plekha5 as a biomarker and potential mediator of melanoma … · yusipu, yugoe, yuhuy, and yuhef...

Biology of Human Tumors

PLEKHA5 as a Biomarker and Potential Mediatorof Melanoma Brain MetastasisLucia B. Jilaveanu1, Fabio Parisi2, Meaghan L. Barr1, Christopher R. Zito1,3,William Cruz-Munoz4, Robert S. Kerbel4, David L. Rimm2, Marcus W. Bosenberg5,Ruth Halaban5, Yuval Kluger2, and Harriet M. Kluger1

Abstract

Purpose: Approximately 40% of patients with metastatic mel-anoma develop brain metastases. Our purpose was to identifygenes aberrantly expressed inmelanoma that might be associatedwith propensity for brain homing.

Experimental Design:We studied gene expression profiles ina cell line model of brain metastasis (cerebrotropic A375Br cellsvs. parental A375P cells) and compared them with profiles ofpatients who developed early brain metastases and who didnot. A tissue microarray containing 169 metastatic melanomacases with variable time to brain metastasis was constructed tofurther study marker expression by quantitative immunofluo-rescence. An in vitromodel of the blood brain barrier (BBB) wasgenerated to evaluate potential mediators of brain metastases.

Results:PLEKHA5wasdifferentially expressed inboth theA375cell line model and patient samples subjected to gene expression

profiling. At the protein level, by quantitative immunofluores-cence, PLEKHA5 was associated with decreased brain metastasis-free survival. PLEKHA5 overexpression was not associated withother metastatic sites. Knockdown of PLEKHA5 decreases theviability of A375Br cells, inhibits BBB transmigration and invasionin vitro. Similar results were found with YUMUL cells, culturedfrom a patient with overwhelming brain metastases. PLEKHA5knockdown did not affect the viability of A375P cells.

Conclusions: PLEKHA5 expression in melanoma tumorswas associated with early development of brain metastases.Inhibition of PLEKHA5 might decrease passage across the BBBand decrease proliferation and survival of melanomacells both in the brain and in extracerebral sites. Clin CancerRes; 21(9); 2138–47. �2014 AACR.

See related commentary by Eisele et al., p. 1978

IntroductionThe incidence of melanoma is constantly increasing (1), and

the prognosis can be poor once the disease metastasizes.Melanoma is relatively resistant to classic chemotherapy andradiotherapy, but in recent years, a number of promisingsystemic therapies have been approved for this disease (2).These include ipilimumab (anti-CTLA4 antibodies), vemurafe-nib and dabrafenib (selective inhibitors of mutant BRAF),trametinib (MEK inhibitor), or nivolumab and pembrolizu-mab, (PD-1 inhibitors) and with these newer drugs, the mediansurvival for patients with metastatic melanoma has started toimprove, providing new hope of prolonged disease response in

subsets of patients with metastatic disease (2). These drugs,however, have been less extensively studied in patients withbrain metastases.

Of all solid tumors, melanoma metastasizes to the brain withthe highest frequency; approximately 40% of patients with met-astatic melanoma develop brain metastases during the course oftheir illness, and brain metastases are found in up to 75% atautopsy (3). Brain metastases have historically been associatedwith a median overall survival of only 2.5 to 4 months in olderseries, but recent series report a median survival of 8 to 10.6months, likely due to the impact of improved brain imagingmodalities, frequent surveillance, and improved local centralnervous system (CNS) therapies, particularly stereotactic radio-surgery (4). The only standard melanoma drug known to pene-trate the blood brain barrier (BBB) is temozolamide. Dabrafenibalso has intracranial activity; response rates in brain metastasesto these drugs are: <10% and 31% to 39%, respectively, with amedian progression-free survival of 1.2 and 4 months, respec-tively (5, 6). Ipilimumab induces response in untreated brainmetastases at a frequency similar to extracerebral metastases (7).Most drugs are only studied in the brain metastasis populationafter they have been fully studied in patients with extracerebralmetastases in phase III trials, delaying access to newer therapies forthe brain metastasis population. This is due to historical poorsurvival in this population, limited studies of drug blood brainbarrier penetration in tumors, and limited understanding of thebiology of brain metastases.

The present dearth of knowledge about the biology andmolec-ular basis of melanoma brain metastasis is a major obstacle to

1DepartmentofMedicine, SectionofMedicalOncology,YaleUniversitySchool of Medicine, NewHaven, Connecticut. 2Department of Pathol-ogy, Yale University School of Medicine, New Haven, Connecticut.3Department of Biology, School of Health and Natural Sciences, Uni-versity of Saint Joseph,West Hartford, Connecticut. 4Department ofMedical Biophysics, Sunnybrook Research Institute, BiologicalSciences Platform, University of Toronto, Toronto, Ontario, Canada.5Department ofDermatology,YaleUniversity School ofMedicine,NewHaven, Connecticut.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Harriet M. Kluger, Section of Medical Oncology, YaleUniversity, 333 Cedar Street,WWW213, NewHaven, CT 06520. Phone: 203-737-2572; Fax: 203-785-3788; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-0861

�2014 American Association for Cancer Research.

