A Molecular Role for Lysyl Oxidase in Breast Cancer Invasion1

[CANCER RESEARCH 62, 4478–4483, August 1, 2002] A Molecular Role for Lysyl Oxidase in Breast Cancer Invasion1

Dawn A. Kirschmann,2 Elisabeth A. Seftor, Sheri F. T. Fong, Daniel R. C. Nieva, Colleen M. Sullivan, Elijah M. Edwards, Pascal Sommer, Katalin Csiszar, and Mary J. C. Hendrix Department of Anatomy and Cell Biology, Holden Comprehensive Cancer Center at The University of Iowa, The Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1109 [D. A. K., E. A. S., D. R. C. N., C. M. S., E. M. E., M. J. C. H.]; Institut de Biologie et Chimie des Prote´ines, Lyon Cedex 07, France [P. S.]; and Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822 [S. F. T. F., K. C.]

ABSTRACT LOX is a copper-dependent amine oxidase that initiates the cova- lent cross-linking of collagens and elastin in extracellular matrices We identified previously an up-regulation in lysyl oxidase (LOX) ex- (reviewed in Refs. 3, 4). It is secreted as a M 50,000 glycosylated pression, an extracellular matrix remodeling enzyme, in a highly invasive/ r proenzyme, which is proteolytically processed by procollagen C pro- metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this teinase (bone morphogenic protein-1) into a mature, biologically study, we demonstrate that the mRNA expression of LOX and other LOX active Mr 32,000 form (3, 4). The formation of collagen/elastin family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and cross-links by LOX leads to an increase in tensile strength and LOXL4] was observed only in breast cancer cells with a highly invasive/ structural integrity, and is essential for normal connective tissue metastatic phenotype but not in poorly invasive/nonmetastatic breast function, embryonic development, and wound healing (3, 5). Conse- cancer cells. LOX and LOXL2 showed the strongest association with quently, aberrant LOX expression or enzymatic activity leads to invasive potential in both highly invasive/metastatic breast cancer cell disease. Decreases in LOX expression or activity have been associated lines tested (MDA-MB-231 and Hs578T). To determine whether LOX with such heritable connective tissues disorders as Ehler-Danlos syn- is directly involved in breast cancer invasion, LOX antisense oligo- drome, cutis laxa, and Menkes’ syndrome (6–10). Increases in LOX nucleotides were transfected into MDA-MB-231 and Hs578T cells, and expression contribute to the development of fibrotic diseases such as found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated arteriosclerosis, scleroderma, and liver cirrhosis, diseases that involve controls. In addition, treatment of MDA-MB-231 and Hs578T cells with connective tissue remodeling (11–14). ␤-aminopropionitrile (an irreversible inhibitor of LOX enzymatic Although the ECM maturation activity of LOX has long been activity) decreased invasive activity. Conversely, MCF-7 cells transfected with thought to be its sole function, more recent evidence implicates the the murine LOX gene demonstrated a 2-fold increase in invasiveness that was involvement of LOX in many important biological functions other reversible by the addition of ␤-aminopropionitrile in a dose-dependent than collagen/elastin cross-linking. LOX has been shown to induce manner. In addition, endogenous LOX mRNA expression was induced motility and migration in monocytes, vascular smooth muscle cells, when MCF-7 cells were cultured in the presence of fibroblast conditioned and fibroblasts (15–17). In addition, LOX may play a role in cell medium or conditioned matrix, suggesting a role for stromal fibroblasts in growth/differentiation as its expression is up-regulated by a number of LOX regulation in breast cancer cells. Moreover, the correlation of LOX growth factors and steroids (3, 4, 18). Moreover, a role for LOX in up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal transcriptional gene regulation and cellular transformation has also melanoma cell lines. These results provide substantial new evidence that been implicated as evidenced by localization of LOX protein and LOX is involved in cancer cell invasion. enzymatic activity to cell nuclei (19), potential utilization of histone H1 as a substrate (20), activation of the collagen III ␣1 promoter (21), and a putative tumor suppressor in nontumorigenic revertants of INTRODUCTION ras-transformed fibroblasts (reviewed in Ref. 3). In this study, we test the hypothesis that LOX expression contrib- In 2002, breast cancer is predicted to account for 31% of all of the utes to cellular invasive ability. The data demonstrate that LOX and diagnosed cancers in women, more than twice that of the second most other LOXL family members are specifically expressed by invasive/ common cancer (lung cancer; Ref. 1). Because metastasis is a major metastatic cancer cells compared with poorly invasive/nonmetastatic challenge in cancer management, it is critical to determine molecular cancer cells, and that modulation of LOX expression and function in markers that definitively distinguish tumors of nonmetastatic potential breast cancer cells affects in vitro cellular invasive activity. from those with metastatic potential. To identify such markers, a clearer understanding of the metastatic progression in breast cancer is MATERIALS AND METHODS required. To this end, we identified an up-regulation in LOX3 expression in invasive/metastatic breast cancer cell lines compared with Cells and Culture Conditions. MCF-7 cells were kindly supplied by Dr. poorly invasive/nonmetastatic breast cancer cell lines (2). F. Miller (Karmanos Cancer Institute, Detroit, MI), and MDA-MB-231, T47D, and Hs578T cell lines were obtained from the American Type Culture Col- Received 2/15/02; accepted 5/23/02. lection (Manassas, VA). The cutaneous melanoma A375P and C8161 cell lines The costs of publication of this article were defrayed in part by the payment of page were kind gifts from Drs. I. J. Fidler (M.D. Anderson Cancer Center, Univer- charges. This article must therefore be hereby marked advertisement in accordance with sity of Texas, Houston, TX) and F. L. Meyskens, Jr. (University of California, 18 U.S.C. Section 1734 solely to indicate this fact. Irvine Cancer Center, Orange, CA), respectively. The uveal melanoma cell line 1 Supported by DAMD17-99-1-9225 (to D. A. K.), University of Iowa College of Medicine Grant (to D. A. K.), NIH/National Cancer Institute CA59702, Order of Eastern OCM-1 was kindly provided by Dr. J. Kan-Mitchell (Karmanos Cancer Insti- Star Breast Cancer Research Fund (to M. J. C. H.), NIH CA776580, and AR47713 tute), and M619 and C918 uveal melanoma cell lines were a kind gift from (to K. C.) Dr. K. Daniels (University of Iowa, Iowa City, IA). Human Rb foreskin 2 To whom requests for reprints should be addressed, at Department of Anatomy and fibroblasts were a generous gift from Dr. Gregory Goldberg (Washington Cell Biology, 1-100 BSB, University of Iowa, 51 Newton Road, Iowa City, IA 52242- Ј Ј Ј 1109. Phone: (319) 335-7655; Fax: (319) 335-7770; E-mail: dawn-kirschmann@ University, St. Louis, MO). Rat prostate cancer cell lines (5 P, 5 A, and 5 B) uiowa.edu. were derived and established from the R-3327 Dunning tumor in our labora- 3 The abbreviations used are: LOX, lysyl oxidase; Rb, foreskin fibroblast cell line; tory. Cell lines were maintained as described previously (22) and were har- ␤ ␤ LOXL, lysyl oxidase-like; ECM, extracellular matrix; APN, -aminopropionitrile; RT- vested when cultures were ϳ80% confluent. PCR, reverse transcriptase-PCR; MICS, membrane invasion culture system; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hLOX, human LOX; mLOX, murine LOX; RNA Isolation. Total RNA was isolated from breast cancer cell lines using aa, amino acid; cm, conditioned medium; cmtx, conditioned matrix. TRIzol RNA isolation reagent (Invitrogen), according to manufacturer’s spec- 4478

