loss of the nuclear pool of ubiquitin ligase chip/stub1 in ... · cancer patients revealed...

Molecular Cell Biology

Loss of the Nuclear Pool of Ubiquitin LigaseCHIP/STUB1 in Breast Cancer Unleashes theMZF1-Cathepsin Pro-oncogenic ProgramHaitao Luan1,2,3,4, Bhopal Mohapatra1,2,3, Timothy A. Bielecki1, Insha Mushtaq1,5,Sameer Mirza2, Tameka A. Jennings1, Robert J. Clubb1,Wei An1,2,3, Dena Ahmed6,Rokaya El-Ansari6, Matthew D. Storck1, Nitish K. Mishra2,3, Chittibabu Guda2,3,Yuri M. Sheinin5,7, Jane L. Meza7,8, Srikumar Raja1, Emad A. Rakha6,Vimla Band1,2,3,7,9, andHamid Band1,2,3,5,7,9

Abstract

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone forHSP90/HSC70, and its expression is reduced or lost in severalcancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss ofnuclear but not cytoplasmic CHIP to predict more aggressivetumorigenesis and shorter patient survival, with loss of CHIP intwothirdsofErbB2þandtriple-negativebreast cancers(TNBC)andin one third of ERþ breast cancers. Reduced CHIP expression wasseen in breast cancer patient-derived xenograft tumors and inErbB2þ and TNBC cell lines. Ectopic CHIP expression in ErbB2þ

lines suppressed in vitrooncogenic traitsand in vivoxenograft tumorgrowth. An unbiased screen for CHIP-regulated nuclear transcrip-tion factors identifiedmany candidateswhoseDNA-binding activ-ity was up- or downregulated by CHIP. We characterized myeloid

zinc finger 1 (MZF1) as a CHIP target, given its recently identifiedrole as a positive regulator of cathepsin B/L (CTSB/L)-mediatedtumor cell invasion downstream of ErbB2. We show that CHIPnegatively regulates CTSB/L expression in ErbB2þ and other breastcancer cell lines. CTSB inhibition abrogates invasion and matrixdegradation in vitro and halts ErbB2þ breast cancer cell line xeno-graft growth. We conclude that loss of CHIP remodels the cellulartranscriptome to unleash critical pro-oncogenic pathways, such asthe matrix-degrading enzymes of the cathepsin family, whosecomponents can provide new therapeutic opportunities in breastand other cancers with loss of CHIP expression.

Significance: These findings reveal a novel targetable pathway ofbreast oncogenesis unleashed by the loss of tumor suppressor ubi-quitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524–35. �2018 AACR.

IntroductionThe ubiquitin-proteasome system (UPS) plays diverse roles in

normal cellular homeostasis. Ubiquitination involves two ubi-quitin-activating (E1) enzymes, a small group of ubiquitin-conjugating (E2) enzymes and a large repertoire of ubiquitinligase (E3) enzymes. E3s dictate substrate specificity and comprisetwo broad groups: the human papilloma virus E6-interacting(HECT) domain and the Really Interesting New Gene (RING)finger domain protein families (1). Consistent with roles of UPSin oncogenesis, certain cancers, for example, multiple myeloma,are clinically treated with proteasome inhibitors (1). Recent focushas shifted to substrate-specific elements of the UPS, such as E3s,with MDM2 inhibitors currently in clinical trials for severalmalignancies (www.clinicaltrials.gov). Notably, mutations ofE3s, such as CBL (2) or FBW7 (3) convert them into oncogenes.

The C-terminus of HSC70-interacting protein (CHIP)/STIP1homology and U-Box containing protein 1 (STUB1) is a U-boxsubfamily RING finger E3 that functions as a negative co-chaperone for the HSP90/HSC70 chaperone to regulate proteinquality control by targeting unfolded or misfolded proteins forproteasomal degradation (4–9). CHIP also targets many matureproteins for ubiquitination and proteasomal degradation ordegradation-independent regulation (10).

CHIP targeting of oncogenic drivers/co-drivers, such as ErbB2,has prompted analyses of CHIP expression in human tumors.Analysis of matched normal and tumor tissues from 27 breast

1Eppley Institute for Research in Cancer and Allied Diseases, University ofNebraska Medical Center, Omaha, Nebraska. 2Department of Genetics, Univer-sity of Nebraska Medical Center, Omaha, Nebraska. 3Department of Cell Biologyand Anatomy, University of Nebraska Medical Center, Omaha, Nebraska.4Department of Molecular Biology, College of Basic Medical Sciences, JilinUniversity, Changchun, Jilin, China. 5Pathology and Microbiology, College ofMedicine, University of Nebraska Medical Center, Omaha, Nebraska. 6Depart-ment of Pathology, University of Nottingham and Nottingham University Hos-pitals NHS Trust, City Hospital Campus, Nottingham, United Kingdom. 7Fred &Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha,Nebraska. 8Department of Biostatistics, College of Public Health, University ofNebraska Medical Center, Omaha, Nebraska. 9Biochemistry and MolecularBiology, University of Nebraska Medical Center, Omaha, Nebraska.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

H. Luan, B. Mohapatra, and T.A. Bielecki are the co-first authors of this article.

Current address for T.A. Jennings: Department of Biology, University of Arkan-sas, Fayetteville, Arkansas; current address for R.J. Clubb, Department ofOncology, Novartis Molecular Diagnostics LLC., Cambridge, Massachusetts;current address for Y.M. Sheinin, Department of Pathology, University ofMinnesota, Minneapolis, Minnesota; and current address for S. Raja, Robert HLurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois.

Corresponding Authors: Hamid Band, University of Nebraska Medical Center,986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 402-559-8572; Fax: 402-559-8270; E-mail: [email protected]; and Vimla Band,[email protected]

doi: 10.1158/0008-5472.CAN-16-2140

�2018 American Association for Cancer Research.

