[CANCER RESEARCH 63, 1657–1666, April 1, 2003]

Lysyl Oxidase-related Protein-1 Promotes Tumor Fibrosis and Tumor Progressionin Vivo1

Gal Akiri, Edmond Sabo, Hagit Dafni, Zehava Vadasz, Yelena Kartvelishvily, Noga Gan, Ofra Kessler, Tzafra Cohen,Murray Resnick, Michal Neeman,2 and Gera Neufeld2,3

Department of Cell Biology and Anatomy, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, 31096, Israel [G. A., Z. V., Y. K., N. G.,O. K., G. N.]; Department of Pathology, Carmel Medical Center, Haifa, 34632, Israel [E. S., M. R.]; Department of Biological Regulation, Weizmann Institute of Science,Rehovot, 76100, Israel [H. D., M. N.]; and Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Carmel Medical Center, Haifa, 34632, Israel [T. C.]

ABSTRACT

The lysyl oxidase gene family members function as extracellular matrixmodulating enzymes. We have found that another member of this family,lysyl oxidase related protein-1 (LOR-1), is highly expressed in metastaticbreast cancer-derived cell lines but not in the nonmetastatic estrogen-dependent MCF-7 cells. Furthermore, LOR-1 expression in periductaltumor cells of breast carcinomas is significantly correlated with increasedtumor malignancy. MCF-7 cells expressing recombinant LOR-1 formedestrogen-dependent tumors that developed much slower than tumorsderived from empty vector-transfected MCF-7 cells. The cells of theseLOR-1-expressing tumors were surrounded by a high concentration ofdense collagen fibers, and the tumors contained many fibrotic foci. Induc-tion of fibrosis in vivo by lysyl oxidase-like enzymes has never beenobserved before and suggests that LOR-1 may function as an autonomousinducer of fibrosis. The appearance of fibrotic foci in spontaneous breastcancer tumors is correlated with poor prognosis and metastasis, and we,therefore, examined the invasiveness of the LOR-1-expressing tumors.LOR-1-expressing MCF-7 cells invaded the pseudocapsules surroundingthe tumors. In contrast, vector-transfected MCF-7 cells did not invade thepseudocapsules. This observation suggests that LOR-1 enhances the ma-lignancy of the tumors. Furthermore, the LOR-1-expressing tumor cellsinvaded blood vessels, nerves, and muscles adjacent to the tumor, indi-cating that the LOR-1-expressing MCF-7 cells acquired metastatic prop-erties. We conclude that LOR-1 promotes tumor fibrosis and tumorinvasiveness simultaneously, which indicates that these two processes maybe associated.

INTRODUCTION

LO4 (protein-lysine 6-oxidase, EC 1.4.3.13) is a copper-dependent,secreted amine oxidase that oxidatively deaminates the �-amino groupof specific peptidyl lysine and hydroxylysine residues of collagen andof lysine in elastin. The resulting peptidyl aldehydes condense spon-taneously with peptidyl aldehydes located in close proximity, or withunreacted �-amino groups, to form inter- and intramolecular cross-linkages stabilizing the fibrous forms of collagen and elastin (1).However, this oxidative activity does not display high specificitybecause LO can also oxidize additional lysine-rich proteins such ashistone-H1, as well as various lysine-rich synthetic peptides (2, 3). Itwas recently observed that LO can be translocated into cell nuclei andthat it can regulate, by an as yet poorly understood mechanism, theexpression of collagen-3A1, indicating that LOs may have additional

functions (4, 5). LO is synthesized as an inactive proenzyme that isactivated by the products of the BMP-1 gene (6).

Recently, several additional LO family members were identified.These new family members are characterized by the presence of aconserved LO-like domain containing conserved copper binding andcatalytic domains at their COOH termini. These new LOL genesinclude LOL (7, 8), LOR-1 (or LOXL2; Ref. 9), LOR-2 (or LOXL3;Ref. 10), and LOXC (or LOXL4; Ref. 11). LOR-1, LOR-2, andLOXC differ with respect to LO and LOL in that they possess a muchlonger NH2-terminal domain, suggesting that they constitute a distinctsubclass of LOs, and that their functions may differ fundamentallyfrom those of LOL and LO (12). LOR-1 was initially identified as agene the expression of which is up-regulated in senescent fibroblastsand in adherent tumor-derived cells (9). It was also found to be highlyexpressed in reproductive tissues (13). It was demonstrated thatLOL is also processed into its active form by bone morphogeneticprotein-1, raising the possibility that the proteins encoded by the otherfamily members may also be activated by proteolytic digestion aftersecretion.

The transition from a localized tumor to an invasive and metastatictumor represents a landmark in the development of malignant diseasebecause it is usually associated with a markedly worse prognosis. Theunderstanding of the processes that govern this transition is therefore,of prime importance. It was recently observed that, in breast cancer,the transition from a localized to an invasive/metastatic tumor isassociated in many cases with the formation of fibrotic foci anddesmoplasia (the presence of unusually dense collagenous stroma)within the primary tumor (14, 15). There are also some indicationsthat a similar correlation may exist in other types of cancers such asin colon cancer and in pancreatic cancer (16, 17). These observationsrepresent apparent paradoxes at first glance because invasiveness haslong been associated with the destruction of ECM by ECM-degradingenzymes like metalloproteases (18, 19) and heparanase (20). How-ever, it is possible that the deposition of excess ECM may stimulate,in turn, expression of matrix-degrading enzymes that will contributeunder certain circumstances to tumor invasion. In fact, there is someevidence that an increase in ECM deposition can, indeed, influencethe production of ECM-degrading enzymes (21, 22).

