Differential Osteopontin Expression in Phenotypically Distinct Subclones of Murine Breast Cancer Cells Mediates Metastatic Behavior*

Cancer progression depends on an accumulation of metastasis-supporting cell signaling molecules, which target signal transduction pathways and, ultimately, gene expression. One such molecule, osteopontin (OPN), represents a key molecular signaling event in tumor progression and metastasis. However, the transcriptional regulatory mechanisms that underlie OPN expression in the setting of breast cancer have not been well studied. In this regard, we have examined the differential transcriptional regulation of OPN in the murine mammary epithelial tumor cell lines, 4T1 and 4T07, which are sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. These lines are phenotypically heterogeneous in their metastatic behavior. 4T1 hematogenously metastasizes to the lung, liver, bone, and brain, whereas 4T07 is highly tumori-genic but fails to metastasize. The tumor growth and metastatic spread of 4T1 cells closely mimics stage IV breast cancer. We demonstrate that a Ras-independent, phosphoinositide-3 kinase-dependent, c-Jun N-terminal kinase-dependent phosphorylation of c-Jun results in binding of an AP-1 c-Jun homodimer to the OPN promoter in 4T1 cells. This differential up-regulation of OPN gene transcription and protein expression in 4T1 cells conveys in vitro correlates of a metastatic phenotype. These results provide new insight into the transcriptional study, when 4T1 and 4T07 cells are compared, we demonstrate that a phosphoinositide-3 kinase (PI3K)- and JNK-dependent phosphorylation of c-Jun differentially up-regulates OPN gene transcription in 4T1 cells to convey in vitro correlates of a metastatic phenotype. These results provide new insight into the transcriptional regulation of OPN as a key mediator of metastatic behavior in malignancy. fonyl fluoride, pH 7.4) and centrifuged at 12,000 (cid:1) for at The protein concentration was determined by the Bio-Rad protein assay kit; the protein samples were separated by 4–20% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Amer- sham Biosciences) by semi-dry transfer (Bio-Rad). The membranes were probed with the following primary antibodies fo r 1 h atroom temperature: goat OPN Ab (R&D Systems, Minneapolis, MN), rabbit double-stranded oligonucleotides their respec- tive complementary oligonucleotides, P]ATP Ci/mmol) T4 polynucleotide kinase G-50 native Tris-borate/EDTA visual- ized autoradiography. specific competitive binding assays, oligonucleotides 20-fold molar In nonspe- cific competitive binding assays,

Cancer progression depends on an accumulation of metastasis-supporting genetic modifications and physiological alterations. These physiological changes are often regulated by cell signaling molecules, which target signal transduction pathways and, ultimately, gene expression. One such molecule, osteopontin (OPN) 1 functions as both a cell attachment protein and a cytokine that signals through two cell adhesion molecules: ␣ v B 3 -integrin and CD44 (1)(2)(3). Initially discovered as an inducible, tumor-promoter gene, OPN is an acidic hydrophilic glycophosphoprotein, is overexpressed in human tumors, is the major phosphoprotein secreted by malignant cells in patients with advanced metastatic cancer, and has been implicated in tumor cell migration and metastasis. Data suggest that OPN overexpression represents a key molecular event in tumor progression and metastasis (4 -11). OPN is not typically mutated to achieve gain-of-function activation; rather, it is differentially transcriptionally regulated by multiple response elements in its promoter. However, the mechanisms that underlie the transcriptional regulation of OPN expression in metastatic biology are largely unknown.
In this regard, we have examined the differential transcriptional regulation of OPN in the murine mammary epithelial tumor cell lines 4T1 and 4T07. These are thioguanine-resistant sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice (12). Although they share a common origin, these lines are phenotypically heterogeneous in their metastatic behavior. 4T1 hematogenously metastasizes to the lung, liver, bone, and brain, whereas 4T07 is highly tumorigenic but fails to metastasize. The tumor growth and metastatic spread of 4T1 cells closely mimics stage IV breast cancer. The potential role of OPN in regulation of metastatic behavior in these cell lines has not been previously examined. In this study, when 4T1 and 4T07 cells are compared, we demonstrate that a phosphoinositide-3 kinase (PI3K)-and JNK-dependent phosphorylation of c-Jun differentially up-regulates OPN gene transcription in 4T1 cells to convey in vitro correlates of a metastatic phenotype. These results provide new insight into the transcriptional regulation of OPN as a key mediator of metastatic behavior in malignancy.