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cancer patients revealed progressive loss of CHIP mRNA express-ion with tumor progression; CHIP knockdown in CHIP-highMCF-7 (ERþ) cells increased and CHIP overexpression in CHIP-lowMDA-MB-231 (triple-negative; TN) cells suppressed oncogen-ic traits in vitro and xenograft tumor growth and metastasis in vivo(11). Another study of 33 normal and 127 breast tumor samplesalso showed an inverse relation of CHIP mRNA levels withincreasing grade and Nottingham Prognostic Index (12). A thirdstudy of 183 patients found an inverse correlation between ErbB2andCHIP protein levels, with reduced CHIP expression associatedwith tumor progression (13). Thus, CHIP appears to function as asuppressor of breast cancer tumorigenesis. However, the extent ofloss of CHIP expression in breast cancer subtypes is unknown.

Mechanistically, CHIP can target cell surface, cytoplasmic,nuclear or secreted proteins for ubiquitin-dependent degradation(10). Relevant to breast cancer, CHIP targets ERRB2 (14–16),SRC-3 transcriptional coactivator (11), macrophage inhibitoryfactor (17), protein kinase 6/breast tumor kinase (18), and actinregulatory protein profilin-1 (19). However, how loss of CHIPpromotes oncogenesis remains unclear.

Here, we assessed both nuclear and cytoplasmic CHIP expres-sion in a well-annotated cohort of >900 breast cancer specimens.We show that a majority of ERBB2þ and TN, and a minority (butsizeable proportion of overall cases) of ERþ breast cancers showloss of CHIP expression, and that loss of the nuclear and not thecytoplasmic CHIP expression predicts features of tumor progres-sion and invasion and shorter patient survival. Loss of CHIPexpression in ERBB2þbreast tumor cell lines unleashed a programof increased tumor cell invasion and migration in vitro andtumorigenesis in vivo, as reported with a TN cell line model(11). Given the importance of the loss of nuclear CHIP, weperformed an unbiased screen of the impact of low versus highCHIP levels on DNA-binding activities of cellular transcriptionfactors in ERBB2þ and TN breast cancer cell lines and identifiedmany shared CHIP-regulated transcription factors. Here, we focusonCHIP regulation ofmyeloid zincfinger 1 (MZF1), as it has beenidentified as a keymediator of pro-invasive signaling downstreamof ERBB2 through upregulation of the expression of matrixdegrading cathepsin B and L enzymes (20). We show the CHIPdependence of this pathway in cell lines representing variousbreast cancer subtypes and demonstrate that chemical targeting ofCTSB inhibits the ERBB2-driven tumorigenesis. We conclude thatloss of CHIP expression unleashes a MZF1-dependent and CTSB/L-mediated pro-oncogenic pathway in breast cancer.

Materials and MethodsDetailed methods are presented in Supplementary Methods.

Cell cultureCulture of HEK-293T cells, ErbB2-overexpressing breast cancer

cell lines SKBR3 and BT474 (ATCC) and 21MT1 (21), ER/PRþ

MCF7 (ATCC) and TN MDA-MB-231 (ATCC) breast cancer celllines, and their lentiviral infection to derive CHIP-overexpressingor CHIP shRNA-expressing lines are described in SupplementaryMethods. Cell lines were regularly tested for Mycoplasma andcontinuously cultured for no more than 3 months.

Antibodies and reagentsRabbit anti-CHIP antibody used for blotting has been

described previously (15). Other primary antibodies or secondary

conjugates for fluorescence or immunoblotting analyses aredescribed under Supplementary Methods.

Cell lysis, immunoprecipitation, and immunoblottingCells lysates were prepared in RIPA or Triton X-100 lysis buffers

(as indicated in figure legends) and used for immunoprecipita-tion and immunoblotting analyses as described in SupplementaryMethods.

Quantitative real-time PCRTotal RNA was extracted using TRIzol reagent (Invitrogen),

reverse transcribed using the Quantitative real-time PCR Kit (cat.204141, Qiagen) and used for real-time qPCR with primersdescribed in Supplementary Methods.

Cell growth assaysCell growth was analyzed as cumulative proliferation over 5

serial weekly passages and by soft agar anchorage-independentgrowth assay as described under Supplementary Methods.

Transwell migration and invasion assaysThemigration or invasion of cells was analyzed using uncoated

or Matrigel-coated Transwell chambers as described under Sup-plementary Methods.

Extracellular matrix degradation assayThis assay was carried out using QCM Gelatin Invadopodia kit

(catalog no. ECM670, EMD Millipore) according to the manu-facturer's protocol, as described in Supplementary Methods.Extracellular matrix (ECM) degradation, seen as focal loss offluorescent signal ("holes") in the labeled gelatin layer, wasquantified using Image J (NIH, Bethesda, MD).

Xenograft tumorigenesisA total of 106 cells inMatrigel (BDBiosciences) were implanted

in the mammary fat pad of female NSG mice (The JacksonLaboratory) primed with subcutaneous estrogen pellet(0.72 mg/60-day release pellets; Innovative Research of America)and tumor growth monitored weekly for 10 weeks. After eutha-nasia, tumors were imaged, and formalin-fixed and paraffin-embedded for further analyses. Details are presented in Supple-mentary Methods.

Protein/DNA arrayA protein/DNA combo-array kit (catalog no. MA1215,

Affymetrix) was used to simultaneously screen 345 transcriptionfactors for DNA-binding activity per vendor protocol withsignals detected using chemiluminescence (See SupplementaryMethods).

Electrophoretic mobility shift assayElectrophoretic mobility shift assay (EMSA) was carried out

using an EMSA kit (catalog no. 20148, Life Technologies) asdescribed in Supplementary Methods.

Cathepsin B/cathepsin L activity assayCathepsin B/L (CTSB/L) activity was assayed using the

Magic Red CTSB/L Activity Kit (catalog no. 937 & 941,Immunochemistry Technologies) according to the manufac-turer's protocol (See Supplementary Methods). Experiments

Loss of CHIP E3 Unleashes MZF1-Cathepsin Axis

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were run in triplicates and repeated thrice. Ten random fieldsper well were imaged. Fluorescence intensity was quantifiedusing ImageJ (NIH).