Two LO family members, LO and LOL, were found to be ex-pressed in areas of fibrogenesis in noninvasive in situ ductal breastcarcinomas (23). During the preparation of this report, it was reportedthat metastatic breast cancer cell lines express LO, LOL, and LOR-1,whereas nonmetastatic breast cancer-derived cell lines, such asMCF-7 cells, do not, and that LO-expressing MCF-7 cells displayincreased invasiveness in in vitro invasiveness assays (24). Thesestudies do not provide an explanation for observations that have noteda link between fibrosis and metastasis (14, 15). Here we provideevidence indicating that LOR-1 expression in nonmetastatic MCF-7cells induces massive deposition of dense collagen fibers and theformation of numerous fibrotic foci in tumors that develop after theimplantation of these cells in nude mice. These changes were accom-

Received 10/9/02; accepted 1/27/03.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the German-Israeli binational science foundation (GIF) andby a grant from the Israel Cancer Research Fund (ICRF; to G.N.), and by grants from NIH(RO1 CA90471) and the Israel Science Foundation (to M. N.).

2 The laboratories of Drs. Neeman and Neufeld contributed equally to the guidance ofthis work.

3 To whom requests for reprints should be addressed, at Department of Cell Biologyand Anatomy, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute ofTechnology, P.O. Box 9469 Efron St. Haifa, 31096, Israel. Phone: 972-4-8295425; Fax:972-4-8523947; E-mail: gera@tx.technion.ac.il.

4 The abbreviations used are: LO, lysyl oxidase; LOL, LO-like protein; LOR-1,LO-related protein-1; LOR-2, LO-related protein-2; ECM, extracellular matrix.

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panied by an increase in the invasiveness of the MCF-7 cells, althoughthe LOR-1-expressing cells were still estrogen dependent.

MATERIALS AND METHODS

Materials. Estrogen pellets (17�-estradiol, 0.72 mg/pellet, 60-day release)were from Innovative Research. The Masson’s trichrome staining kit waspurchased from Bio-Optica Corp.; reverse transcriptase and G418 were fromLife Technologies, Inc.; hygromycin B, tetracycline hydrochloride, and thereticulin staining kit, as well as the anti-collagen type I antibody (cloneCOL-1), were from Sigma. Restriction enzymes, and T4 ligase were from NewEngland Biolabs. The bacterial expression vector pQE-30 and the nickelaffinity column were obtained from Qiagen. [32P]dATP was purchased fromNEN. Monoclonal anti-cytokeratin-7 antibodies (CAM 5.2) coupled to FITCwere acquired from Becton Dickinson. Anti-FITC antibodies conjugated toalkaline phosphatase were purchased from Roche. CAS blocking solution,antibody diluant reagent solution, citrate, and EDTA antigen retrieval bufferswere purchased from Zymed.

Cell Culture. The MCF-7 breast cancer cells were kindly given to us byDr. Hadasa Degani (Weizmann Institute, Rehovot, Israel). The MDA-MB-435breast cancer cell line was kindly provided by Dr. Israel Vlodavsky (Technion,Haifa, Israel). The MDA-MB-231 cells were given to us by Dr. MichaelKlagsbrun (Harvard University, Boston, MA). These cell lines were routinelycultured in DMEM supplemented with gentamicin, amphotericin, glutamine,and 10% FCS. Human umbilical vein-derived endothelial cells were isolatedand cultured as described previously (25). Tissue culture media, sera, and cellculture supplements were from Biological Industries, Kibbutz Beth-Haemek,Israel, or from Life Technologies, Inc. MCF-7 TetOff cells (Clontech), con-taining the tetracycline trans-activator (tTA) were grown in DMEM containing10% Tet system-approved FCS (Clontech), in the presence of 100 �g/mlG418, 150 �g/ml hygromycin B, and 1 �g/ml tetracycline.

Cell Proliferation Assays. Empty vector-transfected MCF-7 cells or LOR-1-producing MCF-7 cells, derived from different clones, were cultured inDMEM supplemented with 10% FCS, 2 mM glutamine, and antibiotics. Forproliferation assays, cells were plated in 24-well dishes at 20,000 cells/well.The cells were counted in a Coulter counter after 4 days.

Cloning of the Human LOR-1 and LOR2 cDNA. Total RNA (4 �g) fromhuman umbilical vein-derived endothelial cells (for LOR-1) or melanoma cells(for LOR-2) was reversed transcribed using moloney murine leukemia virusreverse transcriptase (Life Technologies, Inc.) as described previously (26).The cDNA was amplified using the expand long high-fidelity PCR system(Roche). The primers used for amplification of the LOR-1 cDNA were:5�-CGCAAGCTTGGATCCGGGATGGAGAGGCCTCTGTGC (containing aHindIII restriction site) and 5�-CGCTCTAGAGGATCCTTACTGCGGGGA-CAGCTGGTTG (containing a XbaI restriction site). The primers used foramplification of the LOR-2 cDNA were: 5�-GCCATGCGACCTGTCAGT-GTC and 5�-GGGCAGTGGCACTTAGAT.

The 2.3-kb cDNA fragment of LOR-1 was subcloned into the pGEM-TEasy vector (Promega) by T-A cloning. When sequenced, it contained threepoint mutations compared with the published sequence of LOR-1 (9). Twowere silent mutations but one of these mutations changed amino acid 301(counted from the first methionine) from serine to glycine. This amino acidchange appeared in LOR-1 cDNA that was prepared from several completelyindependent RNA preparations and was, therefore, assumed to represent thecorrect sequence.

