DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro...

DNAJB4 molecular chaperone distinguishes WTfrom mutant E-cadherin, determining their fatein vitro and in vivo

Joana Simoes-Correia1,2,∗, Diana I. Silva1,3, Soraia Melo1, Joana Figueiredo1, Joana Caldeira1,4,

Marta T. Pinto1, Henrique Girao2, Paulo Pereira2 and Raquel Seruca1,5

1IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto 4200-465, Portugal2IBILI – Institute for Biomedical Imaging and Life Sciences, Faculty of Medicine, University of Coimbra, Coimbra

3000-548, Portugal 3ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto 4099-001,

Portugal 4INEB - Instituto de Engenharia Biomedica, University of Porto, Porto 4150-180, Portugal 5Medical Faculty of

the University of Porto, Porto 4200-319, Portugal

Received July 30, 2013; Revised and Accepted November 25, 2013

E-cadherin (Ecad) is a well-known invasion suppressor and its loss of expression is common in invasive carci-nomas. Germline Ecad mutations are the only known genetic cause of hereditary diffuse gastric cancer (HDGC),demonstrating the causative role of Ecad impairment in gastric cancer. HDGC-associated Ecad missense muta-tions can lead to folding defects and premature proteasome-dependent endoplasmic reticulum-associateddegradation (ERAD), but the molecular determinants for this fate were unidentified. Using a Drosophila-based genetic screen, we found that Drosophila DnaJ-1 interacts with wild type (WT) and mutant humanEcad in vivo. DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4), the human homolog of DnaJ-1,influences Ecad localization and stability even in the absence of Ecad endogenous promoter, suggesting apost-transcriptional level of regulation. Increased expression of DNAJB4 leads to stabilization of WT Ecadin the plasma membrane, while it induces premature degradation of unfolded HDGC mutants in theproteasome. The interaction between DNAJB4 and Ecad is direct, and is increased in the context of the unfoldedmutant E757K, especially when proteasome degradation is inhibited, suggesting that DNAJB4 is a molecularmediator of ERAD. Post-translational regulation of native Ecad by DNAJB4 molecular chaperone is sufficientto influence cell adhesion in vitro. Using a chick embryo chorioallantoic membrane assay with gastric cancerderived cells, we demonstrate that DNAJB4 stimulates the anti-invasive function of WT Ecad in vivo.Additionally, the expression of DNAJB4 and Ecad is concomitantly decreased in human gastric carcinomas.Altogether, we demonstrate that DNAJB4 is a sensor of Ecad structural features that might contribute to gastriccancer progression.

INTRODUCTION

E-cadherin (Ecad) is the main component of Adherens Junctionsin the epithelia and acts as the master regulator of cell–cell ad-hesion. In cancer, it has been extensively described as a tumorsuppressor since it suppresses cell invasion, and its loss is afrequent characteristic of invasive carcinomas (1). We havepreviously shown that Ecad expression can be efficiently regu-lated by post-translational mechanisms, such as endoplasmic

reticulum-associated degradation (ERAD) (2) or traffickingderegulation (3) leading to cancer progression. It is now clearthat post-translation regulation of Ecad may account for itsloss in invasive carcinomas (4,5). In hereditary diffuse gastriccancer (HDGC), where germline CDH1 (the gene encodingEcad) mutations are found in 30% of the cases, missense muta-tions lead to partial or complete loss of Ecad protein expression,frequently without affecting the RNA levels (6). In this clinicalsetting, approximately half of the missense mutations described

∗To whom correspondence should be addressed at: IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Rua RobertoFrias, s/n, 4200-465 Porto, Portugal. Tel: +351 225570700; Fax: +351 225570799; Email: [email protected]

# The Author 2013. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2014, Vol. 23, No. 8 2094–2105doi:10.1093/hmg/ddt602Advance Access published on November 29, 2013

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in the literature lead to Ecad structural destabilization, a criterionassociated with loss of function and cancer development (7).Accordingly, the identification of the molecular partners with arole in Ecad structural recognition and regulation assume signifi-cant biological relevance.

Molecular chaperones are the main regulators of proteinhomeostasis and their function include the capacity to regulateprotein folding, aggregation and degradation (8). DNAJ proteinsbelong to the HSP40 family and are known to transfer substratesto Hsp70 chaperones, stimulating their ATPase domain and con-ferring specificity to this family of chaperones (9). Humans have41 different DNAJ encoding genes, among which type B pro-teins, that contain an extreme N-terminal J-domain, a glycine/phenylalanine-rich region, a cysteine-rich region, and a variableC-terminal domain. DNAJB1 (classical Hsp40) is heat inducibleand cooperates with HSPA1A (Hsp70) and HSPA8 (Hsc70) inluciferase refolding in vitro and in vivo (10,11). Interestingly,it has recently been proposed that Hsp40 proteins can be asso-ciated to the Endoplasmic Reticulum and act in misfolding rec-ognition of transmembrane proteins (12). Although there is ahigh homology between DNAJB1 and DnaJ (Hsp40) homolog,subfamily B, member 4 (DNAJB4), the latter is not as signifi-cantly induced by heat shock, and is thought to act as a house-keeping HSP40/DNAJ (13). It has been demonstrated thatDNAJB4 has little impact in refolding and aggregation suppres-sion (14), but its binding to unfolded substrates has beendescribed (9,15). Thus, the evidences suggest that DNAJB4 isnot only important under cellular stress, but also in control con-ditions, and can be involved in misfolding recognition of itsputative substrates.

