braf inhibition decreases cellular glucose ......easytag express35s protein labeling mix...

Cancer Biology and Signal Transduction

BRAF Inhibition Decreases Cellular GlucoseUptake inMelanoma inAssociationwithReductionin Cell VolumeNicholas Theodosakis1, Matthew A. Held1, Alexander Marzuka-Alcala2, Katrina M. Meeth1,Goran Micevic1, Georgina V. Long3,4, Richard A. Scolyer3,5, David F. Stern1, andMarcus W. Bosenberg1,2

Abstract

BRAF kinase inhibitors have dramatically affected treatment ofBRAFV600E/K-driven metastatic melanoma. Early responsesassessed using [18F]fluorodeoxyglucose uptake-positron emissiontomography (FDG-PET) have showndramatic reduction of radio-tracer signal within 2 weeks of treatment. Despite high responserates, relapse occurs in nearly all cases, frequently at sites of treatedmetastatic disease. It remains unclear whether initial loss of18FDG uptake is due to tumor cell death or other reasons. Here,we provide evidence of melanoma cell volume reduction in apatient cohort treated with BRAF inhibitors. We present datademonstrating that BRAF inhibition reduces melanoma glucose

uptake per cell, but that this change is no longer significantfollowing normalization for cell volume changes. We also dem-onstrate that volume normalization greatly reduces differences intransmembrane glucose transport and hexokinase-mediatedphosphorylation. Mechanistic studies suggest that this loss of cellvolume is due in large part to decreases in new protein translationas a consequence of vemurafenib treatment. Ultimately, ourfindings suggest that cell volume regulation constitutes an impor-tant physiologic parameter that may significantly contribute toradiographic changes observed in clinic. Mol Cancer Ther; 14(7);1680–92. �2015 AACR.

IntroductionCancer cells frequently display high rates of fermentative gly-

colysis relative to benign cells under normoxic conditions (1–3), acharacteristic often referred to as "aerobic glycolysis" that involvesincreased consumption of glucose. The benefits of this includeincreased precursors for macromolecule synthesis, generation ofreducing agents, and production of adequate ATP for growth (4–7). Evidence of aerobic glycolysis can be observed clinically usingthe glucose analog radiotracer 18-fluorodeoxyglucose (18FDG)with positron emission tomography (PET) imaging, reflecting arelative increase in glucose uptake in tumors compared withmostnormal tissues. It is often used in tandemwith tumor dimensionalmeasurements, with classification of therapeutic responsesdefined by a set of guidelines known as Response EvaluationCriteria in Solid Tumors (RECIST) based on changes in the sumofthe longest diameters of all computed tomography (CT) or MRI-

measured lesions. Interestingly, during the initial phases of tar-geted therapy, the FDG-PET response may be greater than thedegree of tumor shrinkage noted with CT imaging (8). Together,FDG-PET and RECIST serve as useful methods for tracking theefficacyof therapies in primary andmetastatic tumors (9, 10). Thisoccurs dramatically in melanoma tumors driven by oncogenicBRAF treated with BRAF inhibitors such as vemurafenib anddabrafenib (8, 11–13).

Approximately half of human melanomas harbor activatingmutations at amino acid V600 in the protein kinase BRAF, leadingto constitutive activation of the MAPK pathway (14, 15). TheBRAF inhibitors vemurafenib and dabrafenib suppress down-stream activity of the MAPK pathway in the majority of BRAF-mutant tumors (16, 17). Patients treated with vemurafenib ordabrafenib typically show decreased FDG-PET signal within 2weeks, often before significant reduction in tumormass (8, 13). Inroughly 40%of patients, less than 30%maximal tumor shrinkageis ultimately observed on therapy, yet most tumors will havemarkedly decreased FDG-PET signal with a progression-free sur-vival benefit (12). Subsequent increases in 18FDG uptake intreated tumors tightly correlate with emergence of resistance tovemurafenib, occurring between 2 and 18 months in mostpatients (11). Curiously, many tumors that become PET negativeon therapy often regain 18FDG positivity later in the same loca-tion, likely signifying recurrence of residual disease at those sites.Both the precise mechanism for this early decrease and themechanism behind subsequent increase of glucose uptake atrelapse have yet to be conclusively determined.

In cancer cells, transmembrane GLUT carrier proteins thatfacilitate diffusion across the plasma membrane (18), and hexo-kinase enzymes I–IV (19), which phosphorylate glucose in the

1Department of Pathology,Yale School of Medicine, New Haven, Con-necticut. 2Department of Dermatology,Yale School of Medicine, NewHaven, Connecticut. 3Melanoma Institute of Australia, Sydney, NewSouth Wales, Australia. 4Discipline of Pathology, The University ofSydney, Sydney, New South Wales, Australia. 5Discipline of Medicine,The University of Sydney, Sydney, New South Wales, Australia.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Nicholas Theodosakis, Yale School of Medicine, LMP 15York Street, 5038A, New Haven, CT 06520-8059. Phone: 203-737-3402; Fax:203-785-6869; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0080

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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cytosol, are known to contribute to 18FDG trapping. Only HKIand HKII are expressed to a significant degree in melanoma (20).HKII is known to support aerobic glycolysis, with its activityvarying in response to changing glucose levels (21). HKII is alsotied to regulation of apoptosis through binding to the mitochon-drial membrane porin VDAC (22). Interestingly, high levels ofHKII have been observed in a variety of actively proliferatingcancer cells (21, 23).While reduction of glucose uptakewith BRAFinhibitors in melanoma has been noted (5, 11, 24), previousmechanistic explanations have focused mainly on changes inGLUT-mediated transport (24), with studies specifically investi-gating changes in HK levels mostly confined to MEK inhibitors(25). Here we investigate how the metabolic and morphologicproperties of melanoma cells on therapy interact to produce theobserved changes in 18FDG uptake.

Materials and MethodsHuman melanoma tissue samples

Paired excision biopsies of 10 pretreatment (PRE) andcorresponding early during treatment (EDT) biopsies at 3 and15 days after initiation of BRAF inhibitor therapy wereobtained in patients with BRAF-mutant metastatic melanomaas previously described (8, 16). Maximum standardizeduptake values (SUVmax) from 18FDG-PET imaging wereobtained on day 15 of treatment (8) when available. Apoptoticcell counts represent average numbers of pyknotic tumor cellsacross 5 different 1 mm2 areas per slide. Pyknotic cells weredefined by hyperchromatic and shrunken nuclei with cyto-plasmic compaction. Areas of ischemic, geographic necrosiswere not included.