GST fusion protein pulldown analysesCell lysate pulldown analyses of CHIP-MZF1 interactions using

GST-CHIP, GST-CHIP-H260Q/P269A mutant, GST-DU-box-CHIP (U-box domain deleted mutant) and GST-DTPR-CHIP[tetratricopetide repeat (TPR) domain deleted mutant CHIP]fusion proteins were done as described previously (see Supple-mentary Methods; ref. 15).

Human and animal subjectsThe use of human tissues was approved by Nottingham

Research Ethics Committee 2 under the title "Development ofa Molecular Genetic Classification of Breast Cancer," and incompliancewith current ethical and legal guidelines of theUnitedKingdom. All experiments in this study were conducted in accor-dance with the 1975 Helsinki declaration and its later amend-ments or comparable ethical standards. There waswritten consentobtained from patients at the time of collection of the material.Mouse studies were preapproved by the UNMC InstitutionalReview Board Committee, in compliance with Federal and Stateguidelines.

IHC analysis of breast cancer tissue microarraysTissue microarrays (TMA) corresponding to a well-annotated

971 breast cancer patient cohort at the University of NottinghamHospital Breast Unit and a commercial TMA (BR20837, fromUS Biomax, Inc.) were analyzed by IHC staining (see Supplemen-tary Methods for details) with a previously described (22)anti-rabbit CHIP antibody that was further validated (Supple-mentary Fig. S1).

ResultsReduced nuclear CHIP expression in breast cancer correlateswith tumor progression, invasion, and poorer patient survival

As previous small studies (11–13) have not clarified whichbreast cancer subtypes lose CHIP expression, we performed IHCanalysis of formalin-fixed and paraffin-embedded TMAs from awell-annotated cohort of 971 breast cancer patients (Supplemen-tary Table S1; refs. 23–27) using an established rabbit anti-CHIPantibody (15) further validated inWestern blottingwith lysates ofCHIP-depleted or ectopic CHIP-expressing cell lines (Supplemen-tary Fig. S1). Initial IHC of a commercial breast cancer TMA (208samples) detected both cytoplasmic and nuclear CHIP withintumor cells and in surrounding normal epithelium (Fig. 1A). Inthe 971-sample TMA, 423 and 314 samples, respectively, were

Figure 1.

Reduced nuclear CHIP staining in primary breast cancer TMA specimens. A, Anti-CHIP IHC staining of a representative breast cancer specimen (top) versusnormal breast tissue (bottom) to show cytoplasmic and nuclear staining. B, Representative pictures depicting low or high nuclear versus cytoplasmic CHIPstaining patterns in breast cancer TMAs. C, Kaplan–Meier analysis correlating breast cancer–specific survival (BCSS) with moderate/high versus negative/lownuclear (left) or cytoplasmic (right) CHIP staining in TMA samples. Number of patients in each group are shown in parentheses next to captions. P valuesare indicated inside each box.

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evaluable for cytoplasmic and nuclear staining, with multiplestaining patterns: low cytoplasmic/lownuclear, high cytoplasmic/low nuclear, low cytoplasmic/high nuclear; or high cytoplasmic/high nuclear (Fig. 1B; Supplementary Table S1). Loss of nuclearCHIP staining showed a significant positive correlation withbiochemical markers of tumor progression and metastasis,reduced ER and PR staining, altered cytokeratins (CK18, CK19),increased expression of early epithelial–mesenchymal transitionmarkers (N-cadherin, P-cadherin) and expression of EGFR familyproteins (EGFR, ErbB2, and ErbB4; Supplementary Table S2).Only CK18 (P ¼ 0.001) and ErbB2 (P ¼ 0.016) expressioncorrelated with reduced cytoplasmic CHIP staining (Supplemen-tary Table S2). Lower nuclear but not cytoplasmic CHIP stainingshowed a significant positive correlation with clinicopathologicfeatures of tumor progression (higher tumor size, tumor grade,pleomorphism, and mitotic status; Supplementary Table S1).Kaplan–Meier analysis revealed that low nuclear but not thecytoplasmic staining pattern correlated significantly with poorerbreast cancer–specific survival (Nuclear CHIP, P ¼ 0.003 vs.cytoplasmic CHIP, P ¼ 0.469; Fig. 1C). Using the previouslyestablished ER, PR, and ErbB2/Her2 expression status of the TMAsamples (24–28), low CHIP expression was seen in two thirds ofErbB2þ and TN cases and about one third of ERþ cases.

CHIP suppresses tumorigenic traits in ErbB2þ breast cancer celllines

Validating IHC results, most ErbB2þ and TN breast cancer celllines showed lower CHIP mRNA and protein levels comparedwith ERþ lines (Supplementary Fig. S2A and S2B); the ERþ and TNcell line results confirm aprevious report (11).Western blotting of12 breast cancer patient-derived xenograft (28) lysates confirmedthe lower or absent CHIP expression in TN, ErbB2þ and some ERþ

breast cancers (Supplementary Fig. S2C). The CHIP expressionstatus in the commercial TMA was consistent with these results(Supplementary Fig. S2D).

To gain mechanistic insights into how the loss of nuclear CHIPpromotes oncogenic progression, we utilized ErbB2þ breast can-cer cell lines (BT474, SK-BR3, and 21-MT1) as primary models,confirming key findings in TN (MDA-MB-231) and ERþ (MCF-7)lines. We engineered the ErbB2þ and TN lines to stably over-express Myc-CHIP (CHIP-hi lines; or vector control; CHIP-lolines) and MCF-7 cells with CHIP shRNA (CHIP-lo) to depletethe endogenous CHIP expression (or a control shRNA; CHIP-hi;Supplementary Fig. S1A). Of note, Myc-CHIP is seen as multiplebands, some migrating considerably slowly while others comi-grating with endogenous CHIP; the authenticity of these bands asMyc-CHIP is demonstrated by anti-Myc immunoblotting (Sup-plementary Fig. S1C). As CHIP expression in MDA-MB-231 cellsis known to suppress oncogenic attributes (11), these servedas controls.