ClinicalCancerResearch

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progress against this disease. Identifying new biomarkers is nec-essary to enable future development of targeted therapies fortreatment of melanoma brain metastases.

Distant metastasis is a multiple-step process which includeslocal invasion from the primary site, intravasation into blood orlymphatic flow, survival in the circulation, extravasation into atarget organ, and regrowth. The natural barriers to disseminatedcancer cells are unique to each organ and the ability of metastaticcells to adapt to different environments is distinct (8). This hasbeen demonstrated in studies of metastatic subpopulations ofcells with different organotropisms, which have uncoveredunique gene signatures associated with breast cancer metastasisto specific organs, such as bones, lungs, or brain (9–11).

In melanoma, a few studies have identified moleculesinvolved in the pathogenesis of the disease and mediators ofinvasion or angiogenesis. For example, in preclinical models,elevated levels of VEGF-A were found to cause deterioration ofthe BBB function, vascular hyperpermeability, and rapid growthof melanoma brain metastasis, while increased activity of HSPE(heparanase), an enzyme that degrades polymeric heparansulfate molecules which are major constituents of the endothe-lial cell layer, enhances the invasiveness of melanoma cells tothe brain (12, 13). The JAK-STAT signaling pathway has beenshown to have an important role in melanoma brain metastasisspecifically through STAT3 activation and/or SOCS-1 (suppres-sor of cytokine signaling-1) downregulation (14, 15). In ananalysis of melanoma patient specimens, PI3K-AKT pathwayactivation was found in brain metastases compared with extra-cerebral sites (16, 17). More recently, Gap junction proteins(Connexins) were shown to mediate early events in brainmetastasis, such as tumor cell extravasation and blood vesselcooption (18). Endothelin receptor B (EDNRB) was found to bea mediator of spontaneous brain metastasis, while overexpres-sion of this protein also exacerbated the extracerebral metastasis(19). Profiling of primary melanoma specimens revealed aseven miRNA signature that can predict brain metastasis (20).

We hypothesized that melanoma cells with the ability to crossthe BBB, and grow and survive in the CNSmicroenvironment, aremolecularly distinct from those that cannot. Genes associatedwith CNS tropism might be harnessed for drug targeting in

patients with cerebrotropic melanoma. In this study, we analyzedcell line models and patient specimens to identify additionalbiomarkers of cerebral metastasis in melanoma and conductedfunctional studies for further characterization.

Materials and MethodsCell lines

YUGANK, YUMUL, YUGEN, YUSIK, YUKOLI, YUMUT, YUSIV,YUKRIN, YUSIT, YUMAC, YURIF, YUKADI, YUCOT, YURDE,YUSIPU, YUGOE, YUHUY, and YUHEF are early passage (<20)cell cultures derived from tumors of patients treated at YaleUniversity (New Haven, CT). Cells in culture were tested formutations in BRAF and NRAS by Sanger sequencing. PTEN losswas also assessed by Western blot analysis. Mutation patternswere compared with the tumors fromwhich they were derived forauthentication. Cells underwent less than 20 passages before usein these studies. After 20 passages, cells were discarded and a vialfrom an early passage from the cell bank was used. A375P andA375Br were kindly provided by Dr. S. Huang from M.D. Ander-son Cancer Center in Houston and profiled within 6 months ofreceipt. A375P is a human melanoma cell line derived from alymphnodemetastasis, which fails to produce brainmetastases inmice. A375Br is a highly cerebrotropic derivative, described pre-viously, derived by repeat injection of parental cells in the carotidartery of mice and repassaging of cells recovered from the brain(14, 15). In contrast with A375P, A375Br cells yield brain metas-tasis in 100% of mice. Additional information is provided in theSupplementary Materials.

Patient samples for gene expression studiesWith Institutional Review Board approval, we reviewed charts

of patients with metastatic melanoma treated at our institutionbetween 2005 and 2011. For our discovery studies, we identifiedpatients with available extracerebral metastatic tumors forexpression profiling who had been closely followed with serialsurveillance MRI imaging every 3 months or less, and selectedpatients who developed CNS metastases within 6 months ofdiagnosis of metastatic disease and who did not develop brainmetastases for over 18 months. Age at diagnosis ranged from20 to 80 years (mean, 55.4 years) . The cohort included 65%males and 35% females. Specimens included metastases fromseveral sites: lymph nodes, skin, soft tissue, and visceral meta-stases. The two groups were balanced for gender, age, site ofprimary lesion, and presence of lung metastases.

Gene expression profiling and data analysisRNA extraction, quality control measures, and data prepro-

cessing are described in the Supplementary Materials. Datahave been deposited in NCBIs Gene Expression Omnibus,(accession number: GSE60464).