It has been demonstrated that DNAJB4 acts as a tumor sup-pressor in non-small cell lung cancer model, while inhibitingproliferation, anchorage-independent growth, motility and inva-sion and promoting apoptosis (16). The invasion suppressor roleof DNAJB4 partially relies on the induction of Ecad expressionat the transcriptional level, supposedly due to the downregula-tion of the transcriptional repressor Slug (17). There are noreports exploring the potential of DNAJB4 as a molecular chap-erone of Ecad.

We used a Drosophila-based genetic screen to demonstratethat DnaJ-1 (DnaJ-like-1) interacts with human Ecad in vivo.We confirmed that DNAJB4 is the closest human homolog ofDnaJ-1 and investigated the mechanism through which thisDNAJ protein can regulate Ecad expression. To achieve this,we used gastric cancer cell lines and germline Ecad missensemutations associated to HDGC, where Ecad loss is a cancer ini-tiating event. In our cell models, DNAJB4 regulates Ecad sub-cellular localization, influencing its expression at the plasmamembrane (PM). Interestingly, we demonstrate that this regula-tion is independent of the proximal promoter of Ecad, occurs atthe post-translational level, and that DNAJB4 directly interactswith Ecad, acting as its molecular chaperone. DNAJB4 distin-guishes native WT from unfolded Ecad mutants, determiningtheir divergent fate. It recognizes unfolded Ecad for degradationin the proteasome, and significantly reduces the half-life of amisfolded mutant Ecad identified in a HDGC family (2). In theother hand, it promotes the folding of WT Ecad, resulting inincreased PM expression and induction of cell–cell adhesion.The biological significance of DNAJB4 in a gastric cancer cellmodel was analyzed, and we demonstrate that it stimulates the

anti-invasive function of Ecad but depends on Ecad to act asan anti-invasive molecule in vivo. These results corroboratethe observed concomitant decrease of Ecad and DNAJB4 ex-pression in human gastric carcinoma samples. Our results dem-onstrate that DNAJB4 is a molecular chaperone of Ecad that isable to sense Ecad folding, leading to stabilization of a nativelyfolded Ecad, or to degradation of its unfolded counterpart, deter-mining the invasiveness of gastric cancer cells in vitro andin vivo.

RESULTS

Drosophila DnaJ-1/Ubp64E regulate human E-cadherinin vivo

We used a previously described Drosophila transgenic model(18) expressing human wild-type Ecad (hEcad WT) or twoHDGC-associated pathogenic mutants (hEcad A634V andV832M) in the developing retina (Fig. 1A). By combiningthese strains with a collection of GS lines expressing differentDrosophila genes in the same tissue context, we identified thelines capable of modifying the eye phenotype (19). Using this ap-proach, we could identify new genes that interact with Ecadwithin a tissue context. Among the genes that were found tointeract with WT and mutant Ecad, we found a GS strain thatexpresses DnaJ-1 and Ubp64E. The simple overexpressionof DnaJ-1/Ubp64E does not interfere with the eye morpho-logy, but significantly disrupts the phenotype of the eyesoverexpressing human Ecad (Fig. 1A). We quantified the areasof the fly eyes (Fig. 1B) and found that DnaJ-1/Ubp64E signifi-cantly disrupt WT or mutant Ecad eyes, but reaches higher sig-nificance in the interaction with mutant Ecad, reflecting ahigher reproducibility of the phenotype, which indicatesincreased specificity of this interaction in the context of amutant Ecad.

We used Uniprot Protein Blast Similarity search to determinethe putative human orthologues of DnaJ-1/Ubp64E. We foundthat the closest human orthologues of this pair of genes areDNAJB4 and USP47, respectively. There is high similaritybetween DNAJB4 and DnaJ-1 (55% identity, with 70% posi-tives) and between UBP47 and Ubp64E (50% identity, with65% positives), as analyzed by protein sequence alignment(Supplementary Material, Fig. S1A and B).