Although no significant differences in cell proliferation wereobserved over a single passage, confirming previous MDA-MB-231 results (11), cumulative cell proliferation over multiplepassages was modestly but significantly lower in CHIP-hi versuscontrol cells (Fig. 2A). CHIP-high cells exhibited a significantlylower number of colonies in soft (Fig. 2B), significantly reducedTranswell migration (Fig. 2C), and significantly reduced invasionthrough Matrigel (Fig. 2D) compared with CHIP-lo control cells.

Analysis of CHIP-lo versus CHIP-hi MDA-MB-231 cells, ascontrols, confirmed the expected reduction in primary tumorgrowth and lung metastasis upon CHIP overexpression (Supple-

mentary Fig. S3A–S3B; ref. 11). Notably, the CHIP-hi BT474 cellsformed significantly smaller xenograft tumors compared withthose with CHIP-lo cells (Fig. 3A and B). Exogenous CHIP over-expression in tumorswas confirmedbywestern blotting (Fig. 3C).Histologically, the CHIP-hi BT474 tumors showed lower nuclearatypia and mitotic index (Fig. 3D and E), and reduced Ki67þ

proliferating cells (Fig. 3F and G) compared with CHIP-lo BT474tumors. No significant difference in immunostaining for theapoptotic marker cleaved caspase-3 was noted (Fig. 3F and H),suggesting a primarily cytostatic impact of CHIP overexpression.These studies support the tumor-suppressor role of CHIP inErbB2þ breast cancer as suggested by our clinical–pathologicalanalyses.

Identification of nuclear targets of CHIP in breast cancerAs loss of nuclear but not cytoplasmic CHIP expression pre-

dicted tumor progression and poor patient outcomes, we assessedthe cognate DNA-binding activities of 345 cellular transcriptionfactors in nuclear extracts of control versusCHIP-hi ErbB2þBT474cells, using a commercially available array platform. The DNA-binding activity inCHIP-hi over CHIP-lo extracts was expressed asa fold ratio. Subsets of transcription factors showed changes inDNA-binding activities outside of the arbitrary 3-fold cutoff value,representing potential direct or indirect targets of negative orpositive regulation by CHIP (Fig. 4A, left). These (SupplementaryTable S3) included known CHIP targets, such as p53 and NFkB(29, 30), validating the approach. Analysis of CHIP-hi versusCHIP-lo (control) MDA-MB-231 cells (Fig. 4A, bottom) showedthatmost CHIP targetswere sharedwith theBT474 cellmodel (seeSupplementary Table S4; Fig. 4A), although the extent of changesdiffered. Immunoblot analysis of a sample of known and noveltranscription factors identified as CHIP targets (NFkB p69/p60,Stat3, FOXM1, MZF1) confirmed their downregulation inCHIP-hi versus CHIP-lo BT474 and MDA-MB-231 cell lines(Fig. 4B and C). Thus, our unbiased screen identified novelCHIP-targeted transcription factors whose deregulated functionupon loss of CHIP expression may contribute to tumorprogression.

MZF1 is a direct target of CHIP-mediated ubiquitination anddegradation

We focused further on MZF1, whose DNA-binding activitywas downregulated in CHIP-hi BT474 and MDA-MB-231 cells(Fig. 4A; MZF1 highlighted in white), as it was recently iden-tified as a nexus of ErbB2 signaling that culminates in tumorcell invasiveness through increased transcription of matrix-degrading enzymes cathepsin B (CTSB) and L (CTSL; ref. 20).Electrophoresis mobility shift assay (EMSA) using sequencescorresponding to MZF1-binding sites on the CD34 gene pro-moter (31) confirmed reduced binding activity (shifted bands)in CHIP-hi versus CHIP-lo BT474 cells (Fig. 4D). The MZF1mRNA level was reduced in CHIP-hi versus CHIP-lo cells BT474cells (Fig. 4E), suggesting the regulation of upstream modula-tors of MZF1 transcription as part of CHIP-dependent reduc-tion in DNA-binding activity. MZF1 protein level was reducedin CHIP-hi versus CHIP-lo cells, and antiubiquitin blotting ofMZF1 immunoprecipitations showed increased MZF1 ubiqui-tination in CHIP-hi versus CHIP-lo BT474 cells (Fig. 4F).Cotransfection of CHIP and MZF1 in HEK-293T cells revealedCHIP dose-dependent MZF1 ubiquitination (Fig. 4G, top) andreduction in protein level (Fig. 4G, bottom). Notably, although






ErbB2 levels in CHIP plus ErbB2 cotransfected HEK-293T cellsincreased upon treatment with MG132 (proteasomal inhibitor;Supplementary Fig. S4D and S4E), as expected (14, 15), MG132treatment failed to rescue the MZF1 levels in CHIP cotrans-fected cells (Fig. 4H and J), suggesting the possibility that MZF1degradation occurs through chaperone-mediated autophagyin lysosomes as demonstrated for CHIP targets such asHIF1a (32). Indeed, CHIP-induced reduction in MZF1 levelswas partially rescued by a short treatment of cells with thelysosomal inhibitor bafilomycin A1 (Fig. 4I and K). Finally,depletion of endogenous CHIP by shRNA knockdown inMCF-7 cells led to upregulation of MZF1 and CTSB/L proteinlevels (Supplementary Fig. S4B). These results support theconclusion that MZF1 is a bona fide target of CHIP, involving

indirect transcriptional downregulation and CHIP-dependentubiquitination and lysosomal degradation of MZF1.