Cell culture and generation of acquired resistant linesAll specimenswere collectedwith patients' informed consent in

accordance with the Health Insurance Portability and Account-ability Act (HIPAA) under a Human Investigations Committeeprotocol. Authentication and mutational status were confirmedvia expression profiling and Sanger sequencing as describedpreviously (26). Cell lines were grown inOPTI-MEM (Invitrogen)supplemented with 1% penicillin–streptomycin and 10% FBSmaintained in a 37�C incubator maintained at 5% CO2. All linesutilizedwere derived fromsurgicalmelanoma resections andwereprovided by Dr. Ruth Halaban (Yale University; ref. 27) as part ofthe YSPORE tissue bank and passaged less than 6 months.Parental YUMAC and YUSIT1 lines were continuously grown invemurafenib (up to 5 mmol/L) until they exhibited resistance togrowth inhibition by vemurafenib and were designated YUMACrand YUSIT1r. Parental lines were redesignated YUMACs andYUSIT1s. Acquired resistance was verified over a 72-hour timeperiod and quantified using the CyQUANT NF Cell ProliferationAssay Kit (Life Technologies).

Cell size analysisCellmediandiameter of nonadherent cellswas calculatedusing

a Z2 Coulter Counter (Beckman Coulter, Inc.) calibrated withpolystyrene size-standardized beads (Spherotech). Volumes ofspherical cells in suspension were calculated from these measure-ments. Adherent cell area, both in vitro and from paraffin-embed-ded sections, was calculated by polygonal selection using ImageJ(v. 1.46r, NIH, Bethesda, MD) using 20 separate cells judged to berepresentative of the entire population. Patient sample images

were taken using anOlympus BX41microscope equippedwith anoptical micrometer.

3H-O-methylglucose transmembrane glucose transport assayTo quantify transmembrane glucose transport, we adapted a

previous protocol using the nonphosphorylatable tritiated glu-cose analog 3-O-methylglucose (3-OMG; ref. 28). Cells werecounted by hemocytometer and resuspended in glucose-freeDMEM supplemented with varying concentrations of glucosealong with a ratio of 1.8 mCi of 3-OMG (PerkinElmer) permmol glucose for varying lengths of time. Uptake was haltedwith a quenching cocktail of 300 mmol/L phloretin (Sigma-Aldrich). Four washes were performed with the quenchingcocktail before the residual pellet was lysed and added toUltima Gold Scintillation Cocktail (PerkinElmer). Counts perminute were assessed with a Beckman Coulter LS 6500 LiquidScintillation Counter (Beckman Coulter). Cell number was alsocorrected for using the CyQUANT NF Cell Proliferation Assayafter washing. Results were also corrected for cell size usingtriplicate measurements of cell volume by Coulter Counter asdescribed above.

Hexokinase activity assayTotal cellular hexokinase activity was assessed using an

existing protocol (29), adapted from an earlier protocol(30). Cells were counted by hemocytometer. A volume of lysatecontaining 2.5 � 105 cells or 30 mg of total protein was thenadded to a 96-well optical plate (Thermo Fisher Scientific)followed by an assay cocktail composed of 50 mmol/L trietha-nolamine buffer, 19 mmol/L Adenosine 50-Triphosphate Solu-tion, 100 mmol/L magnesium chloride, 14 mmol/L b-nicotin-amide adenine dinucleotide phosphate, 125 units/mL glucose-6-phosphate dehydrogenase enzyme solution, and varyingconcentrations of glucose. Plates were read using a SpectramaxM3 microplate reader (Molecular Devices) and maximumvelocities computed using SoftMax Pro 6.2.2 (MolecularDevices). Correction for alterations in protein content per cellwas performed using results of the Bradford assay in triplicateor CyQUANT NF Cell Proliferation Assay.

Flow cytometryTo measure glucose uptake, pellets were then resuspended in

PBS supplemented with 300 mmol/L 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG; CaymanChemical) for 10 minutes. After a single wash, pellets werestained using the BD Pharmingen Apoptosis Detection Kit IIaccording to the manufacturer's protocol (BD Biosciences).Samples were analyzed with the BD LSRII flow cytometer toat least 10,000 events per sample. Compensation for spectraloverlap between 2-NBDG and propidium iodide was appliedfor each experiment. Each line was treated independently, andgates were fixed based on negative control signals. Plots weregenerated using FlowJo 9.6.2.

ImmunoblottingImmunoblots were conducted with the following primary

antibodies all used at 1:1,000: Hexokinase II (cat. no. 2867; CellSignaling Technology), b-actin (cat. no. 4970; Cell SignalingTechnology), b-tubulin (cat. no. 2128; Cell Signaling Technolo-gy), GSK3B (cat. no. 9315), p-GSK3B S9 (cat. no. 9323; Cell

BRAF Inhibition Alters Glucose Uptake and Morphology

www.aacrjournals.org Mol Cancer Ther; 14(7) July 2015 1681




Signaling Technology), p-p90RSK T573 (cat. no. 9346; Cell Sig-naling Technology), RSK1/RSK2/RSK3 (cat. no. 9355; Cell Sig-naling Technology), hsp60 (cat. no. sc-1052; Santa Cruz Biotech-nology), GLUT1 (cat. no. 07-1401; Millipore), GLUT3 (cat. no.NBP1-89762; Novus Biologicals) and the secondary antibodyAnti-rabbit IgG, HRP-linked Antibody (cat. no. 7074S; Cell Sig-naling Technology).

Radiographic studiesAfter drug treatment, cells were incubated in cysteine and

methionine-free DMEM (Life Technologies) for 1 hour before1-hour incubation with the same media supplemented withEasyTag EXPRESS35S Protein Labeling Mix (PerkinElmer). Afterlysate collection with or without immunoprecipitation, sampleswere separated by SDS-PAGE and read using a Storm 860 phos-phorimager (GMI Inc.).