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. LYSYL OXIDASE IN BREAST CANCER INVASION ifications. Polyadenylated RNA was isolated from total RNA by binding to centrifuged (2000 rpm, 10 min), filtered (0.22 ␮m) to remove residual cells and oligodeoxythymidine cellulose (Becton Dickinson, Bedford, MA), as de- cellular debris, and stored at Ϫ20°C until use. Conditioned matrices were scribed previously (23). generated by coating six-well tissue culture plates with 122 ␮g of rat tail Semiquantitative RT-PCR Analysis. Reverse transcription of total RNA collagen I (BD Bioscience) and polymerizing with 100% ethanol for 5 min at from breast, prostate, and melanoma cancer cell lines was performed using the room temperature. Matrices were rehydrated with 1 ml of PBS for 5 min at Advantage PCR kit according to manufacturer’s instructions (BD Clontech, room temperature, and 5 ϫ 105 Rb fibroblasts were added to each well in

Palo Alto, CA). PCR amplification was performed with LOX- and LOXL2- complete medium and cultured for 3 days at 37°C, 5% CO2. Wells were treated Ј specific primers: hLOX [accession S78694 forward: 5 -GATCCTGCTGATC- with 20 mM NH4OH for 10 min at room temperature to remove the cells and CGCGACAA-3Ј (bases 500–520); reverse: 5Ј-GGGAGACCGTACTG- subsequently washed three times with sterile water and two times with PBS. GAAGTAGCCAGT-3Ј (bases 843–868)], rat LOX [accession U11038 MCF-7 cells (5 ϫ 105 cells/well) were then added to conditioned matrices for forward: 5Ј-CAACCCGGAAATTACATTCTAAAGGTCAGTG-3Ј (bases an additional 3 days. Total RNA was harvested by adding 1 ml of TRIzol 1376–1405); reverse: 5Ј-CCTTCCTCAGCACCAAGTGTATCC-3Ј (bases reagent to each well. 1986–2009)], and hLOXL2 [accession NM_002318 forward: 5Ј-GGCCGC- Ј Ј CACGCGTG GATC-3 (bases 2093–2110); reverse: 5 -CCCAAGGGTCAG- RESULTS GTAGCAGCCCC-3Ј (bases 2752–2774)]. Annealing temperature and number of amplification cycles were optimized at 64°C and 30 cycles for LOX, and LOX Expression in Breast Cancer Cell Lines. Using differential 68°C and 27 cycles for LOXL2, respectively in a Robocycler gradient 96 display analysis, we identified previously an up-regulation in LOX thermocycler (Stratagene, La Jolla, CA) under the following conditions: 1 mRNA expression in MDA-MB-231 (highly invasive/metastatic) cycle at 94°C for 1 min; 27 or 30 cycles at 94°C for 1 min, 64°Cor68°C for compared with MCF-7 (poorly invasive/nonmetastatic) breast cancer 2.5 min, 72°C for 1 min; and 1 cycle at 72°C for 5 min. GAPDH primers (BD cell lines (2). Here we confirm, by RT-PCR analysis using LOX- Clontech) were used as controls for PCR amplification. PCR fragments were ligated into the pCR2.1-TOPO sequencing vector as per manufacturer’s in- specific primers, that LOX expression is up-regulated specifically in structions (Invitrogen). Plasmid DNA was isolated and subjected to DNA breast cancer cell lines with a highly invasive/metastatic potential sequence analysis using fluorescent Sanger-based dideoxy sequencing on an (Hs578T and MDA-MB-231) and not in poorly invasive/nonmeta- ABI 373A Automated Sequencer (University of Iowa DNA Facility). Two static breast cancer cell lines (T47D and MCF-7; Fig. 1A), suggesting plasmids were sequenced, and each showed 100% identity to the reported LOX a putative role for LOX in breast cancer cell invasive and metastatic DNA sequences. potential. RNA Hybridization Analysis. Northern blot analysis was used to deter- In addition, the up-regulation of LOX mRNA expression observed mine LOXL, LOXL2, LOXL3, and LOXL4 expression in breast cancer cell in highly invasive/metastatic breast cancer cells does not appear to be 32 lines, as described previously (2). P-labeled LOXL- (24), LOXL2- (25), limited to just LOX but is observed with other members of this LOXL3-, (1.2 kb of the 5Ј region), and LOXL4-specific (26) cDNA were used growing amine oxidase family. As shown in Fig. 1B, mRNA expres- as probes. Blots were exposed to a phosphor screen for 12–36 h and then scanned using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). sion of LOXL, LOXL2, and the newest identified members of the No cross-hybridization between LOXL family members was observed. Stable and Transient Transfections. A mammalian expression vector containing the mLOX gene (27) was stably transfected into MCF-7 cells using LipofectAMINE reagent according to manufacturer’s instructions (Invitrogen). Transient transfections with phosphorothioate-modified LOX-specific antisense (5Ј-CCAGGCGAAGCGCATCAC-3Ј) or sense (5Ј-GTGATGCGCT- TCGCCTGG-3Ј) oligonucleotides (Integrated DNA Technologies, Coralville, IA) were performed using LipofectAMINE reagent according to manufacturer’s specifications. Briefly, breast cancer cell lines (5 ϫ 105 cells/well) were seeded onto six-well tissue culture plates. Cells were washed with Opti-MEM medium (Invitrogen) and incubated with 1 ␮M sense or antisense oligonucleotide/ LipofectAMINE complexes or with LipofectAMINE alone. After 5 h, serum containing medium was added and cultures were incubated an additional 24 h at 37°C, 5% CO2 before harvesting cells for in vitro invasion analysis. Cy5-labeled LOX antisense oligonucleotides were subjected to the same conditions to determine the transfection efficiency for each cell line, which were approximately: 95% for MDA-MB-231, 90% for T47D, 80% for MCF-7, and 50% for Hs578T cells. In Vitro Invasion Analysis. Analysis and quantitation of the in vitro invasive phenotype of breast cancer cell lines was performed using the MICS as described previously (28). Where indicated, breast cancer cells were pre- treated with 100 or 200 ␮M ␤APN (Sigma, St. Louis, MO) for 24 h before and during invasion analysis. Statistical significance was evaluated by ANOVA using Microsoft Excel 2000 spreadsheets. Immunofluorescence. mLOX-FLAG-transfected MCF-7 cells (5 ϫ 104 cells/well) were seeded onto 12-mm round glass coverslips in a 24-well tissue culture plate. Cells were fixed with ice-cold methanol for 5 min and blocked with PBS (pH 7.4) containing 1% BSA (Sigma) for1hatroom temperature. Anti-FLAG M2 antibody (20 ␮g/ml; Sigma) was added to fixed cells for 1 h at room temperature. A rhodamine-conjugated goat antimouse IgG secondary Fig. 1. Expression of LOX and LOXL mRNA in breast cancer cell lines. A, RT-PCR antibody (ICN Pharmaceuticals, Aurora, OH) was added and incubated at analysis of LOX mRNA expression in poorly invasive/nonmetastatic breast cancer cell room temperature for 1 h. Between each step, cells were gently washed three lines (MCF-7 and T47D) and in highly invasive/nonmetastatic breast cancer cell lines times with PBS. Coverslips were mounted onto glass slides for analysis by (Hs578T and MDA-MB-231) using LOX-specific PCR primers. GAPDH-specific primers fluorescence microscopy. were used as a control for equal loading. B, RNA hybridization analysis of LOX, LOXL, LOXL2, LOXL3, and LOXL4 mRNA expression in MCF-7, T47D, Hs578T, and MDA- Cm and Cmtx. Cm from MCF-7 cells and Rb fibroblasts were harvested MB-231 cell lines using random primed, 32P-labeled LOX, LOXL, LOXL2, LOXL3, and from ϳ1 ϫ 106 cells grown in complete medium for 3 days. The cm was LOXL4-specific probes. ␤-actin expression was used as a control for equal loading. 4479