The tetratricopetide repeat domain of CHIP is required forinteraction with MZF1

The TPR domain of CHIP has been shown to mediatebinding to most interacting partners such as HSC70 and HSP90(4, 15). Given the CHIP-MZF1 coimmunoprecipitation(Fig. 4G), we used GST fusion proteins of intact CHIP or itspoint or deletion mutants for pulldown of MZF1 from lysatesof HEK-293T cells transiently transfected with MZF1 to map theCHIP domain that mediates its interaction with MZF1. Com-pared to the negative control GST, GST-CHIP robustly pulleddown MZF1 from cell lysates (Fig. 4L). Notably, the U-box

Figure 2.

Suppression of in vitro oncogenicattributes upon CHIP overexpression inCHIP-lo ErbB2þ breast cancer cell lines.A, Cumulative proliferation of CHIP-locontrol (MSCV-puro) versus CHIP-hi(MSCV-CHIP) 21MT1, BT474 and SKBR3cell lines. Data points representcumulative cell numbers with each serialpassage.B,Anchorage-independent cellgrowth in soft agar after 3 weeks ofculture. Y-axis, number of colonies per2,500 seeded cells. C, Transwell cellmigration assay. A total of 2,000 cellswere added in top chamber andmigration scored after 24 hours.Y-axis, number of migrated cells perhigh-power field. D, Matrigel-coatedTranswell invasion assay. A total of2,000 cells were added in top chamberand migration scored after 24 hours. Y-axis, number of migrated cells per high-power field. Data in each figurerepresent mean � SEM of threeexperiments, each in triplicates;� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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point mutant (GST-CHIP-H260Q/P269A) and the U-boxdomain deleted CHIP mutant (GST-DU box-CHIP) showedunimpaired MZF1 pulldown, whereas no pulldown was seenwith the TPR domain deleted CHIP (GST-DTPR-CHIP; Fig. 4L).These results establish that CHIP binds to MZF1 through itsTPR domain.

CHIP is a negative regulator of MZF1-dependent andCTSB/L-mediated ECM degradation

CTSB/L are key regulators of tumor invasion, angiogenesis andmetastasis (33), and their MZF1-dependent transcriptional upre-gulation was shown to mediate in vitro tumor cell invasion uponErbB2 overexpression in breast cancer cells (20). We thereforeasked whether CHIP regulation of MZF1 translates into controlof CTSB/L expression. EMSA with a DNA probe from a knownMZF1-binding site in the CTSB promoter (31) confirmed reducedDNA-binding activity in the nuclear extracts of CHIP-hi versuscontrol ErbB2þ cells (Fig. 5A), with reduced CTSB and CTSLmRNA and protein levels (Fig. 5B, D, and E), reduced CTSB/Lenzymatic activities (Fig. 5G and I) measured with a fluorescentsubstrate (34), and reduced FITC-labeled gelatin degradation (Fig.6A and C; refs. 35, 36). Thus, the MZF1/CTSB/L pro-invasion

signaling axis is negatively regulated by CHIP in ErbB2þ breastcancer cells.

Consistent with CHIP-dependent reduction in the DNA-binding activity of MZF1 in CHIP-hi versus CHIP-lo MDA-MB-231 cells (Fig. 4A), reduced mRNA and protein levels ofCTSB/L (Fig. 5C and F), reduced CTSB/L activity (Fig. 5H and J)and reduced florescent gelatin degradation (Fig. 6B and D)were observed in CHIP-hi versus CHIP-lo MDA-MB-231 cells.Increased CTSB/L expression was also seen in CHIP KD versuscontrol MCF7 cells (Supplementary Fig. S4B). Ectopic over-expression of MZF1 in CHIP-hi as well as CHIP-lo MDA-MB-231 cells increased the florescent gelatin degradation (Fig. 6Eand F). Finally, treatment of CHIP-lo 21MT1 cells (high CTSB/Lactivity; Fig. 6A and C) with CA074, a specific inhibitor of CTSB(37), abrogated the fluorescent gelatin degradation, andmarkedly inhibited the Transwell invasion of 21MT1(Fig. 7A–C) and MDA-MB-231 (Fig. 7D–F) breast cancer celllines. Thus, we conclude that loss of CHIP expression acrossbreast cancer subtypes upregulates the pro-invasive MZF1-CTSB/L signaling axis.

To assess whether the upregulation of CTSB expressionand activity as a result of CHIP downregulation contributes to

Figure 3.

Reduced xenograft growth of CHIP-overexpressing BT474 cells in nude mice. A, Tumor volume of CHIP-lo (MSCV-puro) control and CHIP-hi (MSCV-CHIP)BT474 xenografts over time. Mean� SEM, N ¼ 6 per group. �� , P < 0.001. B, Photograph of resected CHIP-lo (top) and CHIP-hi (bottom) BT474 cell xenografts.C,Western blotting to visualize the endogenous and overexpressed (Myc-tagged) CHIP in resected BT474 xenografts. HSC70, loading control. D, Representativehematoxylin and eosin staining and mitotic events (red circles) of CHIP-lo (left) and CHIP-hi (right) BT474 xenograft tumor sections. E, Quantification of mitoticevents of sections depicted in D. Mean � SEM, n ¼ 3; �� , P < 0.001. F, Ki67 (brown) and cleaved caspase-3 (red) costaining of CHIP-lo (left) and CHIP-hi(right) BT474 xenograft tumor sections. G and H, Quantification of Ki67- (G) and cleaved caspase-3- (H) positive cells depicted in F. Mean� SD, n¼ 4. � , P < 0.05;ns, not significant.






Figure 4.