RNA extraction and quantitative PCRTotal RNAwas extracted using anRNeasyMini kit (Qiagen) and

reverse transcribed using a Transcriptor First Strand cDNA Syn-thesis Kit (Roche) using both Oligo dT and random hexamers.Quantitative PCR was carried out using FastStart Universal SYBRGreen Master (Rox; Roche) relative to GAPDH levels on a Ste-pOnePlus Real-Time PCR System (Life Technologies) and foldchanges were calculated using StepOne Software v 2.0.

ResultsCellular volume reduction occurs in clinical melanomasamples in response to BRAF inhibition

SUVmax values from18FDG-PET imaging in our paired biopsy

PRE and EDT cohort were obtained on day 15 of therapy (8, 16).Evaluation of cellular size in the samples demonstrated a signif-icant decrease in cellular and cytoplasmic volume followinginitiation of BRAF inhibitor therapy (Fig. 1A and B) without asignificant decrease in average number of nuclei per tumor cross-sectional area (Fig. 1C). Contraction of cytosolic volume withoutloss of nuclei consequentially led to an increase in averageintercellular distance in most patients (Fig. 1D). There was notan associated increase in the average number of apoptotic cells permm2 in the majority of cases (Fig. 1E).

BRAF inhibition alters glucose uptake in melanomaAfter noting themorphologic changes, we evaluated changes in

glucose metabolism in vitro to determine whether the phenotypesare linked. To quantify total glucose uptake changes in vitro, weincubated patient-derived melanoma cell lines harboring activat-ing mutations in BRAF with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), a nonradioactive,fluorescent, nonmetabolizable form of glucose similar to[18F]fluorodeoxyglucose, that is trapped within cells upon phos-phorylation by hexokinase (31). Melanoma lines sensitive tovemurafenib, defined elsewhere as showing at least 50% growthinhibition at or below 3 mmol/L drug (26) showed a significantdecrease in 2-NBDG uptake per cell measured by flow cytometryafter 12-hour exposure to vemurafenib, withmaximum reductionin median fluorescent intensity observed by 72 hours (Fig. 2A).This was not associated with a significant increase in cell death asmeasured by Annexin-V and propidium iodide flow cytometry(Supplementary Fig. S1A).

Patients with acquired resistance to mutant BRAF inhibitorsshow elevated SUVmax, relative to normal tissue even on BRAF

inhibitors (11). To investigate this characteristic, we generatedacquired vemurafenib-resistant clones by chronically growingtwo sensitive lines in vemurafenib and assessing 2-NBDGuptake. Drug resistance was verified by determination ofIC50 for proliferation at 72 hours (Supplementary Fig. S1B andS1C). As expected, 2-NBDG uptake in acquired resistant celllines was maintained in the presence of vemurafenib (Fig. 2B).Moreover, intrinsically vemurafenib-resistant mutant BRAF-driven melanoma lines and non-BRAF–mutant lines, showedequivalent or enhanced 2-NBDG uptake relative to vemurafe-nib-sensitive lines (Fig. 2C, Supplementary Table S1). These invitro observations suggest that sensitivity to vemurafenib isclosely associated with glucose uptake per cell, and phenoco-pies what is seen by 18FDG imaging in melanoma tumors frompatients. Moreover, we observed that glucose uptake in sensi-tive lines was completely restored 72 hours after removal ofvemurafenib, further suggesting that glucose uptake loss is areversible effect of BRAF inhibition rather than a result ofapoptosis (Fig. 2D). These results demonstrate that vemurafe-nib greatly reduces glucose uptake per cell within 72 hours ofincubation with drug.

Vemurafenib-induced reduction in glucose uptake is notaccounted for by alterations in transmembrane glucosetransport

The intracellular trapping of 2-NBDG after phosphorylationby hexokinase precludes its use as an accurate indicator ofGLUT function. Therefore, we utilized the nonphosphorylata-ble tritiated glucose analog 3-O-methylgucose (3-OMG) toquantify transport rate changes independent of hexokinaseactivity. Normalizing to cell count, we observed a mild decreasein uptake in vemurafenib-treated vemurafenib-sensitive celllines that was not observed in acquired vemurafenib-resistantcells (Fig. 3A). This was not associated with reduced proteinlevels of two of the major GLUT transporter enzymes known tobe highly active in melanoma, GLUT1 and GLUT3 (32) (Fig.3B). Accordingly, we did not observe a significant change intransporter mRNA levels in sensitive or resistant lines (Fig. 3C).These data indicated that changes in overall glucose transportare insufficient to explain the decrease in glucose uptake inthese BRAF-inhibited cells.

Reduction of hexokinase II protein and overall hexokinaseactivity per cell is observed in sensitive but not acquiredresistant melanoma lines treated with vemurafenib

We next investigated whether reduced hexokinase activitycontributes to BRAF inhibitor induced reduction in cellular 2-NBDG levels. We first performed an in vitro total hexokinaseactivity assay in sensitive and resistant lines, normalizing to cellcount. Vemurafenib-sensitive lines showed a significantdecrease in hexokinase activity per cell after 72-hour vemur-afenib treatment, in contrast to acquired vemurafenib-resistantderivatives, which showed no significant change (Fig. 4A).Consistent with previous literature identifying HKII as theisoenzyme most intimately tied to cellular growth and prolif-eration of tumor cells (21, 23), we observed reduction of HKIIprotein starting at approximately 6 to 8 hours. Marked reduc-tion of HKII levels was seen by 72 hours in sensitive lines (Fig.4B), which corresponded with the decrease in glucose uptakeobserved by 2-NBDG flow cytometry. Melanoma lines with

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Mol Cancer Ther; 14(7) July 2015 Molecular Cancer Therapeutics1682




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Figure 1.BRAF inhibition induces a significant decrease in cell volume in treated humanmelanoma tumors. A significant reduction in mean cytoplasmic volume is observed inthe majority of biopsies of patient tumors after commencement of BRAFi therapy (A and B), without a significant decrease in mean number of nuclei perHPF (C). D, the majority of cases showed a large increase in average intercellular distance after commencement of therapy. E, a significant increase in number ofapoptotic nuclei was not observed in the majority of cases. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s., not significant.