Generation of Polyclonal Rabbit Antibodies against Human LOR-1. AcDNA fragment containing nucleotides 1641–2253 of LOR-1 was amplifiedusing the expand high-fidelity PCR kit. The primers used were 5�-ACATG-CATGCCCTGACCTGGTCCTCAATGC and 5�-CCCAAGCTTGGAAC-CACCTATGTGGCAGTT. The LOR-1 cDNA fragment was subcloned intothe pGEM-T Easy vector (Promega) by T-A cloning. The 613-bp LOR-1cDNA fragment was cut with SphI and HindIII and ligated into the bacterialexpression vector pQE-30, which added a 6�His encoding tag to the 5� of thefragment in-frame. The resulting plasmid was used to produce a recombinant,6�His-tagged Mr 23,000 peptide. The peptide was purified from bacterial cellextracts using nickel-affinity chromatography and was further purified usingSDS-PAGE. The gel was electroblotted onto nitrocellulose; the band contain-ing the peptide was cut out, solubilized in DMSO, and used to immunize

rabbits. Antiserum was affinity purified on protein-A Sepharose column fol-lowed by affinity purification on a column to which the recombinant peptidewas coupled using a previously described method (27). The antibody waseluted from the column using 0.1 M glycine at pH 3. The anti-LOR-1 antibodydid not cross-react with the related human LOR-2 as tested by Western blotanalysis of conditioned medium and cell extracts that were taken from Chinesehamster ovary cells overexpressing human LOR-2 (data not shown; Ref. 10).

Transfections. For constitutive expression of LOR-1, the full-lengthLOR-1 cDNA was digested out of the pGEM-T easy vector (Promega) withHindIII and XbaI (which were incorporated into the primers used for thecloning of the LOR-1 cDNA) and ligated into the mammalian expressionvector pcDNA3.1hygro (Invitrogen) to generate the expression vector pcDNA-LOR-1. Empty pCDNA3.1hygro plasmid or pcDNA-LOR-1 plasmid (10–20�g) were stably transfected into MCF-7 cells using electroporation with aBio-Rad gene pulser (960 �F, 0.28 V). Stable transfectants were selected using300 �g/ml hygromycin B. Clones expressing recombinant LOR-1 were ob-tained in two consecutive stable transfections and screened for LOR-1 expres-sion by using our anti-LOR-1 polyclonal antibodies. Conditioned medium wascollected after 48 h from transfected cells and LOR-1 expression was moni-tored using Western blot analysis (Fig. 2A). C6 glioma cells were transfectedand screened for LOR-1 expression as described above.

For inducible expression of LOR-1, full-length LOR-1 cDNA was clonedinto the pTET-Splice vector (Clontech), which enables an inducible expressionunder the control of tetracycline (Tet off system). The pTET-Splice plasmidDNA was digested with HindIII and SpeI and ligated with the 2.3-kb humanLOR-1 cDNA that was cut out of the pCDNA–LOR-1 plasmid with HindIIIand XbaI to yield the pTET-LOR-1 plasmid. This plasmid was cotransfectedinto MCF-7 TetOff cells with pTK-Hygro at a ratio of 20:1, respectively (i.e.,pTET-LOR-1:pTK-Hygro). LOR-1-expressing cells were selected in mediumcontaining 100 �g/ml G418, 150 �g/ml hygromycin B, and 1 �g/ml tetracy-cline. The best clone showing the highest induction levels in the absence oftetracycline and the lowest basal expression levels in the presence of tetracy-cline was designated MCF-7/Tet-LOR-1.

To examine the kinetics of LOR-1 posttranslational processing, MCF-7/Tet-LOR-1 cells were seeded in 24-well dishes and grown to confluence. Themedium was exchanged to tetracycline-free and serum-free medium and ali-quots of conditioned medium were analyzed by Western blot analysis usingantibodies directed against the COOH-terminal domain of LOR-1.

Northern Blot Analysis. Total RNA was extracted from cultured cellsusing Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) accord-ing to the manufacturer’s instructions. Total RNA (15 �g) was loaded on a1.2% agarose gel, and Northern blot analysis was carried out as describedpreviously (28). LOR-1 and LOR-2 32P-labeled cDNA fragments (nucleotides1–660 and 1061–1590, respectively) were used as probes.

Protein Blot Analysis. Serum-free conditioned medium (40 �l) was sep-arated on a 8% SDS-PAGE gel, and the proteins were electroblotted onto anitrocellulose filters using semidry electroblotting. The filter was blocked for1 h at room temperature with TBST buffer containing 10 mM Tris-HCl(pH-7.0), 0.15 M NaCl, and 0.3% Tween 20 supplemented with 10% low fatmilk. The filter was incubated overnight at 4°C with affinity-purified rabbitanti-LOR-1 polyclonal antibody (1:2500) in TBST. It was subsequentlywashed three times in TBST and incubated with goat antirabbit IgG peroxi-dase-conjugated secondary antibodies for 1 h at room temperature. Boundantibody was visualized using the enhanced chemluminescence detectionsystem.

Nude Mice Experiments. Slow-release pellets containing 17�-estradiol(0.72 mg/pellet, 60-day release; Innovative Research) were preimplanted s.c.in female athymic nude mice (CD1), 6–8 weeks old. MCF-7 cells (107

cells/mouse) were injected into the mammary fat pads, and tumor size wasmeasured with a caliper once or twice a week. Mice were sacrificed 4 weeksafter the injection of the MCF-7 cells. In other experiments, the tumors wereexcised when they reached a diameter of 0.8 cm. The primary tumor wasremoved, weighed, fixed in 10% buffered formalin, and embedded in paraffin.

In other experiments, the development of tumors from C6 glioma cellsexpressing recombinant LOR-1 was studied. Injections of empty vector-trans-fected cells or with LOR-1-transfected cells (2 � 105 cells/mouse) wereinjected s.c. into the hind limb. Mice were sacrificed 3 weeks after the injectionof the cells. The primary tumors were removed, fixed in 10% bufferedformalin, and embedded in paraffin for analysis.