Once we have established that DNAJB4 or USP47 could inter-act with Ecad, we decided to determine which of these geneswould be the stronger candidate as Ecad regulator. To do this,we mimicked the Drosophila experiment in vitro, by overexpressing separately DNAJB4 and USP47 in cells with overex-pression of WT or mutant Ecad [Chinese hamster ovary (CHO)cells]. At the protein level, minor differences were observed inEcad total expression when we over expressed USP47, whilethe overexpression of DNAJB4 induces changes in the patternof Ecad expression (Supplementary Material, Fig. S2). Thiswas unexpected, because DNAJB4 has been demonstrated toinduce Ecad expression through transactivation of the proximalpromoter, which is absent in these cell models (16,17). The Dros-ophila models and the stable transduction of Ecad in CHO cellswere controlled under the activity of exogenous promoters, sug-gesting that the regulation of Ecad by DNAJB4 might alsohappen at the post-transcriptional level.

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DNAJB4 directly interacts with E-cadherin and influencesits expression at the plasma membrane

Ecad expression is determinant for the progression of gastriccancer and its loss is demonstrated to be an initiating event inthis type of cancer (20). To investigate whether DNAJB4 regu-lates the expression and subcellular localization of Ecad in thatcontext, we silenced DNAJB4 in a gastric cancer derived cellline (MKN28) that expresses endogenous WT Ecad, and ana-lyzed the total expression of Ecad by WB and its PM localizationby Flow Cytometry. We found that silencing of DNAJB4 leads toa partial decrease in Ecad expression at the PM (Fig. 2A and B)despite the absence of significant differences in the total amountof Ecad in this cell model (Fig. 2C). Conversely, overexpressionof DNAJB4 in the same cell model induces an increase of PMEcad (Fig. 2D). Interestingly, MKN28 cells are described tohave insignificant levels of Slug (21), suggesting that the

regulation of Ecad by DNAJB4 in this gastric cancer cellmodel does not significantly depends on transcriptional regula-tion, but rather occurs at the post-transcriptional level. To testwhether DNAJB4 could regulate Ecad expression and localiza-tion at the post-transcriptional level, we manipulated its expres-sion in CHO cell lines stably expressing Ecad under the controlof the exogenous promoter CMV. We confirmed that DNAJB4stimulates the PM expression of WT Ecad and observed that ithas the opposite effect in the expression of an unfolded mutantEcad that is prematurely recognized by ERAD, E757K(Fig. 2E) (2). Going in the same direction, DNAJB4 decreasesthe PM expression of Ecad in another gastric cancer cell linethat endogenously express a mutant Ecad (Kato III, Supplemen-tary Material, Fig. S3), suggesting that DNAJB4 is able to distin-guish and differently regulate WT and mutant Ecad at thepost-transcriptional level, when Ecad expression is regulatedby its endogenous promoter or an exogenous one.

DNAJB4 acts as a molecular chaperone of E-cadherin

DNAJB4 belongs to the Hsp40-like family of molecular chaper-ones, thought to be the drivers of the functional specificity ofHsp70 machinery, with a major function of binding selected sub-strates, determining their fate (9). Therefore, we hypothesizedthat the post-transcriptional regulation of Ecad by DNAJB4relies on its molecular chaperone function. To address this ques-tion, we first determined whether DNAJB4 directly interactedwith the putative substrate Ecad. Using CHO cell lines stablyexpressing WT or the ERAD-prone mutant E757K we wereable to co-immunoprecipitate both proteins together (Fig. 3Aand B). Interestingly, DNAJB4 exhibits stronger interactionwith the mutant form, indicating that it is retained bound to theERAD substrate. Since molecular chaperones are frequentlydeterminants of the half-life of their substrates, we decided totest whether DNAJB4 could determine the half-life of Ecad.We tested the impact of DNAJB4 overexpression in cells expres-sing WT and one unfolded mutant Ecad, and found that the half-life of the E757K mutant was reduced (Fig. 3C and D), despitethe intrinsic reduced stability of the mutant (8 h in control condi-tions, 4 h with increased expression of DNAJB4). AlthoughDNAJB4 directly interacts with WT Ecad, it does not influenceits half-life (Fig. 3C and D). To determine whether DNAJB4could indeed sense Ecad folding, we used the previouslydescribed HDGC-associated mutant L583R (which is unfolded),and its variant L583I which is structurally tolerated (7). DNAJB4was able to distinguish the unfolded variant (L583R), leading toits destabilization and decreased expression, while it stabilizesthe neutral variant L583I, presumably natively folded(Fig. 3E). The capacity to directly bind WT or mutant Ecad,and the ability to distinguish unfolded Ecad forms leading totheir premature degradation, suggests that DNAJB4 acts as amolecular chaperone of Ecad.

DNAJB4 mediates the recognition of E-cadherinfor degradation in the proteasome

We have previously shown that a significant number ofHDGC-associated Ecad missense mutants exhibit structuraldefects and are thus destabilized and prematurely degraded inthe proteasome (2,7). To further determine whether DNAJB4

Figure 1. DnaJ-1/Ubp64E interact with WT and mutant human E-cadherin invivo. (A and B) A Drosophila genetic screen identifies the pair DnaJ-1/Ubp64Eas E-cadherin interactors. (A) Representative images of the phenotypes obtainedfrom overexpression of different hEcad forms (WT and HDCG-associatedmutants A634V and V832M) in the fly eye, in comparison with a normal eye(top). Overexpression phenotypes of the putative E-cadherin interactorsDnaJ-1/Ubp64E crossed with hEcad strains, in comparison with the phenotypeof overexpressing DnaJ-1/Ubp64E alone (bottom). (B) Graphic representationof the areas of the flies eyes as in (A) and upon crossing hEcad with a GFPstrain, as control. (∗) P , 0.05; (∗∗) P , 0.01; (∗∗∗) P , 0.005.