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Figure 2.Vemurafenib causes a decrease in glucose uptake in drug-sensitive but not resistant lines. A, treatment with 3 mmol/L vemurafenib (Vem) causes a decrease inglucose uptake as assessed by flow cytometry using the fluorescent glucose analog 2-NBDG reaching maximum decrease by 72 hours. B, acquired resistantlines (e.g., YUMACr) that have been serially passaged in 5 mmol/L vemurafenib show amuch lower decrease in glucose uptake than drug-na€�ve lines (e.g., YUMACs).C, changes in glucose uptake with BRAF inhibition mirror resistance status and are genotype specific. D, glucose uptake loss due to vemurafenib treatment isreversible. All cells were incubated in 3 mmol/L vemurafenib or DMSO vehicle for 72 hours. After 72 hours, the pulse-chase group had vemurafenib-supplementedmedia aspirated, was washed with PBS, and resupplied with normal media supplemented with vehicle.

Theodosakis et al.





acquired resistance to vemurafenib or those without BRAFmutations showed no significant differences in HKII proteinlevels upon BRAF inhibition (Fig. 4C).

Quantitative measurement of HKII mRNA showed virtuallyno change after 72 hours of vemurafenib treatment in sensitiveor resistant lines (Supplementary Fig. S2A), suggesting a post-transcriptional mechanism of HKII loss. We observed a signif-icant loss of HKII protein in innately vemurafenib-resistantlines YUKSI and YUKOLI comparable with sensitive lines (Fig.4D), despite the increased uptake of 2-NBDG observed by flowcytometry (Fig. 1C). We hypothesized that a compensatory

increase in other HK isoforms present in intrinsic resistant linesmight be responsible for the increase of glucose uptake. Inter-estingly, we found that both vemurafenib-sensitive and intrin-sically resistant lines showed a significant rise in HKI levels at72 hours (Fig. 4E). In contrast, all acquired vemurafenib-resis-tant lines as well as non-BRAF–mutant lines showed no sig-nificant change in HKI levels (Fig. 4F), underscoring the com-plex relationship between changes in HK levels and 2-NBDGuptake with BRAF inhibition (Fig. 4G). Furthermore, immu-nohistochemical staining of our biopsy cohort for HKIIrevealed moderate to high levels in all cases but revealed no

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Figure 3.Vemurafenib (VEM) induces a decrease in transmembrane glucose uptake per cell. 3 mmol/L vemurafenib (þ) incubation induces a decrease in transportof the nonphosphorylatable glucose analog 3-OMG normalized to cell count in sensitive lines (A) but not acquired resistant lines relative to DMSO (�) after72 hours (B). C, vemurafenib treatment does not cause a significant decrease in GLUT1 or GLUT3 proteins levels in sensitive or resistant lines by 72 hours. D,vemurafenib does not induce a significant change in GLUT1 or GLUT3 mRNA levels after 72 hours as detected by qRT-PCR.






significant difference between levels in PRE and EDT samples(Supplementary Fig. S2B). These conflicting data suggest thatchanges in HK activity and regulation with vemurafenib treat-

ment do not show a consistent relationship with glucoseuptake, hinting that an additional alteration might be respon-sible for clinical therapeutic responses.

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Figure 4.Vemurafenib (Vem) induces significant loss of HKII in both sensitive and intrinsically resistant lines. A, 3 mmol/L vemurafenib induces a decrease in overallhexokinase activity per cell after 72 hours in sensitive cell lines. Vemurafenib (þ) induces near complete loss of HKII in sensitive (B) but not acquired resistantor non-BRAF cell lines at 72 hours relative to DMSO (�; C). D, intrinsically resistant cell lines show a decrease in HKII comparable with sensitive cell lines. Sensitive and intrinsically resistant lines show a significant increase in HKI with vemurafenib treatment by 72 hours (E) that is not observed in acquired resistantor non-BRAF–mutant lines (F).

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Cell volume decrease predicts extent of reduction of glucoseuptake in vitro

SUVmax alterations measured by 18FDG-PET account for thetotal radioactive signal emitted from a tumor with a particulargross tumor volume. These measurements do not distinguishbetween the relative proportions of cell volume and extracel-lular matrix and edema that comprise the tumor microenvi-ronment. As cellular volume is another major determinant oftotal solute movement into a cell, including glucose, we micro-scopically examined morphologic changes in a wide range ofmelanoma lines using the cell volume measurement functionsof a Z2 Coulter Counter. Over multiple cases, we quantified anaverage volume decrease of 43% across three highly vemura-fenib-sensitive lines: YUMACs, YUSIT1s, and YUGEN8, butthere was no substantial change in cell volume in lines resistantto BRAF inhibition (Fig. 5A and B). Moreover, we observed thatthe intrinsically vemurafenib-resistant lines YUKSI andYUKOLI, which showed mild increase in 2-NBDG uptake withBRAF inhibition, also showed increases in median cell volume.Mean adherent area calculations also supported these trends(Supplementary Fig. S3). Ultimately, when calculated across 13melanoma lines, changes in cell volume following vemurafenibtreatment were found to predict 83% of the variance in NBDGuptake in our model (Fig. 5C, P < 0.001, Student t test). Thisrelationship also showed a strong correlation with vemurafenibconcentrations required to inhibit growth of cells by 50%(GI50), with GI50 in our model predicting 86% of the variancein volume change (Fig. 5D, P < 0.001, Student t test). We foundthe same relationship between cell volume and uptake changeswhen cells were treated with the MEK inhibitor AZD-6244(Supplementary Fig. S4A and S4B), an effect not explained bycell-cycle arrest alone (Supplementary Fig. S4C and S4D).Applying correction for changes in cell volume to calculatedtransmembrane glucose transport (radiolabeled 3-OMGuptake) greatly reduced the perceived decrease in glucosetransport kinetics (Fig. 5E). Similarly, normalization to proteincontent, as opposed to cell number, greatly reduced differencesin pan-HK activity seen in treated versus untreated cells (Fig.5F). Taken together, these data strongly suggest that measuredalterations in glucose transport and phosphorylation aremarkedly influenced by cell volume changes.