Identification of MZF1 as a CHIP-regulated transcription factor in breast cancer cells. A, DNA-binding activities of 345 transcription factors were analyzedin nuclear extracts of CHIP-lo (MSCV-puro control) versus CHIP-hi (MSCV-CHIP) BT474 and MDA-MB-231 cell lines. Y-axis, loge-fold binding in CHIP-hi overCHIP-lo cells. The 3-fold increase (top) or decrease (bottom) in binding (dotted lines)was used as cutoff. MZF1, open symbol.B and C,Validation of CHIP-dependentdownregulation of protein levels of selected transcription factors identified in A by Western blotting of CHIP-lo versus CHIP-hi BT474 (B) or MDA-MB-231 (C)cell lysates. D, Real-time qPCR analysis of MZF1 mRNA levels in CHIP-hi versus CHIP-lo BT474 cells. E, A biotin-labeled double-stranded oligonucleotide withMZF1-specific DNA sequence was used to probe nuclear extracts of CHIP-hi (MSCV-CHIP, C) versus CHIP-lo (MSCV-puro, P) BT474 cells. Two-hundred-foldexcess of the nonlabeled oligonucleotide served as a competitor. F, Immunoblot analysis of MZF1 protein levels in CHIP-hi versus CHIP-lo BT474 cells.G, CHIP-dependent ubiquitination and degradation of MZF1. Anti-MZF1 immunoprecipitations from lysates of HEK-293T cells transfected with GFP-MZF1(1 mg) � Myc-CHIP (1 or 2 mg) were immunoblotted for ubiquitin or MZF1 (top), and whole-cell lysates (bottom) were blotted for MZF1, CHIP, or HSC70(loading control). H and I, HEK-293T cells were transfected with MZF1-GFP (250 ng)�Myc-CHIP (1 mg). Cells were treated with vehicle (�) or proteasomal inhibitorMG132 (50 mmol/L) or lysosomal inhibitor Bafilomycin A1 (100 nmol/L) for 6 or 16 hours, respectively. Lysates were immunoblotted for MZF1 or CHIP. J and K,Quantitation of the MZF1 levels in CHIP-cotransfected cells in the presence of MG132 or Bafilomycin A1, relative to vehicle controls assigned a value of 1.Data shown represent mean � SD of n ¼ 3; � , P < 0.05; ns, not significant. L, The TPR domain of CHIP is required for binding to MZF1. HEK-293T cells transfectedwith MZF1-GFP and their lysates were used for pulldown with GST or the indicated GST–CHIP fusion proteins. Bound MZF1-GFP was visualized by immunoblottingfor GFP (top) or MZF1 (middle). Ponceau Red staining of the membrane shows the relative amounts of fusion proteins used in pulldowns (bottom).

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oncogenesis in vivo, we grewBT474 xenograft tumors to an averageof 0.5 mm3 and treated the mice with CA074 (25 mg/Kg bodyweight, i.p.), trastuzumab (4 mg/Kg, tail vein injection) as astandard ErbB2-targeted therapeutic or the vehicle control, basedon previously used dosages (37–39). CA074 treatment led to amarked and statistically significant inhibition of tumor growth atmultiple time points, comparable to that seen with trastuzumab(Fig. 7G), supporting the conclusion that one mechanism bywhich loss of CHIP promotes breast tumor progression is byeliminating the negative regulation of MZF1 and unleashing thecathepsin expression.

DiscussionLoss of expression of theHSP90/HSC70 co-chaperoneCHIP E3

has emerged as a mechanism to promote tumor progression.Here, we demonstrate that loss of nuclear, and not cytoplasmic,CHIP expression is associatedwith tumor progression and shortersurvival in breast cancer patients and is a feature of about twothirds of ErbB2þ and TN as well as a third of ERþ breast cancers.Importantly, we identify loss of CHIP as a keymechanism to alter

the DNA-binding activities of a substantial subset of cellulartranscription factors and establish that loss of CHIP expressionunleashes a pro-invasion signaling cascade in which the CHIPtarget MZF1 promotes cathepsin B and L expression. Using achemical inhibitor strategy, we show that upregulation of MZF1-CTSB axis due to loss of CHIP expression contributes totumorigenesis.

IHCanalyses of an extensive collectionofwell-annotatedbreastcancer TMAs demonstrated that loss of nuclear CHIP signalscorrelates withmarkers of advanced tumor progression, includinghigher tumor grade, mitotic index, and markers of early EMT(Supplementary Table S1), and significantly shorter breast can-cer–specific and progression-free survival (Fig. 1C). Loss of nucle-ar CHIP was a feature of nearly two thirds of ErB2þ and TNsubtypes, consistent with their poorer intrinsic patient outcomesand higher metastatic odds (40, 41), and about a third of ERþ

breast cancers (Supplementary Table S2). In contrast, loss ofcytoplasmic CHIP expression was only associated with ErbB2positivity and did not predict poor patient survival (Fig. 1C).These results materially extend previous findings using smallerpatient cohorts (11–13), and highlight the subtype preference of

Figure 5.

CHIP levels control the expression of MZF1 target genes cathepsins B and L. A, EMSA analysis with the CTSB promoter sequence in CHIP-hi versus CHIP-loBT474 cells was done as in Fig. 4B. B and C, CTSB/L mRNA expression in CHIP-hi versus CHIP-lo BT474 (B) and MDA-MB-231 (C) cells was analyzed by real-timeqPCR; n¼ 3. D and E, Immunoblotting for CTSB (D) and CTSL (E) protein levels in CHIP-hi versus CHIP-lo BT474 cells. F, IHC analysis of MZF1 (left), CTSB (middle),and CTSL (right) in CHIP-hi versus CHIP-lo MDA-MB-231 cells. G and H, CTSB/L activities in CHIP-hi versus CHIP-lo 21MT1 (G) and MDA-MB-231 (H) cellsanalyzed by the production of a red fluorescent cleavage product. I and J,Quantification of CTSB (top) and CTSL (bottom) activities presented inG andH in CHIP-hiversus CHIP-lo 21MT1 (I) and MDA-MB-231 (J) cells. Each square represents one replicate. Mean � SD. � , P < 0.05; ��, P < 0.01; ��, P < 0.005.