␤APN (a specific irreversible inhibitor of LOX enzymatic activity; Ref. 30), and their in vitro invasive potential was assessed. It is important to note that the inhibition of LOX enzymatic activity by ␤APN is never complete and that ϳ20% of enzyme activity still remains under in vitro conditions (31). A significant inhibition in invasive ability was observed with the addition of ␤APN to Hs578T and MDA-MB-231 cells (Fig. 2B). The inhibition in Hs578T and MDA-MB-231 invasion by ␤APN could not be attributed to ␤APN toxicity, as no difference in the percentage of viability was detected by trypan blue exclusion (data not shown). Moreover, addition of ␤APN to MCF-7 and T47D cells (which do not express LOX) had no effect on invasion compared with untreated controls (Fig. 2B). These results demonstrate that LOX enzymatic activity is required for breast cancer cell invasion of an ECM in vitro. Expression of LOX in MCF-7 Cells Increases Invasive Poten- tial. To determine whether the induction of LOX expression can increase invasive potential, poorly invasive/nonmetastatic MCF-7 cells were stably transfected with a mammalian expression vector containing the mLOX-FLAG fusion protein. The mLOX mature protein has a 94% amino acid identity to the hLOX mature protein. Furthermore, the copper binding (aa 280–290) and active sites (aa 352–368) in mLOX have 100% and 99% amino acid identity to hLOX (aa 286–290 and aa 358–374), respectively, suggesting that mLOX functions similarly to hLOX. In addition, the mLOX-FLAG protein is enzymatically active in 3T6–5 fibroblasts (27). Immunofluorescence staining of mLOX-FLAG-expressing MCF-7 cells with an anti-FLAG antibody demonstrated punctate LOX-FLAG fusion protein expression that localized to the cytoplasm (Fig. 3A). Localization of LOX- FLAG was similar to that observed in 3T6-5 fibroblasts (data not shown). Fig. 2. Suppression of LOX mRNA expression and enzymatic activity inhibits invasive As is shown in Fig. 3B, stable transfection of mLOX in MCF-7 cells potential. A, MCF-7, T47D, Hs578T, and MDA-MB-231 cell lines were transiently significantly increased their in vitro invasive potential by 2-fold transfected with LOX antisense and sense phosphorothioate-modified oligonucleotides compared with untransfected MCF-7 cells. The invasive phenotype (using LipofectAMINE reagent) for 24 h before invasion analysis. Untreated and treated cells were seeded into the upper wells of a MICS chamber, and after 24 h invading cells induced by mLOX expression in MCF-7 cells was reversible, in a were harvested from the lower wells. Percentage of invasion was calculated as the total dose-dependent manner, by addition of ␤APN demonstrating that the ϫ number of invading cells/total number of cells seeded 100. B, MCF-7, T47D, Hs578T, increase in invasion was mediated by catalytically active LOX. These and MDA-MB-231 cell lines were treated with 100 or 200 ␮M ␤APN (an irreversible inhibitor of LOX enzymatic activity) for 24 h before and during in vitro invasion analysis, results show that LOX expression can potentiate cellular invasion; denotes statistical significance by ANOVA (A, P ϭ 0.009 for however, other genes are apparently necessary to obtain an invasive ء .as described above Hs578T and P ϭ 0.017 for MDA-MB-231; B, P ϭ 0.002 for Hs578T and P ϭ 8.0 ϫ 10Ϫ5 for MDA-MB-231); bars, Ϯ SD. potential similar to Hs578T and MDA-MB-231 cells. Up-Regulation of Endogenous LOX and LOXL2 Expression in MCF-7 Cells. To determine whether endogenous LOX expression LOX family, LOXL3 (29) and LOXL4 (26), was also observed in the could be up-regulated in MCF-7 cells by either soluble factors or invasive/metastatic cell lines but not in the poorly invasive cell lines. “cues” obtained from the ECM, MCF-7 cells were cultured for 3 days However, their expression was somewhat disparate between the in- with either Rb fibroblast cm or on a Rb fibroblast cmtx. As is shown vasive/metastatic cell lines (Hs578T and MDA-MB-231). These re- in Fig. 4A, endogenous LOX expression in MCF-7 cells was dose- sults suggest that LOX and LOXL2 have the strongest association dependently up-regulated when cultured with increasing concentra- with an invasive/metastatic phenotype in the breast cancer cell lines tions of Rb fibroblast cm but not in MCF-7 cm. Similarly, endogenous tested. LOX expression was up-regulated in MCF-7 cells that were cultured Transfection of Antisense LOX Oligonucleotides Inhibits Inva- on a Rb fibroblast cmtx. The up-regulation in LOX expression could sion. To directly test whether LOX expression is involved in cellular not be attributed to carry over of LOX RNA or DNA from Rb invasion of an ECM, LOX-specific sense and antisense oligonucleo- fibroblasts, as no LOX expression was detected in Rb cmtx alone. tides were transiently transfected into MCF-7, T47D, Hs578T, and Furthermore, endogenous LOX expression was not induced in MCF-7 MDA-MB-231 cell lines, and their in vitro invasive potential was cells cultured on a collagen I matrix alone (data not shown). In assessed. As shown in Fig. 2A, LOX antisense oligonucleotides but addition, LOXL2 expression was also up-regulated in MCF-7 cells not LOX sense oligonucleotides were capable of significantly inhib- cultured on a Rb fibroblast cmtx (Fig. 4B). These results suggest that iting Hs578T and MDA-MB-231 invasive ability in vitro. No appre- fibroblasts and possibly stromal cells in general can contribute to ciable effect on invasive potential was observed with either LOX LOX and LOXL2 regulation in breast cancer cells. sense or antisense oligonucleotide-treated MCF-7 and T47D cells. LOX and LOXL2 Expression in Other Cancers. To determine These results suggest that LOX expression has an effect on cellular whether the up-regulation of LOX and LOXL2 expression was unique invasion. to breast cancer, other cancer cell lines that are well characterized in ␤APN Inhibition of LOX Activity Inhibits Invasion. To deter- our laboratory were analyzed (32, 33). As shown in Fig. 5, A and C, mine whether LOX enzymatic activity is required for cellular inva- expression of LOX mRNA was absent in poorly invasive cutaneous sion, breast cancer cells were treated with increasing concentrations of (A375P) and uveal (OCM-1A) human melanoma cell lines compared 4480

metastatic breast cancer cells. Furthermore, we demonstrate that an inhibition of LOX expression and enzymatic activity in invasive/ metastatic breast cancer cell lines reduced their invasive potential. Conversely, experimental up-regulation of LOX expression in a poorly invasive/nonmetastatic breast cancer cell line increased invasive potential, which is reduced on inhibition of LOX enzymatic activity. These results are consistent with our hypothesis that LOX enzymatic activity contributes to cellular invasive ability. At first glance our results appear to contradict published literature regarding LOX expression in tumor cell lines; however, an in-depth