Because gene expression can be influenced by the microenvi-ronment, for our initial analysis, we compared samples fromsimilar anatomical sites representing metastases from lymphnodes, soft tissue, and skin for both cerebrotropic and noncer-ebrotropic cases (17 and 25, respectively). Both groups wererepresented by 30% and 36% lymph nodes, by 47% and 40%soft tissue, and 23% and 20% skin, respectively. To test theassociation between gene expression and cerebrotropism, weused the Wilcoxon rank-sum test. We set the significance cutoff

Translational Relevance

In this study,weused an integrated clinical andbench-basedapproach to identify genes aberrantly expressed in metastaticmelanoma samples that might be associated with a greaterpropensity for brain metastasis. Our clinical and mechanisticfindings suggest that PLEKHA5 might be a mediator of thebrain homing phenotype. PLEKHA5 expression was higher inpatients with early brain metastasis compared with patientswithout, in both cerebral- and extracerebral metastases andexpression was associated with a shorter time to developmentof brain metastases. PLEKHA5 seems to be a regulator ofsurvival and proliferation of cerebrotropic melanoma cellsand a mediator of their passage through the blood brainbarrier. PLEKHA5 could therefore be a novel and biologicallyimportant target for pharmacologic inhibition in patients withmelanoma with brain metastases.

Role of PLEKHA5 in Melanoma Brain Metastasis

www.aacrjournals.org Clin Cancer Res; 21(9) May 1, 2015 2139




at 0.05 for q values that were adjusted following the Benjamini–Hochberg correction for multiple testing.

Tissue microarray constructionWe constructed a metastatic melanoma tissue microarray

(TMA) from paraffin-embedded, formalin-fixed tissue blocksfrom 169 patients.

Clinical characteristics. The cohort included 63% males and37% females. Age at diagnosis ranged from 20 to 82 (mean,55 years). Eighty-nine percent had primary cutaneous (non-acral) melanomas, 8% had mucosal or acral-lentiginous mel-anoma, and 3% had uveal melanoma. Of the nonacral cuta-neous melanomas, 43% originated in the trunk, 29% in theextremities, and 28% in the head and neck area. At initialdiagnosis of stage IV disease, 16% had M1a disease, 30%M1b disease, and 55% M1c disease, 31% had elevated lactatedehydrogenase (LDH). Median survival from diagnosis ofstage IV disease was 55 months in patients with M1a diseasecompared with 24 and 22 months in M1b and M1c diseasepatients, respectively. BRAF mutations were found in 43% andNRAS in 19%. Brain metastasis–free survival (BMFS) follow-uptime was up to 187.65 months, with a mean follow-up timeof 19.34 months. Other noncerebral metastases were presentin 50% of patients before or at the time of sample acquisition.In 70% of cases, first cerebral metastasis was diagnosed at orbefore specimen collection. Before sample acquisition, systemictherapy had been administered to 55% of patients.

Specimens included metastases from several sites: lymphnodes, skin, soft tissue, and visceral metastases. Informationabout sites of metastatic disease was only collected frompatients who had imaging to capture involvement of thosesites. A pathologist (D.L. Rimm) reexamined each case andselected a representative region of invasive tumor for coring twocores for each block.

Immunofluorescent staining, automated image acquisition,and analysis

Staining is detailed in Supplementary Material. Automatedimage acquisition and analysis have been previously described(21). JMP version 5.0 software was used (SAS Institute). Organ-specific metastasis-free survival analyses were depicted using theKaplan–Meiermethod. Analyseswere based on sites ofmetastasesat any time during the course of the illness. The associationbetween continuous AQUA scores and other clinical/pathologicparameters was assessed by ANOVA.

BBB transmigration assayFor transmigration assays, we adapted a coculture model of

the BBB which was previously described (11). The in vitro BBBmodel is described in Supplementary Material. PLEKHA5expression was silenced with 40 nmol/L siRNA, and 24 hoursbefore performing transmigration assays, cells were labeledwith CFMDA cell tracker green and recovered in culture.Approximately 50,000 CFMDA-labeled cells were seeded inthe upper well, on top of the endothelial layer and incubatedfor various times. Transmigration was quantified at 8 and 16hours. Depletion of medium of FBS in the upper well to inhibitproliferation did not affect the results. Each experiment was runin triplicate. Inserts were examined microscopically for holes in

the insert. Inserts with holes were discarded. Inserts werewashed with PBS and membranes containing BBB cells as wellas melanoma cells in the process of transmigration were fixedwith 4% paraformaldehyde, mounted on slides, and analyzedmicroscopically. An average of 10 pictures was taken from eachinsert and the number of transmigrated cells were counted foreach insert and averaged for each experiment.

Matrigel basement membrane invasion assayFor invasion assays, we used the BioCoat tumor invasion

24-multiwell Transwell system (Corning). PLEKHA5 expressionwas silencedwith40nmol/L siRNA, and100,000CFMDA-labeledcells were seeded in the upper well in serum-free medium. Thelower well was filled with medium containing 10% FBS. After18 hours, inserts were washed with PBS andmembranes contain-ing invading melanoma cells were analyzed microscopically asdescribed in the BBB transmigration assay.

