AKT (Ser-473), CDK4, PDGFR-β, EGF receptor, c-MET, VEGFR1, VEGFR2, and IGF-1Rβ, which were purchased from Cell Signaling Technology. The β-catenin antibody was purchased from BD Biosciences, and GAPDH was purchased from Sigma. WM1361A and WM1366 cells were treated with 1 μm AZD6244 for 24 h, and the lysate collected was used for phospho-RTK arrays, which were carried out using the phospho-RTK kit from R&D Systems (catalog no. ARY001B), according to the manufacturer's instructions. Cells were plated into 6-well tissue culture plates at 60% confluence and left to grow overnight before being treated with either 1 μm AZD6244 alone, 1 μm crenolanib or the combination of AZD6244 and crenolanib for 120 h before being harvested. In other studies, cells were treated with β-catenin siRNA followed by treatment with either vehicle or AZD6244. Annexin-V staining was performed as described previously (19.Wolf-Yadlin A. Hautaniemi S. Lauffenburger D.A. White F.M. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5860-5865Crossref PubMed Scopus (430) Google Scholar), and apoptosis was assessed via flow cytometry. Animal experiments were conducted under a protocol approved by the University of South Florida Institutional Animal Care and Use Committee (IS0000324). BALB SCID mice (The Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected with 2.5 × 106 cells per mouse (resuspended in 111 μl of L-15 media, 10 mm HEPES, 37.5 μl Matrigel). Tumors were grown to ∼100 mm3 prior to dosing. Mice were treated with either 100 mg of XL888/kg (n = 5) or an equivalent volume of vehicle (10 mm HCl), three times per week by oral gavage. Mouse weights and tumor volumes (L × W2/2) were measured three times per week. Upon completion of the experiment, vehicle and drug-treated tumor biopsies were processed for LC-MRM analysis (as above). Human specimens were procured in the operating theater by fine needle aspiration of resected tumors under a protocol approved by the University of South Florida Institutional Review Board (MCC number 15375); samples were transferred on ice and processed immediately for GeLC-MRM analysis for an exploratory study in the feasibility of assay transfer from preclinical models. WM1366 and WM1361A cells were treated with either 1 μm AZD6244 alone, 1 μm crenolanib alone, or the combination of AZD6244 and crenolanib twice a week for 4 weeks before being fixed in crystal violet and quantified. WM1366 cells were suspended in RPMI 1640 medium supplemented with 5% FBS and plated 200,000 cells per well in a 6-well plate and allowed to adhere overnight. The next day, the media were aspirated, and wells were washed with 1 ml of Opti-MEM and replaced with 1 ml of Opti-MEM. Cells were transfected with 50 nm β-catenin siRNA (Cell Signaling Technology, Danvers, MA) or 50 nm scrambled RNA (Santa Cruz Biotechnology, Santa Cruz, CA) with Lipofectamine, according to the manufacturer's instructions. After 24 h of transfection, wells were supplemented with RPMI 1640 medium, FBS, and/or AZD6244 to make a final concentration of 5% FBS in the wells and a final concentration of 1 μm AZD6244 for MEKi-treated wells. Lysate was created after 24 h of exposure to AZD6244, and Western blotting was performed to analyze β-catenin knockdown efficiency and the associated effects upon pERK, ERK, cyclin B1, and cyclin D1. GAPDH was used as the loading control. Data show the mean of at least three independent experiments. GraphPad Prism 5 statistical software was used to perform the Student's t test, where * indicates p ≤ 0.05; ** indicates 0.05 ≤ p ≤ 0.01; *** indicates p ≤ 0.001, and **** indicates p ≤ 0.0001. An LC-MRM assay was designed to capture all of the HSP family of chaperones and a series of 81 proteins in multiple signaling pathways known to be important for melanoma progression and resistance to targeted therapies. Pathways and processes covered included the cell cycle (CDK1, CDK2, CDK4, cyclin D1, and CHK1), receptor tyrosine kinases (ERBB2 and IGF1R), RAS/MAPK signaling (NRAS, HRAS, KRAS, SOS1, SHC1, BRAF, ARAF, CRAF, MEK1/2, and c-Myc), PI3K/AKT signaling (AKT1, AKT2, AKT3, mTOR, CSK, FAK1, and GSK3β), β-catenin/WNT signaling (APC, axin, β-catenin, and α-catenin), NFκB, Src, and MITF (signaling scheme is shown in Fig. 1). Inhibition of HSP90 is known to be associated with compensatory increases in the expression of the highly abundant chaperone protein HSP70. We began using the LC-MRM platform to analyze the altered expression of chaperones following treatment with HSP90 inhibitor. Increasing concentrations of the HSP90 inhibitor XL888 led to concentration-dependent decreases in the growth of four NRAS mutant melanoma cell lines (M245, M318, WM1361A, and WM1366) (Fig. 2A). In the four cell line models, XL888 (300 nm) induced the expression of HSP70 isoform 1 (HSP71), HSP90α, and HSP90β as measured by LC-MRM (Fig. 2B). Western blot studies confirmed the increases in HSP71 (Fig. 2B). In a second series of experiments, NRAS mutant M245 melanoma cells were grown as xenografts in SCID mice. After tumors were palpable, treatment with XL888 (125 mg/kg) was initiated for 15 days, with drug treatment being associated with significant decreases in tumor volume (p < 0.02) (Fig. 2C). LC-MRM analysis of tumor specimens harvested on treatment showed increased levels of HSP70 isoform 1 expression compared with vehicle controls (Fig. 2D). Total expression levels (in femtomoles/μg total protein) are lower in tumors than the cell line models due to the amounts of protein contributed by the stroma and blood in the mouse tissue. Over 200 proteins have been identified to be clients of HSP90 (27.Taipale M. Krykbaeva I. Koeva M. Kayatekin C. Westover K.D. Karras G.I. Lindquist S. Quantitative analysis of hsp90-client interactions reveals principles of substrate recognition.Cell. 2012; 150: 987-1001Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). The efficacy of HSP90 inhibitors is dependent upon the degradation of client proteins that are essential for tumor survival. As yet, no assays exist that enable multiple client proteins to be monitored simultaneously and evaluated in small clinical specimens. A panel of four NRAS mutant melanoma cell lines (M245, M318, WM1361A, and WM1366) were treated with XL888 (300 nm, 24 h), processed for GeLC-MRM, and analyzed by mass spectrometry (Fig. 3A). XL888 treatment led to a marked decrease in the expression of multiple RTKs, including EGF receptor, IGF-1Rβ, c-MET, and VEGFR1 (Fig. 3A). Unexpectedly, HSP90 inhibition increased expression of VEGFR2 (Fig. 3A). Western blot validation confirmed the decreases in client protein expression identified by LC-MRM, as well as the increased expression of VEGFR2 (Fig. 3B). As the availability of clinical material for analysis is often limited, we next determined whether the LC-MRM platform was sufficiently sensitive to measure the expressi
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