Extrinsic regulation of cell size regulates cellular glucose uptakeindependent of BRAF inhibition

To establish a mechanistic link between cell volume changesand glucose uptake independent of metabolic changes fromvemurafenib, we increased cell volume by decreasing solutiontonicity. We found that decreasing the overall solution tonicityby 50% and 75% during 2-NBDG incubation significantlyincreased cell volume as measured by Coulter Counter (Fig.6A). We also found that changing incubation tonicity concom-itantly increased 2-NBDG uptake (Fig. 6B) under otherwiseidentical conditions without significant cell death or membranedisruption (Supplementary Fig. S5). These findings suggest thatchanges in cell size alone are sufficient to alter glucose uptake inmelanoma. Extending these findings to BRAF-inhibited cells, wefound that incubating cells exposed to vemurafenib for 72 hoursin hypotonic 2-NBDG solutions was sufficient to rescue glucoseuptake relative to untreated controls incubated at normal tonic-ity (Fig. 6C). As a second method for verification of this concept,we treated cells for 24 hours with 50 mg/mL of the ribosomal

translation inhibitor cycloheximide. Cycloheximide treatment ofvemurafenib-sensitive cells produced a decrease in both cellvolume (Fig. 6D) and glucose uptake (Fig. 6E) that was com-parable with the decrease induced by vemurafenib. Cyclohexi-mide treatment in acquired or primary vemurafenib-resistantlines also resulted in marked reduction of cell volume and 2-NBDG uptake that was significantly greater than vemurafenibtreatment in these lines. Cycloheximide treatment of additionallines revealed a positive linear correlation between cell volumeand 2-NBDG uptake (Fig. 6F). Recent studies have demonstratedstrong links between cell volume and changes in total cellularprotein content (19, 33), as well as significant inhibition of 50

cap-dependent translation with BRAF inhibition (34, 35). Thesestudies, in addition to our cycloheximide data, suggested thatchanges in cap-dependent protein translation may be at leastpartially responsible for the noted decrease in cell size andglucose uptake loss. Consistent with this conclusion, we alsonoted a decrease in HKII levels with cycloheximide treatmentthat was comparable with that induced by vemurafenib (Fig.6G), as well as a significant decrease in overall protein synthesisafter 72 hours of drug treatment (Fig. 6H). This underlyingmechanism was also supported by studies showing significantinhibition of assembly of the cap-dependent translation initia-tion complex, as well as decreased HKII de novo translation(Supplementary Fig. S6A and S6B). In parallel, dephosphoryla-tion of ribosomal protein p70S6K and S6 downstream of mTOR,both of which are involved in initiation complex assembly andactivity (36), occurs within 24 hours of treatment, mirroringobserved alterations in cell volume and protein content (Sup-plementary Fig. S7). Additional loss of S6 and p70S6K continuesout to 72 hours, with later loss of upstream Akt phosphorylation,further paralleling increasing loss of glucose uptake and volumeover this timeframe (Supplementary Fig. S8). In line with pre-vious studies (37), loss of S6 phosphorylation was greatlyabrogated in resistant lines, showing a consistent relationshipbetween drug sensitivity, volume, new protein synthesis, and S6activity. The importance of decreases in protein translation overincreased protein degradation was also supported through theinability of the proteasome inhibitors bortezomib or MG132 tosignificantly rescue HKII loss (Supplementary Fig. S9). Thissuggests that targeted destruction of HKII at the proteasome isnot primarily responsible for the observed decreases in HKIIlevels.

DiscussionRECIST criteria are currently widely utilized to evaluate therapy

efficacy. Though response rates are defined by the sum of com-plete and partial responses, it is also apparent that patients withstable disease may derive progression free and overall survivalbenefit from treatment (12). Poor correlation between tumorshrinkage over an initial time window and overall survival isfrequently seen in the melanoma immunotherapy, whereincreases in tumor size can occur before eventual completeregression with survival benefit (6, 38). This suggests that ther-apy-induced changes in tumor volume taken in isolationmay notbe the optimal predictors of patient survival. Incorporation ofadditional data may be needed, necessitating improved under-standing of the mechanism of tumor shrinkage.

Though induction of apoptosis has been assumed to bea principal mechanism of action of BRAF inhibitors, there is






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relatively little evidence that this is the primary mode of effectiveresponse inpatients. At oddswith extensive cell death is the lack ofobservation of tumor lysis syndrome in several large clinical trialsof BRAF inhibitors (1, 2, 9, 10), which might be expected fromtoxic chemotherapy treatments producing comparable RECISTresponses. Evaluation of a series of melanoma patients EDTdemonstrated that geographic tumor necrosis occurred at a higherrate than observed in PRE biopsies (8); however, geographicnecrosis is typically secondary to vascular insufficiency (ischemiaor infarct). A clear increase in individual apoptotic cells was notobserved. In contrast, significant decrease in proliferation (Ki67)and Cyclin D1 levels noted in previous staining of a subset of thiscohort suggest G0–G1 arrest (8). In vitro, high doses of BRAFinhibitors typically induce low levels of apoptosis (0%–10%),with some cell lines (e.g., A375) exhibiting higher rates. Inessentially all BRAF-mutant lines, resistant clones emerge follow-ing chronic treatment in vitro,mimicking the high rate of relapse inmelanoma patients treated with BRAF inhibitors. On the basis ofthese data, tumor shrinkage is likely a complex phenomenon thatintegrates occasional geographic (ischemic) necrosis, cell-cyclearrest, and aminor component of apoptotic cell death. The sumofthese factors does not appear to fully account for and correlatewith clinical tumor shrinkage in BRAF inhibitor–treated melano-ma patients.

Here we provide evidence for BRAF inhibitor–induced reduc-tion in cell volume as a contributing factor in clinical tumorshrinkage and FDG-PET responses. We observed a significantdecrease in cell size in melanoma patient samples EDT, and atrend toward correlation between magnitude of shrinkage andSUVmax decrease. The observed similar level of HKII staining,reduction in cytoplasmic volume, and increase in intercellulardistance early during treatment suggests that decrease in cell sizeprecedes the slow, gradual reduction in tumor size that is typicallyobserved. Thesefindings suggest that early/rapid decrease in FDG-PET signal without corresponding tumor shrinkage as determinedby CT imaging may be due to decreased tumor cellular mass perimaging voxel, intermixed with increased edematous changes.Resolution of the extracellular edematous changes would bepredicted to result in gradual tumor shrinkage associated withresidual to slightly elevated FDG-PET signal, which is typicallyobserved. On a broader scale, these findings raise the possibilitythat imaging-based responses to targeted signal transductioninhibitors may be partially mediated by modulation of cellvolume in addition to cell death.