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could be a direct mediator of the recognition of unfolded Ecad forthe ERAD quality control pathway, we used the ERAD-sensitivemutant E757K. We found that the interaction between DNAJB4chaperone and Ecad is increased in conditions where the proteo-lytic function of the proteasome is inhibited (Fig. 4A), suggest-ing that it preferentially interacts with the degradation-pronefraction of Ecad. We have also previously described thatdimethylsulfoxide (DMSO) is an efficient chemical chaperonethat favors the accumulation of Ecad at the PM, presumably bystabilizing its structure toward a native state and inducing its traf-ficking to, and stability at, the PM (2,3). As a proof of concept,and to confirm that DNAJB4 preferentially binds unfolded

Ecad, we analyzed the interaction between DNAJB4 and Ecadstabilized by DMSO, and found that the interaction betweenthe chaperone and the substrate is decreased if we induce Ecadstability with the chemical chaperone (Fig. 4A). Moreover, ex-pression of DNAJB4 increases the fraction of unfolded Ecaddegraded in the proteasome (Fig. 4B, MG132), but not theamount degraded in the lysosome (Fig. 4B, CQ), suggestingthat it mediates the degradation of ERAD-prone mutant Ecadby the proteasome. Interestingly, DNAJB4 indirectly potentiatesthe chemical chaperone effect of DMSO, stimulating Ecad ex-pression (Fig. 4B), although not by increased interaction withthe substrate (Fig. 4A).

Figure 2. DNAJB4 regulates E-cadherin expression at the transcriptional and post-transcriptional level, determining its expression at the PM. (A–C) MKN28 cellswere transfected with two different siRNA against DNAJB4 and Ecad expression at the PM was analyzed by Flow Cytometry (A and B) or by WB (C). (A) The resultsare the average of three independent experiments. (B) Representative histograms corresponding to Ecad PM expression in control conditions (MKN28) or uponDNAJB4 silencing. (C) 40 mg of total cell extract were analyzed by WB and probed for Ecad and DNAJB4, demonstrating the silencing of DNAJB4. a-tubulinwas used as a loading control. (D) Representative histograms corresponding to Ecad PM expression in control conditions (MKN28) or upon DNAJB4 overexpression;(E) CHO cells stably expressing WT or E757K Ecad were transiently transfected with DNAJB4, the amount of PM Ecad was analyzed by Flow Cytometry, andrepresentative histograms are shown.




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Figure 3. DNAJB4 acts as a molecular chaperone of E-cadherin. CHO cell lines stably expressing WT and E757K Ecad were transfected with DNAJB4. (A and B)Ecad was immunoprecipitated, and the interaction with DNAJB4 was analyzed by WB. Membranes were probed with anti-DNAJB4 and anti-Ecad antibodies. (C andD) 48 h after transient transfection of DNAJB4-V5, cells where incubated with the protein synthesis inhibitor Cicloheximide (CHX) for 0, 2, 4 or 8 h. Ecad expressionwas analyzed by WB, transfection was confirmed with anti-V5, and a-tubulin served as a loading control for quantification purposes. (D) In each time point, Ecadexpression was normalized to the control (0 h, CHX). The results are the average of three independent experiments. Circles represent WT cells and squares representE757K mutant cells. Dashed lines represent cells upon overexpression of DNAJB4. (E) Ecad was either transfected alone or co-transfected with DNAJB4-V5 into theCHO parental cell line, and 48 h later the expression of Ecad, DNAJB4 and a-tubulin was analyzed by WB. Ecad expression was quantified by densitometry and ispresented as the ratio to control conditions (condition without DNAJB4).




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DNAJB4 stimulates the anti-invasive role of nativeE-cadherin and its expression is decreased in gastriccarcinomas

After we have partially elucidated the mechanism wherebyDNAJB4 influences Ecad stability at the post-translational level,we investigated the functional consequences of this regulation.To do this, we used CHO cell models (Ecad-null cells) (22), withstable expression of WT or mutant Ecad subjected to the CMV ex-ogenous promoter, and induced the expression of DNAJB4 bytransient transfection. We observed that increased expression ofDNAJB4 is not sufficient to increase cell adhesion in the absenceof Ecad or in the presence of the non-functional unfolded E757Kmutant Ecad, but induce increased cell aggregation in the presenceofaWTEcad(Fig.5A).Thiswasexpected,becauseit increases theamountofWTEcadin thePM(Fig.2E).Tofurtheranalyzetheroleof DNAJB4 in cell migration, we used the wound healing assay.The results show that, in conditions of Ecad overexpressionwithout transcriptional regulation (due to the lack of the Ecadproximal promoter in these cell models), DNAJB4 is not enoughto further reduce cell migration (Fig. 5B), suggesting that Ecad isthe dominant mobility suppressor.