CHIP loss and the importance of the loss of nuclear CHIP as a pro-oncogenic adaptation in breast cancer.Of note, theCHIP-lowERþ

patients numerically exceed the CHIP-lo ErbB2þ and TN patients,and further studies are needed to assess if these patients belong toa specific molecular subclass. On the basis of previous publica-tions (11) and our results on cell lines (Supplementary Fig. S2D),reduced CHIP mRNA expression provides one mechanism forreduced CHIP levels in tumors. Why nuclear CHIP levels maybeselectively reduced in certain primary tumors is not clear atpresent. Mechanisms such as aberrant nuclear/cytoplasmic trans-port of CHIP or selective degradation of CHIP within the nucleusor cytoplasm will need to be explored as a potential basis for ournew findings.

Analyses in breast cancer cell lines confirmed the predominantloss of CHIP expression in the ErbB2þ and TN subtypes. Usingmatched pairs of ErbB2þ breast cancer cell lines with low endog-enous CHIP expression versus their CHIP-hi derivatives in func-tional assays, we show that CHIP levels are a key determinant ofErbB2-driven cell growth and invasiveness in vitro and xenografttumor growth in nude mice, extending previous findings using

CHIP-reconstituted MDA-MB-231 (TN) and CHIP-depletedMCF-7 (ERþ) lines (11), which served as controls. Although weconfirmed the lack of impact of CHIP restoration on cell prolif-eration in a single passage (11), analyses over multiple passagesrevealed a subtle but significant proliferative disadvantage (Fig.2A), which is profound under anchorage-independent conditions(Fig. 2B). Thus, CHIP serves as a tumor suppressor for ErbB2þ, TNand a proportion of ERþ breast cancers.

Furthering our novel findings that loss of nuclear and notcytoplasmic CHIP correlates with tumor progression, an unbiasedprotein/DNA array screen identified a number of transcriptionfactors whose DNA-binding activities are directly or indirectly up-or downregulated by CHIP (Fig. 4A), vastly expanding the list ofpotential targets of CHIP in the context of cancer (29, 30). Here,we have focused on MZF1, whose activity was downregulated byCHIP, because it was recently identified as amajor transcriptionalactivator of CTSB//L-mediated tumor cell invasion downstreamof ErbB2 in breast cancer cells (42). CTSB/L are overexpressed inprimary breast cancers and CTSB or MZF1 knockdown abrogatedbreast cancer cell invasiveness in vitro (41). Our EMSA analyses

Figure 6.

The CHIP–MZF1–CTSB axis influences tumor cell invasiveness. A and B, Degradation of FITC-labeled gelatin matrix (seen as patchy holes in the uniformgreen matrix) by CHIP-lo versus CHIP-hi 21MT1 (A) and MDA-MB-231 (B) cells seeded for 48 hours and stained for actin-containing invadopodia with phalloidin(red) and for nuclei with DAPI (blue). C and D, Quantification of gelatin degradation presented in A (C) and B (D). Mean � SD; n ¼ 4. E, Restoration ofFITC-labeled gelatin matrix degradation by MZF-1 overexpression in CHIP-hi MDA-MB-231 cells. Stably MZF-1–overexpressing CHIP-lo or CHIP-hi MDA-MB-231cells were analyzed as in A and B. F, Quantification of gelatin degradation is shown in E. Each square represents a replicate. Mean � SD. � , P < 0.05;�� , P < 0.01.G,Western blotting of MZF1 overexpression in CHIP-lo versus CHIP-hi MDA-MB-231 cells.H,Quantification of MZF1 overexpression as shown inG (n¼ 3).

Luan et al.





confirmed the reduced MZF1 DNA-binding activity in CHIP-hibreast cancer cells (Fig. 4D), and we show that MZF1 is botha direct target of CHIP for ubiquitination and destabilization

(Fig. 4F and G), and also indirectly regulated at the mRNA level,potentially due to CHIP regulation of upstream regulators ofMZF1 expression such as MYC (Fig. 4A; ref. 43). Regarding

Figure 7.

CTSB inhibition reduces ErbB2þ breast cancer cell tumorigenesis. A, 21MT1 cells cultured on FITC-gelatin were treated with DMSO vehicle or CTSB inhibitor(CA074; 25 mg/mL) for 48 hours and degradation was analyzed as in Fig. 5G. B, Quantification of FITC-gelatin degradation shown in A. Mean � SD; n ¼ 4.C, 21MT1 cells seeded on Matrigel-coated Transwell invasion membranes were treated with DMSO (control) or CA074 (25 mg/mL) for 24 hours, and cellsthat had invaded through Matrigel to the bottom surface were stained with CyQuant GR fluorescent dye and quantified using a fluorescence reader. Mean � SD;n ¼ 3. D, FITC-gelatin matrix degradation by MDA-MB-231 cells was analyzed after 48 hours culture in DMSO (control) or CTSB inhibitor (CA074; 25 mg/mL)as in Fig. 5G. E, Quantification of FITC-gelatin degradation shown in D. Mean � SD; n ¼ 4. F, Invasion of MDA-MB-231 cells incubated with DMSO (control) orCA074 (25 mg/mL) as in Fig. 6C. Mean � SD; n ¼ 8. G, Groups of 9 nude mice carrying BT474 xenografts (average 0.5 cm3 size) received trastuzumab (viatail vein; 4 mg/kg every 4 days), CA074 (i.p., 25 mg/kg in saline daily), or saline (control), and tumor volumes were monitored every other day. Mean� SEM (n¼ 9;�� , P < 0.001).






indirect transcriptional regulation of MZF1 by CHIP, severaltranscription factors targeted by CHIP, including STAT 1/3 andATF (Fig. 4A–C; and Supplementary Table S3 and S4). MatIn-spector search engine for promoter analysis revealed 5 bindingsites of STAT (STAT1 andSTAT3) and7binding sites of ATFwithintheMZF1 promoter region (www.genomatix.de) and their down-regulation byCHIP could contribute to reducedMZF1 expression.