Recent studies have suggested that MAPK inhibition mayinfluence glucose metabolism in BRAFV600E/K mutant cells byinducing decreases in GLUT transporter or HKII protein levels(5, 11, 24, 25). Decreased metabolic activity has also beenobserved in the formof decreased lactate production (25), oxygenconsumption (39), and metabolic enzyme expression (24): allgenerally normalized to protein mass or cell count. In this study,

we provide evidence in melanoma that cell size alone can signif-icantly regulate glucose uptake in BRAF-mutant melanomas trea-ted with vemurafenib. We did note decreases in glucose uptakeand HK activity per cell; however, following additional normal-ization for cell volume, the changes in HK activity were markedlydiminished. This suggests that while previously demonstratedmetabolic enzyme changes do contribute, they are likely to beonly a part of the mechanism for 18FDG signal loss. This findingmay also help explain the metabolic changes induced by BRAFinhibition in previous reports (11, 24, 25, 40) which have largelyfocused on changes in the first 24 hours after induction of therapywithout exploration of later steady state. Taken together, thesefindings emphasize a broader need for consideration of cell sizechanges in metabolic and biochemical studies, especially inmelanoma biology, where cell populations may consist of mul-tiple subclones with heterogeneous morphologic properties(16, 20).

In this study, we demonstrate an increase in HKI levels in allmelanoma cell lines that show HKII decrease. This could suggestthat HKI may help compensate for changes in HKII in melanomato maintain glycolytic activity, as indicated by our evaluation ofoverall HK activity, and consistent with previous studies in breastand lung cancer (23). Interestingly, we found that intrinsicallyvemurafenib-resistant melanomas show a mild increase in glu-cosemetabolism and decrease in proliferation with treatment, yetdisplay the same HKII/HKI switch seen in sensitive BRAF-mutantmelanomas. Further studies will be required to determine thefunctional effects of upregulation of HKI, as well as the functionalcontributions of individual HK isoforms to glucose uptake.

Evidence provided by this study suggests that total proteintranslationper cell is significantly decreasedwithBRAF inhibition,in agreement with other studies (34, 35). This occurs in parallelwith vemurafenib-induced decreases in p70S6K and S6 activityobserved previously (37), and is largely absent in lines that haveacquired resistance to the drug. The synchronicity of loss of cellvolume, decrease in protein translation, and downregulation ofp70S6K and S6 activity, as well as the correlation of these phe-nomena with drug sensitivity, further support the proposedexplanation for decreases in glucose uptake. Our findings alsoexpand on previous melanoma metabolomics literature, provid-ing a mechanistic explanation for the previously noted down-regulation of multiple key metabolic enzymes (24, 25). Further-more, our study provides evidence for the importance of globalprotein synthesis to maintenance of cell size, and the conse-quences for the clinically relevant characteristic of glucose uptakecapacity when this process becomes dysregulated, emphasizingthe previously established relationship between cell size, cell-cycle progression, and translation (20, 34). Indeed, the G0–G1

cell-cycle arrest andmarkedly reduced bromodeoxyuridine incor-poration we observed in vemurafenib-treated cells also suggeststhat new nucleic acid production is also greatly decreased in the

Figure 5.Vemurafenib (Vem; 3 mmol/L) induces a cellular volume decrease in sensitive lines, which largely accounts for alterations in glucose uptake per cell.A, adherent sensitive but not resistant cell lines show visual decrease in cell size by light microscopy. B, vemurafenib-sensitive but neither acquired norintrinsically resistant cell lines show a decrease in total cell volume with treatment as assessed by Coulter Counter. � , P < 0.05; �� , P < 0.001; n.s., notsignificant. C, changes in cell volume show a strongly significant correlation with changes in measured glucose uptake (r2 ¼ 0.8483, P < 0.01). D, changesin cell size also show a strong correlation with vemurafenib GI50 for mutant BRAF lines (r2 ¼ 0.8568, P < 0.01). E, changes in transmembrane glucosetransport are largely abrogated when corrected for changes in cell volume. F, changes in hexokinase activity are also largely abrogated when normalized towhole lysate protein concentration.






setting of BRAF inhibition; a finding which may reflect broaderchanges in glucose carbon utilization that merits further explo-ration. Nevertheless, the inability of cell-cycle arrest to inde-pendently fully account for decreased size and glucose uptake

suggests that additional MAPK-specific regulation of translationand volume maintenance are also being inhibited. These find-ings taken together suggest a quiescent metabolic phenotype,implying that inhibition of mutant BRAF removes the driving

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Figure 6.Glucose uptake is greatly affected bychanges in cell volume. Decreasingsolution osmolarity while incubatingwith 2-NBDG causes both an increasein cell volume (A) and 2-NBDG uptake(B) that is osmolarity dependent. Theisotonic condition has a totalconcentration of 289 mOsm/L, whilethe weakly and strongly hypoosmoticconditions have concentrations of144.5 mOsm/L and 72.25 mOsm/L,respectively. C, incubation of 3 mmol/Lvemurafenib-treated cells inhypoosmotic 2-NBDG solutionrestored glucose uptake to the samelevel as isotonic 2-NBDG control at 217mOsm/L. D, inhibition of proteintranslation with 50 mg/mLcycloheximide induces a decrease inboth cell volume and glucose uptake(E) at 24 hours with a relationshipsimilar to 3 mmol/L vemurafenib(r2 ¼ 0.8153, P ¼ 0.097; F). G,inhibition of translation withcycloheximide produces comparableloss of HKII to vemurafenibby 24 hours. H, 35S-methioninelabeling shows that incubation ofvemurafenib-sensitive but notresistant lines for 72 hours with 3mmol/L reduced total new proteinsynthesis, � , P < 0.05; �� , P < 0.01;�� , P < 0.001; n.s., not significant.


Theodosakis et al.




force behind neoplastic growth and metabolism without desta-bilizing cells enough to cause apoptosis, levels of which havepreviously been noted to be variable and frequently minimal(39, 41).