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Immunohistochemistry. Formalin-fixed, paraffin-embedded tissues werecut into serial sections of 5 �m each and were used for immunohistochemistry.Sections were deparaffinized by heating to 60°C for 1 h, washed twice withxylene for 5 min, and rehydrated by consecutive washes in 100, 95, and 70%ethanol, followed by a 5-min wash in water. Endogenous peroxidase activitywas inhibited by a 15-min incubation with 3% hydrogen peroxide in methanol,followed by consecutive washes with water and PBS. The sections were thenantigen retrieved by heating them twice for 10 min in a microwave oven to90°C in citrate antigen retrieval buffer (for anticytokeratin antibodies) or inEDTA antigen retrieval buffer (Zymed; for anti-LOR-1 antibodies). Blockingof tissue sections was subsequently done using the CAS blocking solution.After blocking, the sections were incubated with affinity-purified anti-LOR-1antibody (at a dilution of 1:30 to 1:50 in antibody diluant solution), or withmonoclonal FITC-conjugated antihuman cytokeratin-7 antibodies for 1.5 h atroom temperature. The sections were washed three times with TBST, and thedetection of bound antibodies was done using anti-FITC alkaline phosphatase-conjugated antibodies (Roche) at a 1:200 dilution for cytokeratin-7 detection,or with the DAKO Envision detection system (horseradish peroxidase-conju-gated) for LOR-1 detection. Bound antibodies were detected either by using5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (Roche)for the visualization of bound anticytokeratin antibodies or by using 3-amino-9-ethylcarbazole solution (DAKO) for the visualization of bound anti-LOR-1antibodies. The slides were subsequently counterstained by hematoxylin andphotographed. In control experiments, the primary antibodies were omitted.

Histological Stains. Formalin-fixed, paraffin-embedded tissues were sec-tioned, deparaffinized, and rehydrated as described above. H&E staining wasperformed as described previously (29). Masson’s trichrome staining of fibrouscollagens and reticulin staining of collagen type 3 were performed accordingto the manufacturer’s instructions. Sirius red staining of fibrous collagen wasperformed as described previously (30).

Immunocytochemistry. To estimate the amounts of collagen type 1 incultured cells the cells were seeded in 48-well plates and grown to subconflu-ence. They were washed twice with PBS and fixed with 4% paraformaldehydeat room temperature for 15 min. After fixation, cells were washed again twicewith PBS and were permeabilized using 0.25% Triton X-100 for 15 min. Thecells were blocked in PBS containing 1% BSA for 1 h and then washed twicewith PBS. They were incubated for 90 min at room temperature with ananti-collagen type-1 antibody diluted in PBS containing 1% BSA (1:1000).After extensive washes with PBS, bound antibodies were detected using theDAKO Envision detection system.

RESULTS

LOR-1 Is Expressed in Metastatic Breast Cancer-derived CellTypes but not in Nonmetastatic MCF-7 Cells. Desmoplasia andformation of fibrotic foci in breast cancer tumors is associated withthe transition from a localized, relatively benign tumor to an invasive/metastatic tumor (14, 15). LOs contribute to the deposition of collagenby covalently cross-linking collagen monomers (1). To find outwhether expression of LOs is associated with the invasive/metastaticphenotype we screened several human breast cancer derived cell typesfor the expression of LOs. Northern blot analysis revealed that theLOR-1 gene is expressed in the malignant, estrogen-independentMDA-MB-231 and MDA-MB-435 cells (31) but not in estrogen-dependent nonmetastatic MCF-7 cells (Fig. 1A, panel A). LOR-2 onthe other hand was expressed only in MDA-MB-435 cells but not inthe MDA-MB-231 cells nor in MCF-7 cells (Fig. 1A, panel B). Theseexperiments prompted us to investigate in more detail the possiblerole of LOR-1 in the progression of breast carcinoma tumors.

Highly Malignant Cells of Poorly Differentiated (Grade 3)Breast Carcinoma Express LOR-1, whereas Well-Differentiated(Grade 1) Breast Carcinoma Cells Do Not. To determine the ex-pression patterns of LOR-1 in normal breast and in breast carcinomatumors differing in their malignancy, we screened sections derivedfrom human tumors for expression of the LOR-1 protein using anti-LOR-1 antibodies. The expression of LOR-1 in normal human breast

was confined to the epithelial cells of the ducts (Fig. 1B, panel A).Expression of LOR-1 was also detected in intraductal noninvasivecarcinoma cells (ductal carcinoma in situ) in the three tumors thatwere examined (Fig. 1B, panel B). Periductal invasive low-grade(grade 1) breast carcinoma cells form duct-like structures in theperiductal stroma. The tumorigenic cells located in these duct-likestructures did not express LOR-1 in most of the tumors that wereexamined (8 of 11; Fig. 1B, panel C, black arrows). In contrast,intraductal tumor cells that have not migrated out of the ducts pre-sumably still respond to the signals provided by the duct environmentand express LOR-1 (Fig. 1B, panel C, white arrow). However, adecisive majority (13 of 16 tumors) of highly malignant grade-3invasive ductal breast carcinomas, a more aggressive and desmoplas-tic type of tumor that is characterized by a 3-fold higher incidence ofdeath, as compared with grade-1 breast carcinoma, and a 5-fold higherincidence of recurrence after surgery (32, 33) produce large amountsof LOR-1, even though these cells are not associated with ductsanymore (Fig. 1B, panel D). Thus, a significant association existsbetween LOR-1 expression and tumor grade (P � 0.012, as deter-mined by two-tailed Fisher’s exact test). We hypothesize that highlymalignant breast carcinoma cells are able to express LOR-1, eventhough the malignant cells have lost positional signals that are re-quired by less malignant cells so as to enable LOR-1 production.