MATERIALS AND METHODS
Cell Cultures-Mouse mammary tumor cell lines 4T1 and 4T07 (gift from Mark W. Dewhirst, Duke University) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 g/ml), and maintained at 37°C in a humidified atmosphere of 5% CO 2 . For secretedprotein analysis, serum-free DMEM was collected after 24-h incubation and centrifuged at 600 ϫ g for 5 min to remove cellular material; the supernatant was concentrated 100-fold through Ultrafree Centrifugal filters (Millipore, Bedford, MA). For JNK inhibition assays, the selective c-Jun NH 2 -terminal kinase inhibitor SP600125 was purchased from Calbiochem, which was prepared as a stock solution of 90 mM in 99.5% dimethyl sulfoxide. The PI3K inhibitor LY294002 (25 M) was used. Cells in serum-free DMEM were treated with SP600125 at indicated doses for 24 h (see Fig. 4b, unless otherwise indicated, 100 nM was used).
Northern Blot Analysis-Total RNA was isolated using a TRIzol kit according to the manufacturer's instruction (Invitrogen). RNA (10 g) was separated by electrophoresis through denaturing 1.2% agarose gel containing 1% formaldehyde and transferred onto Hybond Nϩ nylon membrane (Amersham Biosciences). The membrane was UV-crosslinked; hybridization was carried out with [␣-32 P]dCTP by random primers DNA labeling system (Invitrogen) to specific activities of 5 ϫ 10 8 cpm/g. A 32 P-labeled 800-bp probe was constructed based upon the murine OPN cDNA sequence (GenBank accession number NM_000610). After hybridization, the membranes were washed and exposed on Kodak OMAT film at Ϫ70°C Transient Transfection and Luciferase Assay-Plasmid and constructs were a gift from Dr. David Denhardt, Rutgers University, and were described previously (13). In brief, deletion constructs of the murine OPN promoter spanning OPN-69 (Ϫ69 to ϩ79), OPN-107 (Ϫ107 to ϩ79), OPN-174 (Ϫ174 to ϩ79), OPN-209 (Ϫ209 to ϩ79), and OPN-258 (Ϫ258 to ϩ79), OPN-512(Ϫ512 to ϩ79), OPN-777 (Ϫ777 to ϩ79), and OPN-Full (Ϫ2100 to ϩ79) were cloned into pGL3-basic luciferase reporter plasmid (Promega, Madison, WI). The point mutant of OPN promoter containing AP-1 binding site was constructed by two-step PCR; the AP-1 binding site TGACACA was mutated to TCATATA and confirmed by DNA sequencing. 4 ϫ 10 5 cells were seeded with antibiotic-free DMEM on each well of 12-well plates the day before transfection. 2 g of plasmid DNA and 4 l of LipofectAMINE 2000, diluted with Opti-MEM, were mixed gently and incubated with cells. Culture medium was changed after 6 h of transfection. Luciferase reporter assays were performed by dual luciferase reporter assay system (Promega) at 36 h after transfection. Cells were washed with phosphatebuffered saline and lysed in lysis buffer according to the manufacturer's instructions. Cell debris was removed by centrifugation at 10,000 rpm for 1 min, 20 l of the supernatant were mixed with 100 l of luciferase substrate, and luciferase activity was measured by luminometer (Turner Designs TD-20/20). Firefly luciferase activity was normalized for transfection efficiency using Renilla reniformis luciferase activity (pRL-SV40). The data are presented as mean -fold luminescence for three independent experiments performed in triplicate.
Nuclear Extracts and Electrophoretic Mobility Shift Assays-Nuclear extract preparation and electrophoretic mobility shift assays were conducted as described previously; all buffers containing the protease inhibitors and dithiothreitol (1 mM) were freshly prepared (13). The cell pellet collected by centrifugation was resuspended in 5 packed cell volumes of ice-cold buffer A containing 10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EDTA, 1.0 mM dithiothreitol, pepstatin A (2 g/ml), and 0.5 mM phenylmethylsulfonyl fluoride, followed by incubation on ice for 20 min. Then 10% Nonidet P-40 was added to 0.5% final concentration. After centrifugation at 150 ϫ g for 5 min, the cell pellets were resuspended in 2 packed cell volumes of ice-cold buffer C containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM dithiothreitol, pepstatin A (2 g/ml), and 0.5 mM phenylmethylsulfonyl fluoride, and vortexed for 20 min at 4°C. Insoluble material was removed by centrifugation at 14,000 rpm; the supernatant containing the nuclear protein was aliquoted and stored at Ϫ80°C until use. For electrophoretic mobility shift assay, wild-type and mutant probes derived from mouse OPN promoter sequence were synthesized, annealed to make double-stranded oligonucleotides with their respective complementary oligonucleotides, and then end-labeled with [␥-32 P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase (Promega), followed by G-50 column purification. The reactions were resolved on 6% native acrylamide gel in 0.5ϫ Tris-borate/EDTA buffer and visualized by autoradiography. In specific competitive binding assays, unlabeled oligonucleotides were added at a 20-fold molar excess. In nonspecific competitive binding assays, unlabeled poly(dI-dC) were used. Supershift assays were performed by preincubating nuclear extracts with rabbit anti mouse c-Jun polyclonal Ab (Santa Cruz Biotechnology).