In summary, our study suggests that cell size is an importantdeterminant of glucose uptake in melanoma and can affectmany of the observed changes in metabolic activity. It alsoprovides a possible explanation for the re-emergence of resis-tant tumors at sites identical to previous metastases. Further-more, these findings underscore the importance of correctingfor physical parameters when undertaking studies in cellularmetabolism, as well as the dynamic interplay between growth,metabolism, and signaling.

Disclosure of Potential Conflicts of InterestG.V. Long is a consultant/advisory board member for GSK, Novartis, and

Roche. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: N. Theodosakis, M.A. Held, A. Marzuka-Alcala,M.W. BosenbergDevelopment of methodology:N. Theodosakis, M.A. Held, A. Marzuka-Alcala,D.F. Stern, M.W. BosenbergAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Theodosakis, M.A. Held, A. Marzuka-Alcala,K. Meeth, G. Micevic, G.V. Long, R.A. Scolyer

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Theodosakis, M.A. Held, A. Marzuka-Alcala,D.F. Stern, M.W. BosenbergWriting, review, and/or revision of the manuscript: N. Theodosakis,M.A. Held, G.V. Long, R.A. Scolyer, D.F. Stern, M.W. BosenbergAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Theodosakis, M.A. Held, G.V. LongStudy supervision: N. Theodosakis, M.A. Held, M.W. BosenbergOther (managed and acquired the human tissue in this article): G.V. Long

AcknowledgmentsThe authors thank Ruth Halaban and Antonella Bacchiochi for providing

melanoma cell lines from the Yale melanoma SPORE tissue bank, as well asCasey Langdon for helping to develop cell lineswith acquired resistance to BRAFinhibition.

Grant SupportThese studies were supported in part by grants from the NIH: Yale SPORE in

Skin Cancer, funded by the NCI P50 CA121974 to R. Halaban and Ruth L.Kirschstein NRSA 1F30CA180591-01 funded by the NCI (to N. Theodosakis).The studywas also fundedby anAmerican SkinAssociation StudentGrant (toN.Theodosakis) and the Yale Cancer Center grant (to M.W. Bosenberg).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 29, 2015; revised April 7, 2015; accepted April 30, 2015;published OnlineFirst May 6, 2015.

References1. McArthur GA, Chapman PB, Robert C, Larkin J, Haanen JB, Dummer R,

et al. Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-upof a phase 3, randomised, open-label study. Lancet Oncol 2014;15:323–32.

2. Falchook GS, Long GV, Kurzrock R, Kim KB, Arkenau TH, BrownMP, et al.Dabrafenib in patients with melanoma, untreated brain metastases, andother solid tumours: a phase 1 dose-escalation trial. Lancet 2012;379:1893–901.

3. Koppenol WH, Bounds PL, Dang CV. Otto Warburg's contributions tocurrent concepts of cancer metabolism. Nat Rev Cancer 2011;11:325–37.

4. Dell' Antone P. Energy metabolism in cancer cells: how to explain theWarburg and Crabtree effects? Med Hypotheses 2012;79:388–92.

5. Sondergaard JN, Nazarian R, Wang Q, Guo D, Hsueh T, Mok S, et al.Differential sensitivity of melanoma cell lines with BRAFV600E mutationto the specific Raf inhibitor PLX4032. J Transl Med 2010;8:39.

6. Hoos A, Parmiani G, Hege K, Sznol M, Loibner H, Eggermont A, et al. Aclinical development paradigm for cancer vaccines and related biologics. JImmunother (Hagerstown, Md: 1997) 2007;30:1–15.

7. Scott DA, Richardson AD, Filipp FV, Knutzen CA, Chiang GG, Ronai ZA,et al. Comparativemetabolic flux profiling of melanoma cell lines: beyondthe Warburg effect. J Biol Chem 2011;286:42626–34.

8. Long GV, Wilmott JS, Haydu LE, Tembe V, Sharma R, Rizos H, et al. Effectsof BRAF inhibitors on human melanoma tissue before treatment, earlyduring treatment, and on progression. Pigment Cell Melanoma Res 2013;26:499–508.

9. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al.Survival in BRAF V600-mutant advanced melanoma treated with vemur-afenib. N Engl J Med 2012;366:707–14.

10. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al.Improved survival with vemurafenib in melanoma with BRAF V600Emutation. N Engl J Med 2011;364:2507–16.

11. Baudy AR, Dogan T, Flores-Mercado JE, Hoeflich KP, Su F, van Bruggen N,et al. FDG-PET is a good biomarker of both early response and acquiredresistance in BRAFV600mutant melanomas treated with vemurafenib andthe MEK inhibitor GDC-0973. EJNMMI Res 2012;2:22.

12. McArthur GA, Puzanov I, Amaravadi R, Ribas A, Chapman P, Kim KB, et al.Marked, homogeneous, and early [18F]fluorodeoxyglucose-positron emis-

sion tomography responses to vemurafenib in BRAF-mutant advancedmelanoma. J Clin Oncol 2012;30:1628–34.

13. Carlino MS, Saunders CA, Haydu LE, Menzies AM, Martin Curtis C Jr,Lebowitz PF, et al. (18)F-labelled fluorodeoxyglucose-positron emissiontomography (FDG-PET) heterogeneity of response is prognostic in dab-rafenib treated BRAF mutant metastatic melanoma. Eur J Cancer 2013;49:395–402.

14. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutationsof the BRAF gene in human cancer. Nature 2002;417:949–54.

15. Garnett MJ, Marais R. Guilty as charged: B-RAF is a human oncogene.Cancer Cell 2004;6:313–9.

16. Rizos H,Menzies AM, Pupo GM, CarlinoMS, Fung C, Hyman J, et al. BRAFinhibitor resistance mechanisms in metastatic melanoma: spectrum andclinical impact. Clin Cancer Res 2014;20:1965–77.

17. Ravnan MC, Matalka MS. Vemurafenib in patients with BRAF V600Emutation-positive advanced melanoma. Clin Ther 2012;34:1474–86.

18. Killander D, Zetterberg A. A quantitative cytochemical investigation of therelationship between cell mass and initiation of DNA synthesis in mousefibroblasts in vitro. Exp Cell Res 1965;40:12–20.