ResultsGene expression studies of cerebrotropic andnoncerebrotropiccell lines and specimens from patients with and without earlybrain metastases

To identify genes specifically associated with brain homingand less likely to be associated with metastases in general oraggressive metastatic melanoma, we studied gene expressionprofiles of the A375P and A375Br cell line model. These areisogenic cell lines and provide an excellent system to studymolecular determinants of brain metastases. Comparison oftranscript profiles between the parental cell line and its cere-brotropic derivative yielded a relatively small set of differen-tially expressed genes, similar to results of studies done in otherdisease models (11, 22). Supplementary Table S1 shows genesthat had a 3-fold or higher change (over- or underexpressed) inexpression level between A375Br and A375P. We also analyzedtranscript profiles of extracerebral metastases of patients withcerebrotropism (defined here as development of brain metas-tasis within <6 months of stage IV disease), compared withpatients who did not develop brain metastases for >18 months,using the Wilcoxon rank-sum test (Supplementary Table S2).These samples were balanced for age, gender, site of primarylesion, presence of lung metastases, and metastatic site of lesion(using metastases from soft tissue, skin and lymph nodes, butnot from visceral organs). To identify clinically significantcandidate genes to study in the A375P/Br model, we restrictedthe analysis of patient data to those genes that had a 3-folddifference in expression levels in the cell line model. Followingthe Benjamini–Hochberg correction for multiple hypothesestesting of the two-sided Wilcoxon sign-rank test, we found thatPLEKHA5 was the only gene that was upregulated in cerebro-tropic patients (adjusted P value ¼ 0.02) and overexpressed inthe A375Br cells.

In Fig. 1A, we show a box plot demonstrating the distribu-tion of expression of PLEKHA5 in extracerebral specimensfrom patients with cerebrotropic melanoma and without.PLEKHA5 expression was higher in the group of patients withearly brain metastases. Fourteen specimens taken from thebrain were also available for analysis. Expression of PLEKHA5in cerebral and noncerebral metastases in patients with earlybrain metastases demonstrated similar distribution (data notshown).

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Clin Cancer Res; 21(9) May 1, 2015 Clinical Cancer Research2140




PLEKHA5 protein expression and correlation with brainmetastasis-free survival

Correlation between mRNA and protein levels cannot beassumed and previous studies have shown that they correlateabout half of the time (23). Furthermore, in order for a gene tobe considered a true mediator of brain homing, we need toconfirm the association between its protein expression andbrain propensity, because most of the biologic functions arecarried out by proteins and not by mRNA. PLEKHA5 was firststudied by Western blot analysis in a panel of melanoma celllines which included the A375P/A375Br cells and a secondisogenic cell line model of cerebrotropism, 113/6-4L and131/4-5B1 cells (24). Differential expression at the mRNA leveltranslated into significant alteration at the protein level; PLE-KHA5 expression was found to be globally higher in the cere-brotropic and brain metastasis-derived cells when comparedwith noncerebrotropic cells. PLEKHA5 was not detected or wasweak in A375P and 113/6-4L parental cells but expressed at highlevels in the cerebrotropic derivatives, A375Br and 131/4-5B1,respectively (Fig. 1B). PLEKHA5 expression was elevated inYUGEN8 and YUKRIN cells, derived from brain metastases,and YUMUL, YUKOLI, YUMUT cultures, all derived from

patients with early melanoma brain metastases. However, PLE-KHA5 was low or minimal in two cerebrotropic cell lines,whereas expression was high in another two noncerebrotropiccell lines.

We then tested the association between PLEKHA5 proteinexpression level and brain propensity using a method of quan-titative immunofluorescence (AQUA). Stainingofour TMAof 169patients with metastatic melanoma with variable patterns ofdisease dissemination was done for AQUA, as depicted inFig. 1C. Staining within a histospot was fairly homogenous andpredominantly membranous and cytoplasmic. At least two coresper patient were included in the TMA and AQUA scores wereaveraged to give oneAQUA score per patient. AQUA scores rangedfrom 2.73 to 101.99 (median: 12.8). Using the Pearson correla-tion test, we compared scores from matching cores includedon the array for each patient and found a high degree of corre-lation; R ¼ 0.88 (P < 0.0001). Unpaired t tests showed thatexpression of PLEKHA5 was higher in patients who developedbrain metastases at some point during their illness (P ¼0.015; Table 1). PLEKHA5 expression neither correlated with anincreased incidence of bone, liver, lung, soft tissue, and skinor lymph node metastases, nor was it associated with the site of

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Figure 1.Association of PLEKHA5 expression anddevelopment of brain metastases. A, box plotshowing that PLEKHA5 expression by DASLassay is higher in patients with cerebrotropicdisease. B, analysis of PLEKHA5 expression byWestern blot analysis. Equal protein gelloading was assessed by probing forexpression of b-actin. YUGANK, YUMUL,YUGEN, YUSIK, YUKOLI, YUMUT, YUSIV, andYUKRIN were all patients with metastaticmelanoma with early melanoma brainmetastases, whereas YURIF, YUKADI, YUCOT,YURDE, YUSIPU, YUGOE, YUHUY, and YUHEFdid not develop brain metastases. The courseof YUSIT andYUMAC is unknown. C, analysis ofPLEKHA5 expression byAQUA. S100protein isused to locate the tumor. Holes are filled tocreate a tumormask andDAPI is used to locatenuclei. Signal from nuclei is subtracted fromthat of the mask to create a cytoplasmiccompartment. Target was measured on ascale of 0–255 within each compartment.D, Kaplan–Meier curves for PLEKHA5demonstrating the correlation betweenmarker expression (dichotomized scores)and BMFS. High PLEKHA5 expression wassignificantly associated with decreased BMFS.






the biopsy, demonstrating that it is elevated in all sites of metas-tases of patients with brain involvement (Table 1).