Fig. 4. Endogenous LOX and LOXL2 expression is induced in MCF-7 cells. A, RT-PCR analysis of LOX mRNA in MCF-7 cells cultured for 3 days either in the presence of 25 or 35% Rb fibroblast or MCF-7 cm, or on a Rb fibroblast cmtx. B, RT-PCR analysis of LOXL2 mRNA in MCF-7 cells cultured for 3 days on a Rb cmtx. Cmtx was generated by culturing Rb fibroblasts on rat tail collagen I for 3 days after which time the cells were Fig. 3. LOX expression induces an invasive phenotype in MCF-7 cells. A, MCF-7 cells removed by 20 mM NH4OH treatment and MCF-7 cells added to the cmtx for an additional were stably transfected with a mammalian expression vector encoding a mLOX-FLAG 3 days. Total RNA was isolated using TRIzol reagent. GAPDH-specific primers were fusion protein [MCF-7(mLOX)] using LipofectAMINE reagent. MCF-7(mLOX) cells used as a control for equal loading. were plated onto 12-mm round glass coverslips, fixed in ice-cold methanol, and stained for FLAG expression using an anti-FLAG M2 antibody and a rhodamine-conjugated secondary antibody. Immunofluorescence was visualized using a Zeiss Axioskop 2 fluorescence microscope. B, MCF-7(mLOX) cells were treated without or with 100 or 200 ␮M ␤APN for 24 h before and during in vitro invasion analysis and the percentage of denotes statistical significance by ء .invasion compared with untreated MCF-7 cells ANOVA [P ϭ 0.003 between MCF-7 and MCF7(mLOX); P ϭ 0.027 between MCF7(mLOX) and MCF7(mLOX) ϩ 200 ␮M ␤APN]; bars, Ϯ SD. with their highly invasive counterparts C8161 and M619, C918, respectively. Similarly, no expression of rat LOX was observed in a poorly invasive rat prostate cancer cell line (5ЈB) compared with highly invasive cell lines 5ЈP and 5ЈA (Fig. 5, B and D). However, similar trends in up-regulation of LOXL2 was only observed in aggressive uveal melanoma cell lines M619 and C918 compared with poorly aggressive OCM-1A. These results suggest that the up-regulation of LOX mRNA expression, and to a limited extent LOXL2, is associated with an invasive phenotype in different cancer cell types.

DISCUSSION It is now widely accepted that the progression of a non-neoplastic cell to a hyperplastic cell and eventually to one that is capable of metastasis requires the stepwise accumulation of many genetic alterations (34). The molecular mechanisms contributing to breast cancer Fig. 5. LOX and LOXL2 expression is associated with an invasive phenotype in other invasion and metastasis are poorly understood; however, the percep- cancers. RT-PCR analysis of LOX and LOXL2 mRNA expression in (A) cutaneous (A375P and C8161) and uveal (OCM-1A, M619, and C918) human melanoma cell lines tion is that additional genetic alterations are required from those and (B) LOX expression in rat prostatic carcinoma cell lines (5ЈP, 5ЈA, and 5ЈB) using involved in tumorigenesis (35). hLOX-, LOXL2-, and rat LOX-specific PCR primers, respectively. GAPDH-specific primers were used as a control for equal loading. Analysis of in vitro invasive potential We identified an up-regulation in LOX expression in invasive/ of (C) human cutaneous and uveal melanoma cell lines, and (D) rat prostatic cell lines was metastatic breast cancer cells compared with poorly invasive/non- performed in a in vitro MICS chamber; bars Ϯ SD. 4481