19. Mir M, Wang Z, Shen Z, Bednarz M, Bashir R, Golding I, et al. Opticalmeasurement of cycle-dependent cell growth. Proc Natl Acad Sci U S A2011;108:13124–9.

20. Wachsberger PR, Gressen EL, Bhala A, Bobyock SB, Storck C, Coss RA, et al.Variability in glucose transporter-1 levels andhexokinase activity in humanmelanoma. Melanoma Res 2002;12:35–43.

21. John S, Weiss JN, Ribalet B. Subcellular localization of hexokinases I and IIdirects the metabolic fate of glucose. PLoS ONE 2011;6:e17674.

22. Arzoine L, Zilberberg N, Ben-Romano R, Shoshan-Barmatz V. Voltage-dependent anion channel 1-based peptides interact with hexokinase toprevent its anti-apoptotic activity. J Biol Chem 2009;284:3946–55.

23. Patra KC, Wang Q, Bhaskar PT, Miller L, Wang Z, Wheaton W, et al.Hexokinase 2 is required for tumor initiation and maintenance and itssystemic deletion is therapeutic in mouse models of cancer. Cancer Cell2013;24:213–28.

24. Parmenter TJ, KleinschmidtM, Kinross KM, Bond ST, Li J, KaadigeMR, et al.Response of BRAF-mutant melanoma to BRAF inhibition is mediated by anetwork of transcriptional regulators of glycolysis. Cancer Discov 2014;4:423–33.






25. Falck Miniotis M, Arunan V, Eykyn TR, Marais R, Workman P, Leach MO,et al. MEK1/2 inhibition decreases lactate in BRAF-driven human cancercells. Cancer Res 2013;73:4039–49.

26. Held MA, Langdon CG, Platt JT, Graham-Steed T, Liu Z, Chakraborty A,et al. Genotype-selective combination therapies for melanoma identifiedby high-throughput drug screening. Cancer Discov 2013;3:52–67.

27. Halaban R, Zhang W, Bacchiocchi A, Cheng E, Parisi F, Ariyan S,et al. PLX4032, a selective BRAF(V600E) kinase inhibitor, activatesthe ERK pathway and enhances cell migration and proliferation ofBRAF melanoma cells. Pigment Cell Melanoma Res 2010;23:190–200.

28. Yamamoto N, Ueda M, Sato T, Kawasaki K, Sawada K, Kawabata K, et al.Measurement of glucose uptake in cultured cells. Curr Protoc Pharmacol2011;Chapter:Unit 12.4.1–22.

29. Enzymatic Assay of Hexokinase; cited 2014. Available from: http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-hexokinase.html

30. Bergmeyer HU, Grassl M, Walter HE. In: Bergmeyer HU, editor. Methods ofenzymatic analysis, 3rd ed., Vol. II. DeerfieldBeach, FL: VerlagChemie; 1983. p222–3. See more at: http://www.sigmaaldrich.com/technical-documents/pro-tocols/biology/enzymatic-assay-of-hexokinase.html#sthash.TFc4cFtw.dpuf.

31. ZouC,WangY, ShenZ. 2-NBDGas afluorescent indicator for direct glucoseuptake measurement. J Biochem Biophys Methods 2005;64:207–15.

32. Park SG, Lee JH, Lee WA, Han KM. Biologic correlation between glucosetransporters, hexokinase-II, Ki-67 and FDG uptake in malignant melano-ma. Nuclear Med Biol 2012;39:1167–72.

33. Kafri R, Levy J, Ginzberg MB, Oh S, Lahav G, Kirschner MW. Dynamicsextracted from fixed cells reveal feedback linking cell growth to cell cycle.Nature 2013;494:480–3.

34. Boussemart L, Malka-Mahieu H, Girault I, Allard D, Hemmingsson O,Tomasic G, et al. eIF4F is a nexus of resistance to anti-BRAF and anti-MEKcancer therapies. Nature 2014;513:105–9.

35. Silva JM, Bulman C, McMahon M. BRAFV600E cooperates with PI3Ksignaling, independent of AKT, to regulate melanoma cell proliferation.Mol Cancer Res 2014;12:447–63.

36. Clark DE, Errington TM, Smith JA, Frierson HF Jr, Weber MJ, Lannigan DA.The serine/threonine protein kinase, p90 ribosomal S6 kinase, is animportant regulator of prostate cancer cell proliferation. Cancer Res 2005;65:3108–16.

37. Kang S, Elf S, Lythgoe K, Hitosugi T, Taunton J, Zhou W, et al. p90ribosomal S6 kinase 2 promotes invasion and metastasis of humanhead and neck squamous cell carcinoma cells. J Clin Invest 2010;120:1165–77.

38. Wolchok JD, Hoos A, O'Day S, Weber JS, Hamid O, Lebbe C, et al.Guidelines for the evaluation of immune therapy activity in solidtumors: immune-related response criteria. Clin Cancer Res 2009;15:7412–20.

39. Haq R, Shoag J, Andreu-Perez P, Yokoyama S, Edelman H, Rowe GC, et al.Oncogenic BRAF regulates oxidativemetabolismvia PGC1alpha andMITF.Cancer Cell 2013;23:302–15.

40. Yamada K, Brink I, Bisse E, Epting T, Engelhardt R. Factors influencing[F-18] 2-fluoro-2-deoxy-D-glucose (F-18 FDG) uptake in mela-noma cells: the role of proliferation rate, viability, glucose trans-porter expression and hexokinase activity. J Dermatol 2005;32:316–34.

41. ParaisoKH, Fedorenko IV, Cantini LP,MunkoAC,HallM, SondakVK, et al.Recovery of phospho-ERK activity allows melanoma cells to escape fromBRAF inhibitor therapy. Br J Cancer 2010;102:1724–30.


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2015;14:1680-1692. Published OnlineFirst May 6, 2015.Mol Cancer Ther Nicholas Theodosakis, Matthew A. Held, Alexander Marzuka-Alcala, et al. Association with Reduction in Cell VolumeBRAF Inhibition Decreases Cellular Glucose Uptake in Melanoma in

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braf inhibition decreases cellular glucose ......easytag express35s protein labeling mix...

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