After we have established the role of DNAJB4 on cell adhe-sion and migration in a cell culture system, we analyzed therelevance of this Ecad/DNAJB4 dominance upon invasion of atissue context, using for this purpose the chick embryo chorio-allantoic membrane (CAM) assay (23). We generated MKN28gastric cancer cells with stable silencing of Ecad (shEcad) anda control cell line (shScramble), and transiently transfectedDNAJB4 in these two distinct cellular contexts ( SupplementaryMaterial, Fig. S4A). The different cell lines were inoculated inthe CAM and, after 3 days of incubation, cell invasion and

vascularization of the CAM were evaluated in response to thedifferent cell lines, as described in Materials and Methods.Labeling of the human cells inoculated in the CAM revealedthat DNAJB4 reinforces the anti-invasive function of WTEcad, but this invasion-suppressor potential is lost if Ecad isnot expressed (silencing by shRNA, Fig. 6), suggesting that inthe gastric cancer model Ecad is the dominant anti-invasivemolecule. Analysis of the neovascularizing potential of thesecells over the CAM shows that DNAJB4 is anti-angiogenic,and that this tumor suppressor feature is also Ecad-dependent(Supplementary Material, Fig. S4B).

Furthermore, using the on-line database Human Protein Atlas(24,25) we analyzed the expression patterns of Ecad andDNAJB4 in human sporadic gastric carcinomas. In normalgastric tissue, DNAJB4 is moderately expressed in more than75% of the cells, in the glands, together with Ecad (Fig. 7, Sup-plementary Material, Table S2). Interestingly, 67% (8 out of 12)of the gastric carcinoma samples exhibit decreased (weak) ex-pression of DNAJB4 (Fig. 7, Supplementary Material, TablesS1 and S2). Although Ecad expression seems to be decreasedin the majority of the carcinoma samples (SupplementaryMaterial, Table S1), only 8% (1 out of 12) are annotated tohave ‘Moderate’ expression, compared with the ‘Strong’ expres-sion obtained for the normal tissue (Supplementary Material,Tables S1 and S2), which is clearly underestimated. Theseresults suggest that decreased DNAJB4 expression convoywith Ecad loss in human gastric carcinoma, corroborating theresults obtained upon silencing of DNAJB4 in the gastriccancer cell line MKN28 (Fig. 2A and B).

Altogether, these results suggest that the post-translationalregulation of native Ecad by its molecular chaperone DNAJB4stimulates cell adhesion in vitro, but the anti-invasive potential

Figure 4. DNAJB4 mediates the recognition of unfolded E-cadherin for degradation in the proteasome. CHO cell lines stably expressing WT and E757K Ecad weretransfected with DNAJB4-V5 and 24 h after transfection cells were treated with MG132 (8 h), 2% DMSO (24 h) or Chloroquine (CQ, 6 h) as indicated. (A) Immu-noprecipitation was performed with anti-Ecad antibody, and WB against Ecad and V5 tag; (B) Total cell extracts were analyzed by WB, probed for Ecad, and the bandsintensities were quantified. The result is representative of three biological replicas.




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of DNAJB4 seems to depend also of the transcriptional regula-tion of Ecad. Our results also demonstrate that Ecad is thedominant invasion-suppressor in the gastric cancer context,reinforcing the need to maintain its stability for prevention ofcancer invasiveness and metastization.

DISCUSSION

The role of Ecad in cell–cell adhesion is well described, and ithas been shown that Ecad loss can drive gastric cancer develop-ment in mice and humans (20). Moreover, germline Ecad muta-tions are the only known genetic cause of HDGC, furtherdemonstrating the causative nature of Ecad loss in gastriccancer (26–28). Germline Ecad mutations strongly predisposecarriers for development of early-onset gastric cancer, andonly rarely associate with lobular breast cancer (29,30), suggest-ing that Ecad assumes a special role in the gastric tissue

environment. Several layers of Ecad regulation have beendescribed to be important in gastric cancer development, includ-ing mutations, deletions, loss of heterozygosity, promoter hyper-methylation or regulation by transcription factors and miRNAs(6). Despite this, it is common to find cases displaying Ecadprotein loss without significant impairment of transcription(stable tumor CDH1 mRNA levels) (31). Accordingly, it islikely that post-translational regulation of Ecad contributes togastric cancer development.