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. LYSYL OXIDASE IN BREAST CANCER INVASION analysis shows some interesting consistencies. For example, LOX was myofibroblasts, and ECM in the newly formed stroma around carci- first identified as a “ras recision gene” in normal cells transformed by nomatous ducts rather than tumor cells. Because myofibroblasts and LTR-c-H-ras. LOX expression was reduced greatly in ras-trans- initiated stromal cells are known to potentiate tumor progression (54, formed NIH 3T3 fibroblasts, and was restored to high levels in cells 55), we investigated the contribution of fibroblasts to the induction of reverted by IFN treatment (36, 37). Other reports confirm the loss of LOX expression in a poorly invasive/nonmetastatic breast cancer cell LOX expression in transformed cells (38–41), in which a decrease in line. Indeed, we observed an increase in endogenous LOX expression LOX activity was noted in malignant cell lines including fibrosar- in MCF-7 cells cultured in the presence of fibroblast-cm, as well as a coma, rhabdomyosarcoma, and choriocarcinoma (42). Csiszar et al. fibroblast-cmtx. Because exogenous LOX expression in MCF-7 cells (43) demonstrate a decrease in LOX expression in colon tumors and has been shown to induce cellular invasion and motility, these results Ren et al. (44) demonstrate a progressive reduction of LOX expres- suggest that soluble factors as well as molecular signals left in the sion with the transition from normal prostate epithelium to malignant ECM by fibroblasts have the potential to influence tumor progression. prostate epithelium. Consistent with these reports, poorly invasive/ Whether these processes are recapitulated in vivo remains to be nonmetastatic breast cancer, uveal and cutaneous melanoma, and rat investigated. prostate cancer cell lines used in our study express little to no LOX. Although not the primary focus of the current study, we also Still other studies show an up-regulation in LOX expression in identified an association between the expression of other members of some malignant tumors (45), some osteosarcoma cell lines (46), and the LOX family and the invasive phenotype of breast cancer cells. In all conventional (clear cell) renal cell carcinoma tumors analyzed particular, LOXL2 demonstrated a similar expression pattern to LOX (47). In addition, Stassar et al. (48) demonstrate that LOX expression in breast cancer cells and was similarly induced in MCF-7 cells is significantly associated with a higher staging in clear cell renal cultured on a fibroblast-cmtx. There is extensive sequence homology carcinoma tissues. Furthermore, Ren et al. (44) reported that in two of between LOXL2 and LOX with regards to the copper binding and three mouse prostate cancer cell lines derived from primary tumors catalytic domains, suggesting similar biological functions. Saito et al. and their matched lung metastasis, LOX expression was decreased in (56) demonstrate that LOXL2 is highly expressed in astrocytoma, the metastatic lesions compared with primary tumors. However, one fibrosarcoma, and cervical adenocarcinoma cell lines. However, sub- of the primary tumors did not express LOX whereas its matched sequent analysis is needed to determine the role of LOXL2 in breast metastases did (44). Taken together these reports also support our cancer invasion and metastasis. observations of LOX up-regulation in aggressive cancer cell lines. In addition to invasive/metastatic breast cancer cell lines, LOX Why is there such disparity in the literature regarding LOX expres- expression was up-regulated in highly invasive human uveal and sion in various cancers? One possibility may be differences in cell cutaneous melanoma, and rat prostate cancer cell lines. The expres- types such as cell origin or differentiation status. Fibrosarcoma, os- sion of LOX in several different types of invasive cancer cell lines teosarcoma, rhabdomyosarcoma, and renal cell carcinoma originate may suggest a general mechanistic role for this gene in cell-cell and from the mesoderm, whereas breast adenocarcinoma cells, melanoma cell-matrix interactions. These new observations raise the possibility cells (neural crest), and choriocarcinoma cells originate from the of using LOX expression in tumor