Our TMA included 11 cases with matched tissue cores from acerebral and extracerebral site or two different extracerebralsites. Comparison of PLEKHA5 AQUA scores by the Pearsoncorrelation test showed a high degree of correlation of PLE-KHA5 expression in these paired samples; R ¼ 0.86 (P <0.0001), suggesting once again that molecular aberrationsassociated with the brain homing phenotype can also be foundin extracerebral samples.

ANOVA analysis or t test was used to test the associationbetween PLEKHA5 expression and other clinical variables. Nocorrelation was found between PLEKHA5 expression and gen-der or age or BRAF/NRAS mutational status (P > 0.05). By Coxunivariate survival analysis, we found no association betweenPLEKHA5 expression and overall survival, once again suggest-ing that PLEKHA5 expression is associated specifically withthe brain homing phenotype and not with aggressive diseasein general.

We next assessed the association between PLEKHA5 levelsand BMFS by Cox univariate analysis. For each patient, BMFS isdefined as the time interval between diagnosis of first distantmetastasis and imaging diagnosis of brain metastasis. We notethat patients in this cohort were under active surveillance fordevelopment of brain metastasis by frequent brain imaging.Patients without brain metastasis were censored at the timeof their last MRI or CT scan. High PLEKHA5 expression wasassociated with decreased BMFS (P ¼ 0.007, HR ¼ 1.3). Tovisually assess the association between PLEKHA5 and BMFS,we dichotomized scores by the median value and performedKaplan–Meier analysis using log-rank statistics. High PLEKHA5expression was associated with decreased BMFS (log-rank P ¼0.02; Fig. 1D). Using the Cox proportional hazards model, wedid multivariable analysis, and found that high PLEKHA5expression remained an independent predictor of short BMFS(P ¼ 0.015). The only other variable associated with BMFS bymultivariate analysis was American Joint Committee on CancerM stage (P ¼ 0.04). All other variables included in the model(age, gender, and LDH) were not associated with BMFS (Sup-plementary Table S3). No association was found betweenPLEKHA5 expression and time to development of lung, liver,or bone metastasis (Supplementary Fig. S1).

PLEKHA5 knockdown in cerebrotropic melanoma cell linesTo address the role of PLEKHA5 in proliferation of cerebro-

tropic melanoma cell lines, we used siRNAs to inhibit expres-

sion of PLEKHA5 and studied their effects on cell viability. At96 hours, three of the four different siRNA clones testedcompletely abrogated expression of PLEKHA5 in A375Br cellsat a concentration of 40 nmol/L (Supplementary Fig. S2).Target knockdown was furthermore confirmed in YUMULcells, which similarly to A375Br cells express high levels ofPLEKHA5 (Supplementary Fig. S3). YUMUL was established inculture from a metastasis of a patient with early and over-whelming brain metastases. PLEKHA5 knockdown inhibitedviability of A375Br and YUMUL cells in culture at each ofthe three siRNA concentrations (Fig. 2). The degree of knock-down for each of the three siRNA concentrations is shownin Supplementary Fig. S3. In contrast, knockdown of PLEKHA5expression did not affect viability of A375P cells (Fig. 2). Ascrambled siRNA was utilized as a negative control and didnot show an effect on proliferation in any of the three cell lines.Assessment of PARP cleavage and Annexin V/propidiumiodide staining showed that PLEKHA5 knockdown does notinduce apoptosis (data not shown). This suggests that thedecrease in cell number (total viable cells) following PLEKHA5silencing is mediated by a reduction in cell growth.

Previous studies by Lin and colleagues showed that astrocytescan have a protective effect onmelanoma cells and prevent themfrom undergoing apoptosis (25). In addition, astrocytes havebeen shown to enhance proliferation of cancer cells throughparacrine signaling (26). To test whether astrocytes could poten-tiate a protective effect and reduce the cytotoxicity of PLEKHA5knockdown, we cultured A375Br cells with conditioned medi-um and in close proximity to astrocytes using a Transwellsystem. PLEKHA5 silencing inhibited viability of A375Br cells,both in the absence and in the presence of astrocytes and theeffect was comparable (Supplementary Fig. S4).

PLEKHA5 and melanoma transmigration through an in vitromodel of the BBB and invasion through Matrigel matrix

Given the association between PLEKHA5 and decreasedBMFS, we sought to determine whether this molecule was amediator of BBB passage. We utilized siRNAs to inhibit expres-sion of PLEKHA5 in the highly cerebrotropic A375Br andYUMUL cells and then tested the ability of these cells totransmigrate through an in vitro model of the BBB (Fig. 3).A375Br cells were more able to transmigrate through the in vitroBBB than their parental A375P cells (Fig. 4). Knockdown ofPLEKHA5 expression inhibited transmigration of A375Brthrough the in vitro BBB more than 4-fold (Fig. 4). Silencing ofPLEKHA5 expression also inhibited transmigration of YUMUL

Table 1. Association between PLEKHA5 and clinical or tumor characteristics

t statistic P

Clinical characteristics Soft tissue and Skin metastasis �1.59 0.117Lymph node metastasis �1.182 0.241Lung metastasis �0.12 0.905Liver metastasis �1.449 0.152Bone metastasis 0.096 0.924CNS metastasis 2.491 0.015

Site of biopsy Soft tissue and skin �1.34 0.182Lymph node �1.517 0.132Viscera 1.122 0.274Brain 1.44 0.155

NOTE: Two independent sample t tests comparing PLEKHA5 expression (continuous AQUA scores) between patients who developedmetastases at specific sites atany time during the course of illness and thosewhodid not, and between specimens of a specific origin and all others. Significant correlations (P <0.05) aremarked inbold.