The Growth of Tumors That Develop from LOR-1-expressingMCF-7 Cells Is Retarded. To determine whether LOR-1 expressioncontributes to the progression of breast cancer tumors to the invasive/metastatic phenotype, we transfected noninvasive MCF-7 cells withan expression plasmid containing the full-length human LOR-1cDNA. Several clones of cells that expressed large amounts of LOR-1were isolated. Two such clones of cells, clones 12 and 24, were usedin additional experiments. The conditioned medium also containedshorter forms of LOR-1 as revealed by Western blot analysis usingantibodies directed against the LOR-1 c-terminal domain. Theseshorter forms may be generated by proteolytic processing in analogyto other members of the LO family (Refs. 6 and 34; Fig. 2A, panel A).Clone 12 cells expressed larger amounts of LOR-1 as compared withclone 24 cells (Fig. 2A, panel A). To make sure that the low molecularweight forms are produced as a result of posttranslational processing,we also expressed LOR-1 in MCF-7 cells under the control of atetracycline-inducible promoter (35). It can be seen that once thetetracycline inhibition is removed, the cells start to produce full-lengthLOR-1, which is then converted into a shorter, Mr 70,000 COOH-terminal-containing form (Fig. 2A, panel B). It is not yet clear whetherall of these forms are enzymatically active.

Parental MCF-7 cells and empty plasmid-transfected MCF-7 cells,as well as cells of the two LOR-1-producing MCF-7 clones, wereinjected into the mammary fat pads of female nude mice and allowedto form tumors. The parental MCF-7 cells as well as all of the clonesof cells that we derived from the parental cells formed tumors onlyin the presence of s.c. estrogen slow-release pellets (data not shown;Ref. 36). Interestingly, the rate at which tumors containing LOR-1-expressing cells developed was inhibited as compared with the rate ofdevelopment of tumors derived from parental (not shown) or emptyvector-transfected MCF-7 cells (Fig. 2B, panel A and B). The signif-icance of the differences in the sizes of the tumors was determinedusing a two-tailed Student’s t test on day 25. The differences in theaverage size of tumors that developed either from clone-12 cells orfrom clone-24 cells in comparison with the average size of tumorsderived from control cells were significant (P � 0.05 for clone-24 andP � 0.001 for clone-12). To determine whether the decreased rate oftumor growth was caused by a slower rate of cell proliferation, wealso compared the proliferation rate of empty vector-transfectedMCF-7 cells with that of clone 12 and clone 24 cells. However, we

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could not detect significant differences in their rates of proliferation(data not shown).

Tumors That Develop from LOR-1-producing MCF-7 CellsContain Many Necrotic and Fibrotic Foci Rich in Collagen De-posits. To understand better why tumors expressing LOR-1 developslowly, we examined H&E-stained sections of control and LOR-1

producing tumors. Interestingly, tumors that develop from clone 12cells (Fig. 3A, panel B) or from clone 24 cells (not shown) containmany necrotic areas, whereas tumors derived from parental (notshown) or empty expression vector-transfected cells (Fig. 3A, panelA) contain very few necrotic areas. Additionally, the LOR-1-express-ing tumors also contained extensive fibrotic areas that contained

Fig. 1. Expression of LOR-1 in breast cancer-derived cell lines and in spontaneous human breast cancer tumors. A, expression of LOR-1 and LOR-2 in human breast cancer-derivedcells. Total RNA was prepared from confluent MCF-7 cells, MDA-MB-231 cells, and MDA-MB-435 cells as described in “Materials and Methods.” Northern blot analysis using aLOR-1-specific cDNA probe (panel A) or a LOR-2 cDNA probe (panel B) was performed as described in “Materials and Methods.” Both probes also bound nonspecifically to the 28Sribosomal RNA, and it can be seen that the RNA amounts loaded in the different lanes are similar. B, Expression of LOR-1 in normal human breast and in breast cancer tumors. Apolyclonal affinity-purified antibody directed against the COOH-terminal domain of LOR-1 was used to detect expression of LOR-1 in tissue sections derived from normal human breast(panel A), ductal carcinoma in situ (panel B), invasive well-differentiated grade-1 ductal carcinoma (panel C) and poorly differentiated grade-3 ductal carcinoma (panel D) as describedin “Materials and Methods.” Shown are representative tissue sections. Strong expression of LOR-1 (red stain) is seen in the epithelium of the normal ducts (panel A, white arrow) andin tumor cells located within ducts (panels B and C, white arrows). No expression of LOR-1 is seen in the duct-like epithelial structures in the periductal stroma of grade-1 ductal breastcarcinoma (panel C, black arrows). In contrast, the tumor cells of grade-3 ductal breast carcinoma exhibit a disordered morphology and express LOR-1 (red stain). Panel A, �100;panels B–D, �200.

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mainly host-derived cells such as fibroblasts rather than MCF-7 cells.The host-derived cells were easily distinguishable from the tumorcells because they did not react with an antibody directed againsthuman cytokeratin 7, a marker of breast epithelial cells (Fig. 3A, panelC) and were only counterstained with hematoxylin (Fig. 3A, panel C;Refs. 37 and 38).

To find out whether tumors derived from LOR-1-expressingMCF-7 cells contain increased concentrations of collagen, we stainedtumor sections with Masson’s trichrome stain, which reacts mainlywith collagen type 1 producing an azure coloring (39). Tumors thatdeveloped from parental or empty vector-transfected MCF-7 cellscontained limited amounts of collagen that was scattered between thetumor cells and did not contain any fibrotic foci (Fig. 3A, panel D,arrows). In contrast, tumors that developed from clone 12 or clone 24LOR-1-producing MCF-7 cells contained much larger amounts ofdense collagen fibers between the tumor cells, indicating that LOR-1induces accumulation and deposition of collagen in the tumors (Fig.3A, panel E, arrows). Furthermore, LOR-1 expression induced theformation of numerous fibrotic foci that appeared choked with colla-gen fibers (Fig. 3A, panel F, white arrow). We have also stainedtumor sections derived from empty vector transfected MCF-7 cellsand from clone 12 and clone 24 cells with Sirius red, a dye which

stains collagen type-1 primarily and, to a much lesser extent, collagentype-3 (30). This method also revealed a large increase in collagendeposition in tumors derived from LOR-1-expressing MCF-7 cells ascompared with tumors derived from empty vector-transfected MCF-7cells (data not shown). Blood vessels within tumors that develop fromvector- transfected MCF-7 cells contain thin fluffy collagen fibersaround blood vessels (Fig. 3A, panel G, white arrow). In contrast,blood vessels found in tumors derived from LOR-1-producing MCF-7cells were sheathed by a dense layer of collagen underneath theendothelial cell layer, as revealed by Masson’s trichrome staining(Fig. 3A, panel H, black arrows). In some of the vessels, there wasalso a second, more distal sheath of thick collagen fibers around thevessels, which was indicative of perivascular fibrosis (Fig. 3A, panelH, white arrow).