cells as a predictive/prognostic ectoderm, and prostate carcinoma cells originate from the endoderm. indicator for cancer staging and progression. However, the observation of LOX expression in renal cell carcinoma and some osteosarcoma cell lines, and a lack of LOX expression in ACKNOWLEDGMENTS choriocarcinoma blurs these delineations between mesoderm versus ecto/endoderm. Another plausible explanation of the disparity in LOX We thank Drs. Richard Seftor, Zhila Khalkhali-Ellis, Carrie Rinker-Schaef- expression may be that an up-regulation in LOX expression is a fer, and Keith Fong for helpful scientific discussions. consequence of an epithelial to mesenchymal transition in aggressive carcinoma cells or a mesenchymal to epithelial transition in aggres- REFERENCES sive melanoma cells. We have shown previously that cancer cells 1. Jemal, A., Thomas, A., Murray, T., and Thun, M. Cancer statistics, 2002. CA Cancer demonstrating an “interconverted” phenotype in which cells display J. Clin., 52: 23–47, 2002. both cytokeratin and vimentin intermediate filaments have an in- 2. Kirschmann, D. A., Seftor, E. A., Nieva, D. R. C., Mariano, E. A., and Hendrix M. J. C. Differentially expressed genes associated with the metastatic phenotype in creased invasive and metastatic potential (32, 49, 50). In the cell lines breast cancer. Breast Cancer Res. Treat., 55: 127–136, 1999. that we have tested, there is a 100% correlation with LOX expression 3. Smith-Mungo, L. I., and Kagan, H. M. Lysyl oxidase: Properties, regulation and multiple functions in biology. Matrix Biol., 16: 387–398, 1997. and an interconverted phenotype. However, transfection of MCF-7 4. Csiszar, K. Lysyl oxidase: a novel multifunctional amine oxidase family. Prog. cells with LOX did not induce a phenotypic epithelial to mesenchymal Nucleic Acid Res. Mol. Biol., 70: 1–32, 2001. transition, as shown in Fig. 3A, suggesting that other genes are 5. Casey, M. L., and MacDonald, P. C. Lysyl oxidase (ras recision gene) expression in human amnion: ontogeny and cellular localization. J. Clin. Endocrinol. Metab., 82: necessary for this process (51). 167–172, 1997. Alternatively, LOX expression could be the result of global genetic 6. Kuivaniemi, H., Peltonen, L., and Kivirikko, K. I. Type IX Ehlers-Danlos syndrome differences, with respect to the availability of LOX substrates, be- and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein. Am. J. Hum. Genet., 37: 798–808, tween normal cells and tumor cells. For instance, disparities in gene 1985. function in different cellular contexts have been noted in microcell- 7. Khakoo, A, Thomas, R., Trompeter, R., Duffy, P., Price, R., and Pope, F. M. Congenital cutis laxa and lysyl oxidase deficiency. Clin. Genet., 51: 109–114, 1997. mediated chromosomal transfer experiments (reviewed in Ref. 35). 8. Yeowell, H. N., Marshall, M. K., Walker, L. C., Ha, V., and Pinnell, S. R. Regulation Hence, the loss of LOX in a cell that normally expresses LOX or the of lysyl oxidase mRNA in dermal fibroblasts from normal donors and patients with gain of LOX in a cell that normally does not express LOX may confer inherited connective tissue disorders. Arch. Biochem. Biophys., 308: 299–305, 1994. 9. Royce, P. M., Camakaris, J., and Danks, D. M. Reduced lysyl oxidase activity in skin tumorigenic and invasive properties, respectively. This premise indi- fibroblasts from patients with Menkes’ syndrome. Biochem. J., 192: 579–586, 1980. cates a complex role for LOX in cancer requiring additional investi- 10. Pinnell, S. R. Molecular defects in the Ehlers-Danlos syndrome. J. Investig. Derma- gation. tol., 79: 90s–92s, 1982. 11. Ooshima, A., and Midorikawa, O. Increased lysyl oxidase activity in blood vessels of In vivo, Peyrol et al. (52, 53) report that LOX is associated with an hypertensive rats and effect of ␤-aminopropionitrile on arteriosclerosis. Jpn. Circ. J., extracellular stromal reaction in breast carcinoma and bronchopulmo- 41: 1337–1340, 1977. 12. Kagan, H. M., Raghavan, J., and Hollander, W. Changes in aortic lysyl oxidase nary carcinoma. They demonstrated that in breast tissues, LOX activity in diet-induced atherosclerosis in the rabbit. Arteriosclerosis, 1: 287–291, expression was most strongly associated with myoepithelial cells, 1981. 4482