Using a recently described Drosophila-based genetic screenwith WT and mutant human Ecad ectopically expressed in thefly eye (19), we searched for new post-translational regulatorsof Ecad expression in vivo. The DnaJ-1/Ubp64E pair stronglyinteracted with human Ecad (WT and mutant), suggesting thattheir human homologs DNAJB4 or USP47 could be efficientmodulators of Ecad expression. We tested the effect of the over-expression of the two human homologs separately, and con-cluded that USP47 does not significantly influence Ecad

Figure 5. Post-translational regulation of E-cadherin by DNAJB4 is sufficient to induce cell adhesion but does not reduce cell migration in vitro. CHO cell lines stablyexpressing WT or mutant (E757K)Ecad, or an empty vector were transiently transfected with DNAJB4-V5 or the V5-tag, as indicated. (A) Slow aggregation assay wasperformed and cells were photographed 48 h after seeding. Representative images are shown. (B) Wound healing assay was performed in CHO WT and cells werephotographed every 2 h, during the 8 h period. The experiment was repeated three times and representative images are shown for time points 0 and 4 h; WC, woundclosure.




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expression, while DNAJB4 modulates Ecad expression in thepresence, or absence, of Ecad proximal promoter.

DNAJB4/HLJ1 is a DNAJ-containing Hsp40-like molecularchaperone that was previously described to act as a tumor sup-pressor gene in non-small cell lung cancer, partially throughthe indirect induction of Ecad expression at the transcriptionallevel (17). Interestingly, it has also been shown that in lungcancer cell models, curcumin induces transcription of

DNAJB4 and consequently increases Ecad expression at theprotein level (32). Because curcumin is described to act as achemical chaperone (33) and is an inducer of the heat shock re-sponse (34), these evidences suggest that the regulation of Ecadby DNAJB4 expression can also occur at the post-translationallevel.

We verified that, in gastric cancer cell models, DNAJB4 isable to induce Ecad expression at the PM. We were surprised

Figure 6. DNAJB4 potentiates the anti-invasive function of WT E-cadherin in human gastric cancer cells, as revealed by a chick embryo chorioallantoic membrane(CAM) assay. MKN28 cells where infected with a shRNA to silence Ecad (sh Ecad) or a scramble control (sh Scramble) and selected by antibiotic resistance. Cellswere subsequently transfected with DNAJB4-V5 when indicated. A human-specific cytokeratin staining allowed the distinction of inoculated cells and the evaluationof cell invasion over the CAM; cells were scored as invasive (1) or non-invasive (0). At least three independent experiments were used for the graphic representation.(B) Representative images of the invasion of the same cells over the CAM. Amplification 20×.

Figure 7. Concomitant decrease of E-cadherin and DNAJB4 expression in human gastric carcinomas. Representative histological samples of one normal gastric tissueand two gastric carcinomas, stained with anti-Ecad and anti-DNAJB4 antibodies. Top and bottom stainings correspond to the same patient, and the age and gender areindicated in the top-right corner. The insight represents a higher magnification of each staining. F, female; M, male.




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to observe that in the presence of unfolded mutant Ecad the effectwas opposite, with the overexpression of DNAJB4 resulting inreduced expression of mutant Ecad at the PM. Owing to the chap-erone nature of DNAJB4 we thought that this could result from adirect post-translational regulation of Ecad. Using cadherin-nullCHO cell lines stably transduced with WT and mutant Ecad (inthe absence of the proximal promoter region) we show thatDNAJB4 directly interacts with Ecad, and regulates the half-lifeof an unfolded mutant Ecad, implying that it acts as its mole-cular chaperone. We used mutants with known energetic penal-ties (as a measure of their unfolding tendency) (7) to validatethe capacity of DNAJB4 to act as a sensor of Ecad structuraldefects, and confirmed that it destabilizes the unfolded mutantL583R while it stabilizes the tolerated synthetic variant L583I.We also demonstrate that DNAJB4 increases the rate of unfoldedEcad degradation in the proteasome, and exhibits increasedbinding to Ecad when the proteasome is inhibited, suggest-ing that DNAJB4 mediates Ecad recognition for prematureproteasome-dependent degradation, supposedly contributingto ERAD of the unfolded Ecad variants. In the opposite direc-tion, when Ecad is stabilized by chemical chaperone treatment,Ecad/DNAJB4 interaction decreases, although the molecularchaperone potentiates the DMSO effect that results in Ecadstabilization (2). This is not surprising, because DMSO was re-cently shown to promote the multiple functions of DNAJB4(35). It is also in good agreement with our previous datashowing that DMSO acts by stimulating trafficking machinery,promoting the expression of Ecad at the PM (3); DNAJB4might be one of the molecular effectors of the chemical chaper-one effect of DMSO, possibly contributing to the recognitionof unfolded substrates, to deliver them to the Hsp70/Hsp90folding cycle. Indeed we observe that DNAJB4 is upregulated

upon proteasome inhibition in different cell lines (data notshown), supposedly as a response to the accumulation of mis-folded proteins.