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cells, although the effect was less pronounced (2-fold). Finally toinvestigate the role of PLEKHA5 in invasion of cerebrotropiccells through reconstituted basement membranes, we utilized aMatrigel matrix Transwell system. We found that PLEKHA5knockdown also inhibited Matrigel invasion of A375Br andYUMUL cells (Fig. 5). The effects of PLEKHA5 silencing onmotility utilizing a wound-healing assay could not be assessedbecause of the low baseline migration of the two cell lines tested(data not shown).

DiscussionOur purpose was to identify candidate genes that might medi-

ate melanoma cerebrotropism. We compared genome-wideexpression analysis of amelanoma cell line derived from a lymphnode metastasis (A375P) and compared it with a cerebrotropicderivative (A375Br) generated by repeated carotid artery injectionin mice. We also profiled tissues from patients who developedearly brain metastases with patients who did not. PLEKHA5 was

significantly upregulated in cerebrotropic patients and over-expressed in A375Br cells. Using a method of quantitativeimmunofluorescence on a larger sample set, we confirmed theassociation between high PLEKHA5 expression and brain pro-pensity (using BMFS as the endpoint). PLEKHA5 expressionwas not associated with development of metastases at other sitesand expression was high in samples from patients with brainmetastases, regardless of the anatomical location of the speci-men assayed. To determine whether PLEKHA5 is mechanisticallyassociated with CNS dissemination and homing, we knockeddown PLEKHA5 expression by siRNA and found that this inhib-ited proliferation of cerebrotropic cells, both alone and in thepresence of astrocytes. Furthermore, we found that transientsuppression of PLEKHA5 diminished transmigration of cerebro-tropic cells across an in vitro BBB model and inhibited theirinvasion through reconstituted basement membranes.

PLEKHA5 belongs to the PLEKHA family (PLEKHA1 -7) andcontains both the PH (pleckstrin homology) and Trp-Trp WWputative domains. The PH domain is shared by proteins with

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Figure 2.Effect of PLEKHA5 silencing on cell viability.A375Br, A375P, and YUMUL cells weretransfected with 10, 20, or 40 nmol/L of eitherPLEKHA5 siRNAor a scrambled, control siRNAfor 96 hours. PLEKHA5 siRNA inhibited cellviability of A375Br and YUMUL cell lines by43% to 69% and 49% to 59%, respectively.Viability of the A375P cell line was lessaffected at all siRNA concentrations. A controlscrambled siRNA did not affect viability in anyof the cell lines. Significant P values from theStudent t test comparing the viability betweentreated versus untreated cells are indicated by� for P < 0.05 or �� for P < 0.001.






phosphoinositide-binding specificities that are key mediators ofcellular events such as signaling, cytoskeleton rearrangement,membrane protein targeting, and vesicular trafficking. PLEKHAproteins are thought to function mostly as adaptor proteinsbecause they lack catalytic subunits (27). PLEKHA5 expression

was found to be elevated in melanoma by Dowler and colleagues(27). More recently multisplicing PLEKHA5 variants have beenidentified in mice and humans, but their functions are poorlycharacterized (28). The L-PLEKHA5 form (1282 amino acids) isthe dominant variant in adulthood and is expressed predomi-nantly in the brain, while a shorter form, S-PLEKHA5 (1116amino acids) is ubiquitous (28). Both variants play a role innormal brain development (28). Full-length PLEKHA5 isexpressed at the plasma membrane and is associated with micro-tubules (29). Increased expression of membrane-associated PLE-KHA5 was found at cell–cell contacts and also at the actin-polymerization site at the leading edge of motile cells in awound-healing assay, suggesting an important role for PLEKHA5in cell migration (29). It is unclear whether PLEKHA5 function isexerted primarily by its PH domain-specific lipid binding affinityor by other yet uncharacterized protein–protein interactionssimilar to other PH-domain containing proteins, which usuallyengage in a variety of protein–protein and protein–lipid interac-tions and formation of membrane-associated signaling com-plexes. Interestingly, previous studies have shown a higher degreeof PI3K-AKT pathway activation in melanoma brain metastaseswhen comparedwith extracranial sites (16, 17). In addition,manylines of evidence suggest possible links between PI3K pathwayactivation and expression or activity of a number of knownmelanoma brain metastasis mediators and their respective path-ways, such asVEGF-A,HSPE, STAT3, andConnexins. For example,HSPE stimulates PI3K and AKT and enhances VEGF expression(30). Connexin43 colocalizes with AKT and its cellular traffickingseems tobedependent onAKT,whereas PI3K andSTAT3 signaling

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Figure 4.Effect of PLEKHA5 silencing on BBB transmigration of cerebrotropic cells.In vitro BBB transmigration activity of A375Br and YUMUL (� anti-PLEKHA5siRNA) was compared with A375P cells. The number of transmigratedcells was summed for ten different fields, averaged for triplicatedeterminations, and plotted as shown. Knockdown of PLEKHA5 expressioninhibited transmigration of A375Br and YUMUL cells through the in vitro BBBapproximately 4- and 2-fold, respectively. Significant P values from theStudent t test comparing levels of transmigration between treated versusuntreated cell lines are indicated by � for P < 0.05.