13. Chanoki, M., Ishii, M., Kobayashi, H., Fushida, H., Yashiro, N., Hamada, T., and 36. Contente, S., Kenyon, K., Rimoldi, D., and Friedman, R. M. Expression of gene rrg Ooshima, A. Increased expression of lysyl oxidase in skin with scleroderma. Br. J. is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras. Science (Wash. Dermatol., 133: 710–715, 1995. DC), 249: 796–798, 1990. 14. Kagan, H. M. Lysyl oxidase: mechanism, regulation and relationship to liver fibrosis. 37. Kenyon, K., Contente, S., Trackman, P. C., Tang, J., Kagan, H. M., and Friedman, Pathol. Res. Pract., 190: 910–919, 1994. R. M. Lysyl oxidase and rrg messenger RNA. Science (Wash. DC), 253: 802, 1991. 15. Lazarus, H. M., Cruikshank, W. W., Narasimhan, N., Kagan, H. M., and Center, 38. Krzyzosiak, W. J., Shindo-Okada, N., Teshima, H., Nakajima, K., and Nishimura, S. D. M. Induction of human monocyte motility by lysyl oxidase. Matrix Biol., 14: Isolation of genes specifically expressed in flat revertant cells derived from activated 727–731, 1994. ras-transformed NIH 3T3 cells by treatment with azatyrosine. Proc. Natl. Acad. Sci. 16. Li, W., Liu, G., Chou, I. N., and Kagan, H. M. Hydrogen peroxide-mediated, lysyl USA, 89: 4879–4883, 1992. oxidase-dependent chemotaxis of vascular smooth muscle cells. J. Cell. Biol., 78: 39. Csiszar, K., Entersz, I., Trackman, P. C., Samid, D., and Boyd, C. D. Functional 550–557, 2000. analysis of the promoter and first intron of the human lysyl oxidase gene. Mol. Biol. 17. Nelson, J. M., Diegelmann, R. F., and Cohen, I. K. Effect of ␤-aminopropionitrile and Rep., 23: 97–108, 1996. ascorbate on fibroblast migration. Proc. Soc. Exp. Biol. Med., 188: 346–352, 1988. 18. Sanada, H., Shikata, J., Hamamoto, H., Ueba, Y., Yamamuro, T., and Takeda, T. 40. Friedman, R. M., Yeh, A., Gutman, P., Contente, S., and Kenyon, K. Reversion by ␤ Changes in collagen cross-linking and lysyl oxidase by estrogen. Biochim. Biophys. deletion of transforming oncogene following interferon- and retinoic acid treatment. Acta, 541: 408–413, 1978. J. Interferon Cytokine Res., 17: 647–651, 1997. 19. Li, W., Nellaiappan, K., Strassmaier, T., Graham, L., Thomas, K. M., and Kagan, 41. Giampuzzi, M., Botti, G., Cilli, M., Gusmano, R., Borel, A., Sommer, P., and H. M. Localization and activity of lysyl oxidase within nuclei of fibrogenic cells. Di Donato, A. Down regulation of lysyl oxidase induced tumorigenic transformation Proc. Natl. Acad. Sci. USA, 94: 12817–12822, 1997. in NRK-49F cells characterized by constitutive activation of ras proto-oncogene. 20. Kagan, H. M., Williams, M. A., Calaman, S. D., and Berkowitz, E. M. Histone H1 is J. Biol. Chem., 276: 29226–29232, 2001. a substrate for lysyl oxidase and contains endogenous sodium borotritide-reducible 42. Kuivaniemi, H., Korhonen, R-M., Vaheri, A., and Kivirikko, K. I. Deficient produc- residues. Biochem. Biophys. Res. Comm., 115: 186–192, 1983. tion of lysyl oxidase in cultures of malignantly transformed human cells. FEBS Lett., 21. Giampuzzi, M., Botti, G., Di Duca, M., Arata, L., Ghiggeri, G., Gusmano, R., 195: 261–264, 1986. Ravazzolo, R., and Di Donato, A. Lysyl oxidase activates the transcription activity of 43. Csiszar, K., Fong, S. F., Ujfalusi, A., Krawetz, S. A., Salvati, E. P., Mackenzie, J. W., human collagene III promoter: possible involvement of Ku antigen. J. Biol. Chem., and Boyd, C. D. Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 275: 36341–36349, 2000. in colorectal tumors. Int. J. Cancer, 97: 636–642, 2002. 22. Kirschmann, D. A., Lininger, R. A, Gardner, L. M. G., Seftor, E. A., Odero, V. A., 44. Ren, C., Yang, G., Timme, T. L., Wheeler, T. M., and Thompson, T. C. Reduced lysyl Ainsztein, A. M., Earnshaw, W. C., Wallrath, L. L., and Hendrix, M. J. C. Down- oxidase messenger RNA levels in experimental and human prostate cancer. Cancer Hs␣ regulation of HP1 expression is associated with the metastatic phenotype in breast Res., 58: 1285–1290, 1998. cancer. Cancer Res., 60: 3359–3363, 2000. 45. Wakasaki, H., and Ooshima, A. Immunohistochemical localization of lysyl oxidase 23. Celano, P., Vertino, P. M., and Casero, R. A., Jr. Isolation of polyadenylated RNA with monoclonal antibodies. Lab. Investig., 63: 377–384, 1990. from cultured cells and intact tissues. Biotechniques, 15: 26–28, 1993. 46. Fuchs, B., Zhang, K., Bolander, M. E., and Sarkar, G. Identification of differentially 24. Kim, Y., Boyd, C. D., and Csiszar, K. A new gene with sequence and structural expressed genes by mutually subtracted RNA fingerprinting. Anal. Biochem., 286: similarity to the gene encoding human lysyl oxidase. J. Biol. Chem., 270: 7176–7182, 91–98, 2000. 1995. 