The functional divergence of the different members of theDNAJ protein family has been described but is not completelyelucidated (9), leaving room for the identification of specificsubstrates corresponding to particular DNAJ members. Wedemonstrated that DNAJB4 directly acts as a molecularchaperone of Ecad and efficiently regulates its expression atthe post-translational level, increasing Ecad-dependent cell–cell adhesion when Ecad is retained in its natively folded state.Interestingly, when Ecad is intrinsically unfolded by mutation,or not expressed, DNAJB4 does not affect the adhesion capacityof our cell models. We used a CAM assay to evaluate the roleof DNAJB4 in the regulation of gastric cancer cell invasion,and we observed that its expression increases the anti-invasivepotential of Ecad-expressing cells, although this feature is lostin the absence of Ecad expression. To evaluate the expressionof Ecad and DNAJB4 in human gastric cancer, we collected allthe available gastric carcinoma samples from Human ProteinAtlas (12 adenocarcinomas, of which 8 correspond to the samepatient with stainings for both proteins). Cancer samples fre-quently exhibit decreased expression of both proteins, whencompared with the normal tissues, suggesting that DNAJB4might also act as a tumor suppressor in gastric cancer, possiblyinterfering with Ecad expression at the transcriptional and post-translational level.

In summary, we suggest that DNAJB4 chaperone is a molecu-lar determinant of Ecad fate, acting as a sensor of Ecad structuraldefects (see proposed model, Fig. 8). DNAJB4 is able to distin-guish between native Ecad and its unfolded counterpart (result-ing from hereditary/sporadic mutation, or unfolding due to a

Figure 8. Proposed model establishing the role of DNAJB4 in the regulation of WT and mutant E-cadherin. DNAJB4 acts as a molecular chaperone of Ecad, bindingdirectly after synthesis, with increased affinity for unfolded mutant Ecad. High levels of DNAJB4 increase the expression of WT Ecad in the PM (B), in comparison tolow DNAJB4 levels (A). In case of an unfolded Ecad, DNAJB4 recognizes the structural defects, and increased levels of DNAJB4 stimulate Ecad proteasome-dependent degradation, leading to decreased Ecad half-life and decreased PM expression (D), when compared with conditions with low DNAJB4 levels (C).




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stress insult during cancer progression), directing Ecad towardthe PM (native Ecad) or to the proteasome for degradation(unfolded Ecad). Owing to the prevalence of Ecad mutationsin hereditary and sporadic forms of diffuse gastric cancer, weenvisage that this dual role of DNAJB4 could be explored fordesigning new therapeutic strategies in attempt to preventEcad loss during cancer progression.

MATERIALS AND METHODS

Drosophila strains and genetic manipulations

The Drosophila genetic screen is detailed elsewhere (19). Gal4/UAS system was used to induce human Ecad expression (WT,A634V and V832M) in the Drosophila eye as previouslydescribed (18) and these flies were crossed with a GS line expres-sing DnaJ-1 (DnaJ-like-1) and Ubp64E (Ubiquitin-specific pro-tease 64E), kindly provided by Jose Felix de Celis. Eyes wereexamined under a Leica compound microscope, and digitalimages were processed and quantified using Adobe Photoshop.Statistical evaluation was done using a Student t-test.

Cell culture, transfections and treatments

CHO cells (ATCC:CCL-61, Spain) were grown in MEM Alpha,MKN28 and KatoIII (IPATIMUP Cell Line Bank) in RPMI(Invitrogen), all supplemented with 10% fetal bovine serum(Hyclone) and 1% penicillin-streptomycin (Invitrogen). CHOstable cell lines were established previously (2) and maintainedin antibiotic selection with 5 mg/ml blasticidin (Invitrogen). Forstable silencing of Ecad, MNK28 were transduced by lentiviralinfection of a previously described shRNA (36) and correspond-ing control (Addgene plasmid numbers 18 801 and 1864); selec-tion was done with 5 mg/ml puromycin (Sigma). For transienttransfections, cells were seeded in 6-well plates and transfected(30% confluence) with 1 mg of vector DNA or 50 nM of siRNA(Qiagen), using Lipofectamine 2000 (Invitrogen). For doubletransfections, we used 0.5 mg of each plasmid. Several plasmidsused were previously described (13) and obtained from Add-gene (numbers 19 472, 19 499, 19 526, 19 445, 19 444);USP47 (ubiquitin-specific peptidase 47) plasmid was previouslydescribed (37) and kindly provided by Garry Melino. MutantsL583R and L583I were previously cloned in a pIRES2-EGFPplasmid (7). Protein synthesis was inhibited with 50 mM ofCycloheximide (Sigma); proteasome with 10 mM MG132 (Cal-BioChem); lysosome with 25 mM Cloroquine; and 2% DMSO(Sigma) was used as chemical chaperone.