Jilaveanu et al.





seems to be interdependent (31, 32). PLEKHA5 clearly hasphosphoinositide-binding specificity and studies are currentlyunderway in our laboratory to assess the role of PI3K in itsmembrane recruitment and to investigate the functional cross-talk between PI3K-AKT signaling and PLEKHA5 in melanomabrain metastasis.

Both, transmigration across the BBB and proliferation andsurvival within the CNS microenvironment are likely to benecessary for the formation of brain metastases. The role ofPLEKHA5 in mediating CNS penetration of melanoma cellsand/or facilitating proliferation and survival in the CNS has notbeen characterized. Its established role in cell migration mightfacilitate BBB transmigration, while binding to its downstreammessengers might result in increased cell survival. Downregula-tion of PLEKHA5 in A375P cells had little effect on prolifera-tion, as baseline levels were low, whereas downregulation inA375Br and YUMUL cells (cerebrotropic with high levels atbaseline) inhibited proliferation. The inhibitory effect of PLE-KHA5 knockdown was also seen in the presence of astrocytes,though this effect was not studied during direct physical contactbetween astrocytes and tumor cells. Our results suggest thatPLEKHA5 might be an important mediator of melanoma brainmetastasis and therefore an attractive target for pharmacologicinhibition in both cerebral and extracerebral metastases inpatients with metastatic melanoma.

PLEKHA5 can be inhibited by emerging gene silencing tech-nologies, such as synthetic U1 adaptors (33). Given that PLE-KHA5 does not have a known critical kinase domain, its functioncannot be inhibited by small-molecule kinase inhibitors. Analternative approach for inhibition is specifically targeting thePH domain of PLEKHA5 protein. This class of inhibitors hasbeen used to selectively bind to the PH domain and inhibit thefunction of proteins such as Akt and PDPK1, resulting in antitu-mor activity (34). This approach has also been proposed forinhibiting PLEKHA7, another PLEKHA familymember, andnoveltarget for inhibition in KRAS-mutated colon cancer (35).

Our gene expression profiles identified a large number ofpotential drug targets phenomenologically associated with earlydevelopment of brain metastases. We note that we focused ourstudies on PLEKHA5 as it was differentially expressed in thecell line model, although other genes of potential importancewere differentially expressed in the human tumors and requirefunctional validation in different models. For example, BCHE(Butyrylcholinesterase) was represented on the Illumina plat-form by two probes, both of which were the most differentiallyexpressed in the human samples, but not in the A375 cell linemodel. BCHE would be an excellent candidate for future studiesusing other models. We also found upregulation of additionalgenes with known roles in neural cell development, such asECE2, NRCAM, SOX13, and STMN1, or genes expressed in thebrain and associated with normal neural function, includingRHEB, SNCA, STXBP6, and CC2D1A (36–44). High expressionof these genes in extracerebral sites further suggests that theymight facilitate the brain homing process. Some of these geneshave also been found to have a role in the development,progression, and dissemination of melanoma or other malig-nant processes. For example, increased expression of NRCAMand STMN1 potentiates proliferation and migration of mela-noma cells, whereas SNCA, a gene primarily expressed in thebrain, but also in various tumors including ovarian, colorectal,and melanoma, is involved in tumorigenesis (45–48).

One of the limitations of this study is that the timing ofresection or biopsy of metastatic sites was not uniform, and notdone in all cases at initial diagnosis of metastatic disease. It isunknownwhether expressionof PLEKHA5changes over time, andthis needs to be evaluated in future studies.

In previous studies, mRNA profiling of matched melanomacranial and extracranial metastases did not identify PLEKHA5as differentially expressed between these two anatomical loca-tions (17, 49). Our paired cerebral and extracerebral samplesindicate that PLEKHA5 expression at the two sites is similar,and PLEKHA5 expression is high in both sites. Our studysuggests that some of the molecular aberrations associated withbrain metastasis can also be evident in other metastatic loca-tions, and that in melanoma, genes mediating the metastaticspread to the brain and brain homing can also mediate theirmetastasis and growth at other visceral sites. This is supportedby studies by Bos and colleagues, who identified biomarkers ofbrain dissemination in extracerebral tumors, and showed thatthese can mediate the metastatic spread of breast cancer cellsto other sites as well (11). This paradigm was also more recentlyreported by Cruz-Munoz and colleagues who utilized a spon-taneous brain metastasis mouse model and showed that over-expression of EDNRB, a mediator of spontaneous melanomabrain metastases, results in enhanced overall metastatic diseaseand increased incidence of spontaneous brain metastases,whereas inhibition significantly suppresses both extracerebraland cerebral metastatic burden (19).