25. Jourdan-Le Saux, C., Tronecker, H., Bogic, L., Bryant-Greenwood, G. D., Boyd, 47. Young, A. N., Amin, M. B., Moreno, C. S., Lim, S. D., Cohen, C., Petros, J. A., C. D., and Csiszar, K. The LOXL2 gene encodes a new lysyl oxidase-like protein and Marshall, F. F., and Neish, A. S. Expression profiling of renal epithelial neoplasms: is expressed at high levels in reproductive tissues. J. Biol. Chem., 274: 12939–12944, a method for tumor classification and discovery of diagnostic molecular markers. 1999. Am. J. Pathol., 158: 1639–1651, 2001. 26. Asuncion, L. A., Fogelgren, B., Fong, K. S. K., Fong, S. F. T., Kim, Y., and Csiszar, 48. Stassar, M. J., Devitt, G., Brosius, M., Rinnab, L., Prang, J., Schradin, T., Simon, J., K. A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an Peteren, S., Kopp-Schneider, A., and Zo¨ller, M. Identification of human renal cell altered scavenger receptor cysteine rich domain. Matrix Biol., 20: 487–491, 2001. carcinoma associated genes by suppression subtractive hybridization. Br. J. Cancer., 27. Seve, S., Decitre, M., Gleyzal, C., Farjanel, J., Sergeant, A., Ricard-Blum, S., and 85: 1372–1382, 2001. Sommer, P. Expression analysis of recombinant lysyl oxidase (LOX) in myofibro- 49. Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B., and Trevor, K. T. Experimental blast-like cells. Connect. Tissue Res., in press, 2002. co-expression of vimentin and keratin intermediate filaments in human breast cancer 28. Hendrix, M. J. C., Seftor, E. A., and Fidler, I. J. A simple quantitative assay for cells results in phenotypic interconversions and increased invasive behavior. Am. J. studying the invasive potential of high and low human metastatic variants. Cancer. Pathol., 150: 483–495, 1997. Lett., 38: 137–147, 1987. 50. Chu, Y-W., Seftor, E. A., Romer, L. H., and Hendrix, M. J. C. Experimental 29. Maki, J. M., and Kivirikko, K. I. Cloning and characterization of a fourth human lysyl coexpression of vimentin and keratin intermediate filaments in human melanoma cells oxidase isoenzyme. Biochem. J., 355: 381–387, 2001. augments motility. Am J. Pathol., 148: 63–69, 1996. 30. Wilmarth, K. R., and Froines, J. R. In vitro and in vivo inhibition of lysyl oxidase by 51. Kiemer, A. K., Takeuchi, K., and Quinlan, M. P. Identification of genes involved in aminopropionitriles. J. Toxicol. Environ. Health, 37: 411–423, 1992. epithelial-mesenchymal transition and tumor progression. Oncogene, 20: 6679–6688, 31. Tang, S. S., Trackman, P. C., and Kagan, H. M. Reaction of aortic lysyl oxidase with 2001. ␤-aminopropionitrile. J. Biol. Chem., 258: 4331–4338, 1983. 52. Peyrol, S., Raccurt, M., Gerard, F., Gleyzal, C., Grimaud, J. A., and Sommer, P. Lysyl 32. Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B., Gardner, L. M., Boldt, H. C., Meyer, oxidase gene expression in the stromal reaction to in situ and invasive ductal breast M., Pe’er, J., and Folberg, R. Biologic determinants of uveal melanoma metastatic carcinoma. Am. J. Pathol., 150: 497–507, 1997. phenotype: role of intermediate filaments as predictive markers. Lab. Investig., 78: 153–163, 1998. 53. Peyrol, S., Galateau-Salle, F., Raccurt, M., Gleyzal, C., and Sommer, P. Selective 33. Luo, J., Sharma, N., Seftor, E. A., De Larco, J. Heidger, P. M., Hendrix, M. J. C., and expression of lysyl oxidase (LOX) in the stromal reactions of broncho-pulmonary Lubaroff, D. M. Heterogeneous expression of invasive and metastatic properties in a carcinomas. Histol. Histopathol., 15: 1127–1135, 2000. prostate tumor model. Pathol. Oncol. Res., 3: 264–271, 1997. 54. Schmitt-Gra¨ff, A., and Gabbiani, G. Phenotypic features of stromal cells in normal, 34. Bechmann, M. W., Niederacher, D., Schnu¨rch, H. G., Gusterson, B. A., and Bender, premalignant and malignant conditions. Eur. J. Cancer, 28A: 1916–1920, 1992. H. G. Multistep carcinogenesis of breast cancer and tumor heterogeneity. J. Mol. 55. Tlsty, T. D., and Hein, P. W. Know thy neighbor: stromal cells can contribute Med., 75: 429–439, 1997. oncogenic signals. Curr. Opin. Genet. Dev., 11: 54–59, 2001. 35. Yoshida, B. A., Sokoloff, M. M., Welch, D. R., and Rinker-Schaeffer, C. W. 56. Saito, H., Papaconstantinou, J., Sato, H., and Goldstein, S. Regulation of a novel gene Metastasis-suppressor genes: a review and perspective on an emerging field. J. Natl. encoding a lysyl oxidase-related protein in cellular adhesion and senescence. J. Biol. Cancer Inst., 92: 1717–1730, 2000. Chem., 272: 8157–8160, 1997.

4483

Dawn A. Kirschmann, Elisabeth A. Seftor, Sheri F. T. Fong, et al.

Cancer Res 2002;62:4478-4483.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/62/15/4478

Cited articles This article cites 53 articles, 14 of which you can access for free at: http://cancerres.aacrjournals.org/content/62/15/4478.full#ref-list-1

Citing articles This article has been cited by 41 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/62/15/4478.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/62/15/4478. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.