These experiments indicate that LOR-1 enhances, on its own, theaccumulation and deposition of collagen in vivo. To find out whetherLOR-1 can promote collagen accumulation in other types of tumors,we expressed LOR-1 in C6-glioma cells. C6-glioma cells do notexpress fibrous collagens, and the tumors that they form on implan-tation in mice are not fibrotic (40). When we implanted C6-gliomacells transfected with empty vector in nude mice, the cells formed, asexpected, tumors that contained insignificant amounts of collagen. In

Fig. 2. The growth of tumors derived from MCF-7 cells expressing recombinant LOR-1 is inhibited. A, expression and posttranslational processing of recombinant LOR-1 in MCF-7cells. Serum-free conditioned medium (40 �l) was collected from confluent MCF-7 cells transfected with empty expression vector (Vec), or from clone 12 (c12) or clone 24 (c24)MCF-7 cells that constitutively express recombinant LOR-1 (panel A). Serum-free conditioned medium (40 �l) was also collected in 24-h intervals from MCF-7/Tet-LOR-1 cells 48 hafter the exchange of their growth medium to tetracycline-free and serum-free medium (panel B). All of the cells were grown to confluence in 24-well dishes. The aliquots of conditionedmedium were separated on an 8% SDS-PAGE gel, and the proteins were electroblotted onto nitrocellulose filters using semidry electroblotting. Western blot analysis using an antibodydirected against the COOH-terminal domain of human LOR-1 was performed as described in “Materials and Methods.” B, in vivo growth rate of tumors derived from control orLOR-1-expressing MCF-7 cells: empty vector-transfected (Vec), clone 24 (c24), and clone 12 (c12). MCF-7 cells were injected into the mammary fat pads of female athymic nudemice as described in “Materials and Methods” (107 cells/mouse). Each cell type was implanted into eight animals. Tumor area was measured at the indicated times. Error bars, theSD of the mean. The experiment was terminated and the mice sacrificed when the tumor reached a diameter of �0.8 cm (panel A). Panel B, representative mice harboring tumors thatdeveloped for 25 days from empty vector-transfected MCF-7 cells (VEC), from clone 24 cells (c24), or from clone 12 cells (c12); arrows, tumors.

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contrast, the tumors that developed from C6-glioma cells expressingrecombinant LOR-1 contained large deposits of collagen fibers asrevealed by Masson’s trichrome staining, especially at the invasivefront of the tumors (data not shown). It therefore follows, that theinduction of collagen deposition by LOR-1 is a general property thatis not specific to MCF-7 cells or to breast cancer. The tumors thatdeveloped from either LOR-1-producing MCF-7 cells or from LOR-1-producing C6-glioma cells also contained much higher concentra-tions of collagen-type 3 fibers (Fig. 3B, panels B and D) in compar-ison with tumors that developed from empty vector-transfected cells(Fig. 3B, panels A and C), as revealed by reticulin staining (41). Inaddition, the collagen-type 3-containing fibers in the tumors thatdeveloped from the LOR-1-expressing cells also appeared to be muchthicker than collagen-type 3 fibers in control tumors (Fig. 3B). Theseresults indicate that LOR-1 can affect simultaneously the depositionof several types of collagen. The source of the deposited collagen isunclear at this point. However, it is likely to be produced by the cellsof the host mice because LOR-1-expressing MCF-7 cells that aregrown in culture do not produce significant amounts of collagen (datanot shown).

MCF-7 Cells Expressing LOR-1 Form Highly Invasive Tumorsin Vivo. It was reported that the appearance of fibrotic foci in breastcancer tumors correlates with their degree of invasiveness (15). Be-cause LOR-1-producing MCF-7 cells form tumors rich in fibroticareas, we also asked whether the LOR-1-expressing cells that formthese tumors acquire invasive properties. Tumors that developed afterthe implantation of empty vector-transfected MCF-7 cells in nudemice were surrounded by a thick pseudocapsule. The border betweenthe tumor and the pseudocapsule in these tumors was sharply defined,and the MCF-7 cells did not migrate out of the tumors into thepseudocapsules (Fig. 4, A and B). We could not detect tumorigeniccells within blood vessels located adjacent to these control tumors, norwere there tumorigenic cells observed in the perineural space ofneighboring nerves and between muscle located near the tumors (Fig.4, A and B). In contrast, MCF-7 cells expressing recombinant LOR-1were able to invade the pseudocapsules of the tumors that developedafter their implantation in nude mice (Fig. 4C, arrows). The LOR-1-expressing tumor cells infiltrated the pseudocapsules of these tumorsand invaded adjacent tissues. Tumor cells invading muscles adjacentto the tumor were identified using antibodies directed against humancytokeratin-7 (Fig. 4D, dark blue stain, black arrows) as well as withan antibody directed against LOR-1 (Fig. 4E, red stain, white arrows).In addition, we have also observed tumor cells invading the vascularsystem (Fig. 4, F and G, arrows) as well as the perineural space ofadjacent nerves (Fig. 4H, arrow). These last characteristics are hall-marks of metastatic tumor cells (42, 43). These observations provideevidence indicating that the production of LOR-1 by breast cancertumor cells contributes to the transition from the noninvasive stage ofbreast cancer to the invasive/metastatic stage.