Flow cytometry

Experiments were performed as described previously (2).Briefly, cells were grown to confluent monolayers, detachedwith Versene (Invitrogen) and resuspended in ice-cold phos-phate buffered saline (PBS) with 0.05 mg/ml CaCl2. Ecad wasprobed in 5 × 105 cells with the extracellular primary antibodyHECD1 (Invitrogen). Cells were washed twice and incubatedwith Alexa Fluor 488 goat anti-mouse (Invitrogen) in the darkfor 30 min. 5 × 104 cells were analyzed in a Coulter EpicsXL-MCL flow cytometer (Beckman-Coulter) and the data wasanalyzed with FlowJo software.

Western blot and immunoprecipitation

Cells lysates were obtained and processed as described else-where (2) and detailed in Supplementary Material. Westernblots were probed with antibodies against Ecad (Invitrogen orBD Biosciences),a-tubulin (Sigma), DNAJB4 (Santa Cruz Bio-technology) or V5 (Invitrogen), and the correspondent HRP-conjugated secondary antibodies, all diluted in 5% non-fat milk(0.5% Tween-20 in TBS), followed by ECL detection (Bio-Rad). Immunoblots were quantified with Quantity One Software(Bio-Rad). For immunoprecipitation, 400 mg of protein werecoupled to anti-Ecad antibody (BD Biosciences), and precipi-tated with Protein G beads (GE Healthcare or Millipore), accord-ing to manufacture instructions.

Cell migration and aggregation assays

For the wound healing assay, CHO cells were transfected asdescribed above, and grown to confluence in 6-well plates. Anartificial wound was created with a yellow Gilson pipette tip,and cells were washed twice with PBS. Phase contrast photo-graphs were acquired with the 10 × objective of a LeicaDMIRE2 microscope. Wound closure was accessed by measur-ing the distance between wound edges at time intervals.

For the slow aggregation assay, 96-well plates were coatedwith 50 ml agar solution (100 mg Bacto-Agar, 15 ml of PBS).Cells were detached with trypsin-EDTA (Invitrogen), resus-pended, and 2 × 104 cells were seeded per well. The plate wasincubated (378C, 5%CO2) and aggregation was evaluated in aninverted microscope (Leica DMIRE2, ×10 magnification)48 h after seeding, and images captured using a digital camera.

Chicken embryo CAM assays

MKN28 were transduced with shRNAs as described above andinoculated over the CAM as detailed in Supplementary Materi-als. Briefly, 1 × 106 cells were placed on top of E10 growingCAM into a 3 mm silicon ring under sterile conditions. After 3days, the embryos were euthanized and the CAM was excisedfrom the embryos, photographed ex ovo, at 20× magnification(Olympus, SZX16 with a DP71 camera). To evaluate angiogen-esis, the number of new vessels (,15 mm diameter) growingradial toward the ring area was counted in a blind fashionmanner. Excided CAMs were fixed (10% neutral-buffered for-malin), paraffin-embedded and stained with hematoxylin–eosin to detected invasive cells. The samples were processedas detailed in Supplementary Materials. Slides were subsequent-ly incubated with mouse monoclonal antibody against pan-cytokeratin (Sigma). Invasion was evaluated under the micro-scope, in a blind fashion manner, by three independent users(scored 1 if invasive, and 0 if not invasive).

Immunohistochemistry

The human tissue samples are part of a large Tissue MicroarrayAnalysis platform, annotated in the Human Protein Atlas data-base (24,25). The samples were collected from surgical speci-mens, in accordance with approval from the local ethicscommittee (Stockholm, Sweden). All antibodies were previous-ly validated. The protocols used for immunohistochemistry and




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digital imaging of the tissues was described previously (25).Immunohistochemistry of Ecad and DNAJB4 from the 12available stomach cancer samples are included as supplemen-tary data (Supplementary Material, Table S1), and the compari-son between the expression of both proteins, in normal andcancer tissues, is summarized in Supplementary Material,Table S2. Representative Ecad and DNAJB4 stainings werechosen from Normal (Patient 2411) and Stomach Cancer(Patients 2105 and 2326) samples, and the antibodiesHPA004812 and HPA028385 were chosen for Ecad andDNAJB4 staining, respectively. All the information about theantibodies, validation data, immunostainings, and data usagepolicies, is available at http://www.proteinatlas.org.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We would like to thank Carla Marques (IBILI, Coimbra, Portu-gal) for the technical support with virus production, and PauloPereira (IBMC, Porto, Portugal) for complementary work andadvices with the Drosophila model.

Conflict of interest statement. The authors disclose no conflictsof interest.

FUNDING

This work was supported by Fundacao para a Ciencia e Tecnolo-gia, Portugal (Projects: PTDC/SAU-OBD/64319/2006, PTDC/SAU-ONC/110294/2009, PTDC/SAU-OBD/104017/2008,NORTE-07-0124-FEDER-000022; PhD fellowships: SFRH/BD/29777/2006 to J.C.; Postdoc fellowship: SFRH/BPD/48765/2008 to J.S.-C.; salary support from Ciencia Program2008 to M.T.P.).

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