We show that MZF1 and CHIP associate with each other(Fig. 4G) and used GST pulldown assays to identify the TPRdomain of CHIP to mediate interaction with MZF1 (Fig. 4L).Notably, our analyses using proteasomal (MG132) and lysosom-al (Bafilomycin A) inhibitors show that CHIP-dependent desta-bilization of MZF1 protein primarily occurs in the lysosome(Fig. 4I). It is likely that the lysosomal degradation of MZF1reflects CHIP-dependent chaperone-mediated autophagy as dem-onstrated for HIF1a and other targets (32). In this regard, MZF1possesses several sequences QSFRQ and KAFRQ (sequences thatidentified 447-451, 548-552, 576-580, 660-664, 688-692 aminoacids) homologous to the motif KFERQ found in other chaper-one-mediated autophagy targets (32). Because intermediarykinases (CDC42BPb, ERK2, PAK4, PKCa) were implicated inMZF1-mediated CTSB/L transcription downstream of ErbB2(41), negative regulation of these kinases or ErbB2 (14, 15) mayalso contribute to CHIP regulation of MZF1.

Our analyses support a key role of loss of CHIP as amechanismto unleash the MZF1-dependent transcriptional network thatcontrols CTSB/L expression (Fig. 5) and extracellular matrix(ECM) degradation (Fig. 6), and to promote oncogenesis (Fig. 2).CTSB is a well-established downstream mediator of invasive/metastatic signaling in various cancers (44, 45), including breastcancer (20, 36). Specific CTSB inhibition with CA074 reduced thematrix degradation and cell invasion in vitro and tumor growthin vivo (Fig. 7), providing further support for loss of CHIPexpression as a key breast cancer adaptation to unleash thepro-oncogenic MZF1-CTSB pathway. The strong effect of CTSBinhibition alone on matrix degradation and xenograft tumorgrowth, the latter comparable with trastuzumab (Fig. 7D), sug-gests that CTSB may be the dominant player in BT474 cells,consistent with strong reported impact of CTSB depletion on invitro invasiveness of ErbB2þbreast cancer cell lines (42).However,further studies to assess the contribution of CTSL and otherpotential targets of MZF1 are warranted. Our in vitro results inTN and ERþ cell lines (Fig. 4) also support the unleashing ofMZF1-CTSB/L pathway in the corresponding breast cancer sub-types, and further studies in these and other tumors with loss ofCHIP expression (10) will be of interest. Of note, The CancerGenome Atlas breast cancer data analysis (eBioportal) did not,however, reveal a positive correlation between CTSB/L andMZF1mRNA expression (in fact we noted moderate negative Pearson

correlation coefficients). We surmise that MZF1 and/or CTSB/Lexpressed in myeloid lineage and potentially other stromal com-ponents may contribute to this discrepancy. Transcriptomic anal-ysis of laser capturesmicro-dissected tumor samples ormulticolorIHC with lineage markers will be necessary to further assess therelationship between MZF1 and CTSB/L expression in relation toCHIP/STUB1 expression in primary tumors.

In conclusion, our studies demonstrate that upregulation ofMZF1/CTSB/L axis is an important pro-oncogenic mechanismunleashed by loss of CHIP expression in breast cancer. Given theestablished roles of cathepsins in matrix remodeling, invasion,angiogenesis, and metastasis (44, 45), future studies to establishthe role and targeting of MZF1-CTSB/L axis in metastatic tumorsettingsmay provide new therapeutic opportunities for breast andother tumors with loss of CHIP expression (30, 46–49).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T.A. Bielecki, J.L. Meza, H. BandDevelopment of methodology: H. Luan, T.A. Bielecki, T.A. Jennings, J.L. MezaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Luan, T.A. Bielecki, R.J. Clubb, M.D. Storck,E.A. Rakha, H. BandAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Luan, T.A. Bielecki, S. Mirza, W. An, D. Ahmed,R.El-Ansari, N.K. Mishra, C. Guda, J.L. Meza, S. Raja, H. BandWriting, review, and/or revision of the manuscript: H. Luan, T.A. Bielecki,W. An, M.D. Storck, J.L. Meza, S. Raja, E.A. Rakha, H. BandAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. An, M.D. Storck, E.A. RakhaStudy supervision: S. Raja, H. BandOther (immunohistochemical scoring): D. AhmedOther (establishing critical collaborations): H. Band

AcknowledgmentsWe thank the Band laboratory members for helpful discussions. This work

was supported by the NIH grants CA116552, CA105489, and CA87986 (toH. Band), and CA96844 and CA144027 (to V. Band); DOD grants W81XWH-11-1-0167, W81XWH-17-1-0616 (to H. Band), and W81XWH-07-1-0351,W81XWH-11-1-0171 (to V. Band); NIGMS (NIH) Institutional DevelopmentAward P30 GM106397; and support to the Confocal, Flow Cytometry,Bioinformatics and other Cores from the NCI Cancer Center Support Grant(P30CA036727) to FPBCC, NIH/INBRE Award (P20GM103427), and fromNebraska Research Initiative. T.A. Bielecki and T.A. Jennings were trainees underaNCICancer Biology TrainingGrant (T32CA009476).H. Luan received aChinaScholarship Council graduate fellowship. B. Mohapatra and W. An receivedUNMC graduate fellowships. T.A. Jennings was a Susan G. Komen Foundationpostdoctoral fellow (KG091363).

Received August 10, 2016; revised January 11, 2018; accepted March 1, 2018;published first March 6, 2018.

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2018;78:2524-2535. Published OnlineFirst March 6, 2018.Cancer Res Haitao Luan, Bhopal Mohapatra, Timothy A. Bielecki, et al. Cancer Unleashes the MZF1-Cathepsin Pro-oncogenic ProgramLoss of the Nuclear Pool of Ubiquitin Ligase CHIP/STUB1 in Breast

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