DISCUSSION

The appearance of fibrotic foci in breast carcinoma tumors hadbeen linked to high malignancy, metastasis, and poor prognosis (14,

15). We have shown here for the first time that overexpression ofLOR-1, a recently identified LO family member, in MCF-7 breastcancer cells and in C6-glioma cells induces the deposition of largeamounts of dense collagen fibers (desmoplasia) in the tumors thatdevelop after the implantation of these cells in nude mice. Theseexperiments demonstrate that the expression of a LO family membersuffices to induce collagen accumulation and fibrosis in vivo. Further-more, we have shown that LOR-1 expression enhances the invasive-ness of the MCF-7 cells, and that LOR-1 is likely to contribute to theemergence of metastasis because the LOR-1-expressing MCF-7 cellsalso display characteristics that are usually associated with metastaticcells, such as the ability to invade the vascular system. Taken together,these observations argue for a role of LOs in the progression of breastcancer and show for the first time that the expression of a singleprotein can induce simultaneously fibrosis and invasiveness, twoprocesses that had been previously observed to be linked in theprogression of spontaneous breast cancer tumors (14, 15).

We have found that cells of well-differentiated grade-1 ductalcarcinomas that have migrated out of the ducts do not usually produceLOR-1. In contrast, malignant grade-3 ductal carcinoma cells locatedoutside of the ducts do produce large amounts of LOR-1. We hypoth-esize that the ducts produce a signal that induces LOR-1 production inthe epithelial cells of the ducts, and that this signal is absent in theperiductal stroma. If our hypothesis is correct, than the well-differen-tiated grade-1 ductal carcinoma cells that are located in the periductalstroma lose their ability to express LOR-1 because the signal thatinduces LOR-1 production is missing, whereas adjacent tumor cellslocated within the ducts still express LOR-1 (Fig. 1B, panel C). Thebasement membrane is known to provide signals that are essential forthe proper differentiation and function of the epithelial cells lining theducts (44, 45). It is, therefore, conceivable that such a localized signalwill be provided by the basement membrane. We hypothesize that,once the carcinoma cells lose contact with the duct environment, theirLOR-1-producing ability is tightly linked to their degree of malig-nancy. Thus, the highly malignant cells of grade-3 ductal breastcarcinomas seem to express LOR-1 in the vast majority of tumorsregardless of their location, whereas the less malignant cells ofgrade-1 ductal breast carcinoma fail to produce LOR-1 in most tumorsonce they lose contact with the duct environment. Interestingly, it waspreviously observed that highly malignant grade-3 carcinomas usuallycontain fibrotic foci, whereas the less malignant grade-1 carcinomasdisplay a lower incidence of fibrotic foci (46).

The differentiated epithelial cells located within the ducts and in thelobules of the breast normally produce LOR-1, as do tumorigenic cellsof in situ ductal carcinomas. We could not detect LOR-1 expressionin the stromal cells surrounding the ducts. This expression patterndiffers dramatically from the reported expression patterns of LO andLOL, which are not expressed by the tumor cells of ductal carcinomasin situ but are expressed by stromal cells surrounding the ducts (23,47). Interestingly, production of LOR-1 by the normal epithelial cellsof the duct or by cells of in situ ductal carcinomas is not accompaniedby fibrosis (data not shown), nor are these cells invasive. We hypoth-esize that LOR-1-induced fibrosis occurs only when LOR-1-produc-

Fig. 3. Tumors arising from LOR-1-expressing cells contain many fibrotic and necrotic foci rich in collagen deposits. A, Tumors derived from LOR-1 expressing MCF-7 cells containmany fibrotic foci and collagen type-1 deposits. Tumors derived from empty vector-transfected MCF-7 cells (panels A, D, and G) or from MCF-7 cells expressing recombinant LOR-1(panels B, C, E, F, and H) were stained with H&E (panels A and B) or with Masson’s trichrome stain (azure stain; panels D–H) as described in “Materials and Methods.” A monoclonalantibody specific for human cytokeratin-7 was used to differentiate between MCF-7-derived tumor cells and host cells as described in “Materials and Methods” (panel C; deep bluestain). Black arrows, necrotic and fibrotic areas (panels A and B), host cells in fibrotic foci (panel C), collagen bundles between cells (panels D and E), and collagen deposits underneathendothelial cells of blood vessels (panel H). White arrows, collagen fibers in fibrotic area (panel F) or the outer thick collagen sheath deposited around blood vessel in LOR-1-expressingtumors (panel H). Similar results were obtained using tissue sections from 4–6 different tumors derived from independent LOR-1 expressing or empty vector-transfected clones of cells.A and B, �20; D–F, �200; C, �40; G–H, �400. B, tumors derived from LOR-1-expressing MCF-7 cells or C6-glioma cells contain a high concentration of collagen type-3 fibers.Tumor sections derived from empty vector-transfected MCF-7 cells (panel A), LOR-1 producing clone 12 MCF-7 cells (panel B), empty vector-transfected C6-glioma cells (panel C)or LOR-1-producing C6-glioma cells (panel D) were stained for collagen type-3 using reticulin stain as described in “Materials and Methods.” �200.

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ing cells come into close contact with stromal collagen-producingcells. Likewise, invasiveness may also be potentiated by LOR-1indirectly, as a consequence of the induction of fibrosis, thus linkingthe two processes. Alternatively, it is possible that invasiveness isinduced by LOR-1 using a separate mechanism, and that normal,LOR-producing cells possess inhibitory mechanisms that hinderLOR-1-induced invasiveness. However, because a link has beenfound between the presence of fibrotic foci in spontaneous breastcancer tumors and poor prognosis (14, 15), it is more likely that thesetwo processes are somehow interdependent. This would seem, at firstglance, to be a paradox, because many studies have established a linkbetween the production of ECM degrading proteases and tumor in-vasiveness (18–20). How then can an enzyme that apparently inducesthe deposition of ECM proteins and is, therefore, expected to inhibittumor invasiveness, enhance invasiveness instead? One possible ex-planation is that the production of a great excess of ECM proteins canin turn trigger expression of degrading enzymes that will then over-come local inhibitory effects of the deposited ECM. Alternatively, the

fibrosis-inducing activity and the invasiveness-enhancing activities ofLOR-1 may be induced by LOR-1 simultaneously and independently.