Our RNA profiling studies were limited by the small cohortsize and the fact that patients had metastases at other sites aswell. Our gene selection was based on differential expressionin the A375Br cells, a BRAF-mutated cell line, and expressionpatterns might be driven by BRAF status. However, there wasno association between BRAF or NRAS mutational status andPLEKHA5 expression in human tumors. A375Br cells are notuniquely brain metastatic because they form extracranial meta-stases as well. Differentially expressed genes might therefore be

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Figure 5.Effect of PLEKHA5 silencing on invasion of cerebrotropic cells.Reconstituted basement membrane invasion of A375Br and YUMUL(� anti-PLEKHA5 siRNA) was compared with A375P cells. The number ofinvading cells was summed for ten different fields, averaged for triplicatedeterminations, and plotted as shown. Knockdown of PLEKHA5expression inhibited Matrigel invasion of A375Br and YUMUL cells.Significant P values from the Student t test comparing levels ofinvasion between treated versus untreated cells are indicated by � forP < 0.05.






associated with metastases and aggressive disease in general,and might not be specific for development of brain metastases.However, our validation studies in a larger cohort of patientsusing BMFS as endpoint, coupled with our functional analyses,provide further confidence that PLEKHA5 is associated with abrain metastatic phenotype. The majority of patients withmelanoma with brain involvement have extracerebral metas-tases as well, and an ideal drug for the brain metastatic pop-ulation would target melanomas at all sites. Moreover, we notethat the expression studies were primarily conducted on extra-cerebral metastases, both for practical reasons (availability of alarge number of specimens for study) and due to the need tocotargeting drivers of both proliferation and survival in CNSlesions and extracerebral sites. Prospective validation of theassociation between PLEKHA5 and development of brainmetastases is warranted and clinically relevant, as this mightbe useful for selection of patients for more frequent brainimaging. In addition, efforts are ongoing in our laboratory todevelop clinically relevant xenograft models to further studythe role of PLEKHA5 in the development of brain metastasesin vivo.

In conclusion, our studies suggest that melanomas that tend tohome to the brain might be preprogrammed to passage throughthe BBB and then growand survive in theCNSmicroenvironment.Mediators of the brain CNS penetration and proliferation can beidentified and potentially inhibited in both cerebral- and extra-cerebral metastases. PLEKHA5 is possibly one such mediator.Although PLEKHA5 likely cooperates with additional moleculesin facilitating brain metastasis, our studies indicate that systemicpharmacologic targeting of PLEKHA5 might affect the brainhoming process and inhibit growth in both cerebral and extra-cerebralmetastatic sites. Further studies arewarranted to elucidatethe specific role of PLEKHA5 in brain homing and to identifyupstream and downstream regulators of PLEKHA5 function,which might be easily druggable.

Disclosure of Potential Conflicts of InterestD.L. Rimm reports receiving a commercial research grant from Novartis and

is a consultant/advisory board member for Genoptix. No potential conflicts ofinterest were disclosed by the other authors.

DisclaimerIts contents are solely the responsibility of the authors and do not

necessarily represent the official view of NIH.

Authors' ContributionsConception and design: L.B. Jilaveanu, H.M. KlugerDevelopment of methodology: F. Parisi, R.S. Kerbel, D.L. Rimm, M.W.Bosenberg, H.M. KlugerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.L. Rimm, H.M. KlugerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.B. Jilaveanu, F. Parisi, C.R. Zito, M.W. Bosenberg,Y. Kluger, H.M. KlugerWriting, review, and/or revision of the manuscript: L.B. Jilaveanu, F. Parisi,D.L. Rimm, M.W. Bosenberg, H.M. KlugerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructingdatabases):M.L. Barr, C.R. Zito,W.Cruz-Munoz, R.S. Kerbel,R. Halaban, H.M. KlugerStudy supervision: L.B. Jilaveanu, M.W. Bosenberg, Y. Kluger, H.M. KlugerOther (tissue facilitation and review): D.L. Rimm

AcknowledgmentsThe authors thank Dr. S. Huang from M.D. Anderson Cancer Center for

providing A375Br and A375P cells.

Grant SupportThis work was supported by CTSA Grant Number KL2 TR000140 (to L.B.

Jilaveanu, PI) from the National Center for Research Resources (NCRR) andthe National Center for Advancing Translational Science (NCATS), compo-nents of the NIH, and NIH roadmap for Medical Research. This work wasalso supported in part by grants from the National Cancer Instituteincluding the K24CA172123 (to H.M. Kluger, PI), Yale SPORE in SkinCancer, P50 CA121974 (to R. Halaban, PI), CTSA grant ULI RR024139from the NCCR (to R. Sherwin, PI), and pilot funding from the Yale CancerCenter (to H.M. Kluger, PI).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 7, 2014; revised August 18, 2014; accepted September 17,2014; published OnlineFirst October 14, 2014.

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2015;21:2138-2147. Published OnlineFirst October 14, 2014.Clin Cancer Res Lucia B. Jilaveanu, Fabio Parisi, Meaghan L. Barr, et al. Brain MetastasisPLEKHA5 as a Biomarker and Potential Mediator of Melanoma

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