We have not been able to detect collagen production by culturedcontrol MCF-7 cells nor could we detect collagen production by theLOR-1-expressing MCF-7 cells grown in conventional cell culture(data not shown). It, therefore, seems likely that the collagen seen intumors developing from LOR-1-producing MCF-7 cells or fromLOR-1-producing C6-glioma cells originates from stromal host cells.It was shown that LO can activate the collagen-3A promoter (5), andit is, therefore, possible that LOR-1 can function as a signalingmolecule to induce collagen synthesis in stromal host cells by aparacrine mechanism.

Interestingly, the development of tumors from LOR-1-expressingMCF-7 cells was inhibited as compared with tumors developing fromcells that do not express LOR-1. LO was shown to behave as a tumorsuppressor that inhibits the expression of ras (48, 49). It is possiblethat LOR-1 expression affects tumor development similarly, althoughthe high-level expression of LOR-1 in grade-3 ductal carcinoma and

Fig. 4. Tumors derived from MCF-7 cells expressing recombinant LOR-1 are invasive. Shown are histological sections of tumors that developed from empty vector-transfectedMCF-7 cells (A and B) or from tumors that developed from MCF-7 cells expressing recombinant LOR-1 (C–H). The tumors developed from cells implanted into the mammary fat padsof nude mice. The histological sections were labeled with a monoclonal antibody specific for human cytokeratin-7 (A–D and F–H; blue purple stain) or with an antibody directed againstLOR-1 (E, red stain) as described in “Materials and Methods.” Black arrows, tumor cells labeled with an antibody directed against human cytokeratin-7. White arrows, tumor cellslabeled with an antibody directed against LOR-1. Counterstaining was performed using hematoxylin (light blue). Tumor cells of control tumors do not invade the capsule nor do theyinvade blood vessels, nerves, or muscles located near the tumor (A and B). In contrast, tumor cells expressing recombinant LOR-1 invade the pseudocapsule of the tumors (C), infiltratebetween adjacent muscles (D and E), invade the tumor-associated vasculature (F and G) and the perineural space of nerves adjacent to the tumor (H). Similar results were obtainedusing tissue sections from four-to-six different tumors derived from independent LOR-1-expressing or empty vector-transfected clones of cells. v, blood vessels; n, nerves; m, musclefibers; c, pseudocapsule of tumor. A–C, F, and H, �100; D, E, and G, �200.

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the lack of an effect on the in vitro proliferation of the LOR-1-expressing MCF-7 cells seems to conflict with this hypothesis. Thesetumors also contained abundant necrotic areas. This observation ispuzzling in that usually tumors that expand quickly are more likely todevelop necrotic areas, because angiogenesis cannot catch up with therapid expansion of the tumor (50). In the case of the LOR-1-express-ing tumors, it is possible that the collagen deposits inhibit angiogen-esis and lead to the formation of necrotic areas. Indeed, the necroticareas were usually observed near the centers of fibrotic foci (see Fig.3A, panels B and F). In addition, we have also seen that tumorscontaining LOR-1-producing cells contained blood vessels that weresurrounded by a thick sheath of collagen fibers. Such coating may alsocontribute to the inhibition of tumor angiogenesis and favor theformation of necrotic areas. Another contributor to the formation ofnecrotic areas in LOR-1-producing tumors may be hydrogen peroxide,a toxic side product of LO activity (51). It was shown that hydrogenperoxide can cause apoptosis and death of MCF-7 cells (52, 53).Hydrogen peroxide concentrations would probably reach their highestconcentrations in the areas in which the largest concentrations ofcollagen can be found, because, in such areas, the activity of LOR-1would be expected to be maximal, and the necrotic areas in the tumorswere indeed located usually at the center of fibrotic foci.

The recent discovery of five distinct enzymes that contain a LO-likecatalytic domain raises questions regarding their substrate specificityand biological roles. The collagen types that were deposited as a resultof the expression of LOR-1 included both collagen type 1 and colla-gen type 3. LO, LOL, and LOX-C are known to oxidize lysineresidues on collagen type-1 (34, 54, 55). LOR-1 can also oxidizelysine residues on collagen type 1.5 It is, therefore, likely that theseenzymes overlap in their biological functions. It is, thus, likely thatadditional enzymes belonging to this family besides LOR-1 contributeto the induction of fibrosis and to tumor progression in breast cancer,as well as in other malignancies in which there is some evidencelinking abnormal fibrosis with tumor progression (16, 17). This pos-sibility is supported by observations that indicate that metastaticbreast cancer-derived MDA-MB-435 cells express both LOR-1 andLOR-2 (Fig. 1), whereas MDA-MB-231 cells were found to expressLO in addition to LOR-1 (24). It is, therefore, likely that the coex-pression of several LO family members may produce synergisticeffects and, thus, enhance fibrosis and invasiveness more potentlythan in cases in which only one LO family member is produced.

To conclude, our experiments indicate that LOR-1 is a potentinducer of tumor fibrosis and an enhancer of tumor invasiveness.Furthermore, the expression of LOR-1 in spontaneous tumors isassociated with higher tumor grade and malignancy. It remains to beseen whether it would be possible to inhibit the progression of breastcancer tumors using LOR-1 inhibitors.

ACKNOWLEDGMENTS

We thank Sharon Soueid for excellent technical assistance. We thank AssafGilad for his help with immunohistochemistry and for helpful discussions.

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LOR-1 INDUCES TUMOR PROGRESSION AND FIBROSIS

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2003;63:1657-1666. Cancer Res   Gal Akiri, Edmond Sabo, Hagit Dafni, et al.  

in Vivoand Tumor Progression Lysyl Oxidase-related Protein-1 Promotes Tumor Fibrosis

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