Biotin-Streptavidin DNA Affinity Assay-Oligonucleotide containing biotin on the 5Ј-nucleotide of the sense strand (high performance liquid chromatography purity synthesized by Sigma Genosys) was used in the pull-down assay. The sequences of the oligonucleotide is as follows: 5Ј-GTGGCAAAAACCTCUTGACACAUTCACTCCACCT-3Ј, which corresponds to positions Ϫ90 to Ϫ58 of the mouse OPN promoter. The oligonucleotide was annealed to its complementary oligonucleotide and bound to Dynabeads M280 following the manufacturer's instructions (Dynal Biotech, Oslo, Norway). Nuclear proteins were isolated from 4T1 and 4T07 cells, as described previously. Protein concentration of the nuclear extract was determined using a Bio-Rad protein assay kit. The nuclear protein was incubated for 1 h at 25°C with biotinylated 33-mer duplex oligonucleotide bound to Dynabeads M280 streptavidin in protein binding buffer [80 mM NaCl, 50 mM Tris-HCL, pH 7.5, 4% (v/v) glycerol, 5 mM MgCl 2 , 0.25 mg/ml poly(dI-dC), 2.5 mM EDTA, 30 M mutant double-strand oligonucleotide, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). The magnetic beads were washed three times with protein binding buffer in 80 mM NaCl containing excess poly(dI-dC) and mutant duplex oligonucleotide competitor DNA; the fractions were eluted with elution buffer (0.5 M NaCl, 50 mM Tris-HCL, pH 7.5, 4%(v/v) glycerol, 5 mM MgCl 2 , 0.25 mg/ml poly(dI-dC), 2.5 mM EDTA, 30 M mutant double-strand oligonucleotide, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) and were stored at Ϫ80°C.
Protein Sequencing-The protein was separated by SDS-PAGE and stained with silver. The individual protein band samples were excised and digested overnight with trypsin. The resulting digest was then injected onto a Microbore high performance liquid chromatography (Beckman 32 K Gold) system, and the fractions were collected. The 10 best fractions were selected for matrix-assisted laser desorption/ionization mass analysis of the intact protein (Applied Biosystems Voyager DE-Pro); subsequently, the best fractions were selected for Edman sequencing (Applied Biosystems Procise 470). The resulting data were manually interpreted and searched using Sequest against the National Center for Biotechnology Information nonredundant data base.
Adhesion Assay-Adhesion assays were performed on 96-well microtiter plates coated with 10 g/ml matrigel. Cells were trypsinized and resuspended in DMEM with 1% bovine serum albumin, 1 mM MgCl 2 , and 0.5 mM CaCl 2 at a concentration of 1 ϫ 10 6 cells/ml. 1 ϫ 10 5 cells (100 l) were added into each well and placed for 30 min at 37°C in 5% CO 2 humidified air incubation. Non-adherent cells were removed by gently washing the wells three times with phosphate-buffered saline with 1 mM MgCl 2 and 0.5 mM CaCl 2 . Adherent cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, followed by rinsing with phosphate-buffered saline, and stained with 0.4% crystal violet for 10 min. After extensive rinsing, the dye was released from the cells by addition of 30% acetic acid, and the microtiter plates were read in a microplate reader (Molecular Devices, Berkeley, CA) at 590 nm. In selected instances, cells were treated for 48 h with 100 nM SP600125, or transfected for 12 h with sense (S) and antisense (AS) oligonucleotides for OPN and JNK. The sequence for the antisense OPN oligonucleotide was 5Ј-GACAACCAAGCCCTCCCAGA-3Ј, whereas that of antisense JNK1/2 was a combination of JNK1 and JNK2 (JNK1AS, 5Ј-CTCTCT-GTAGGCCCGCTTGG-3Ј; JNK2AS, 5Ј-GTCCGGGCCAGGCCAAAGT-C-3Ј).
Cell Migration and Invasion Assay-The migration and invasion assay were carried out in a Boyden Chamber system (Corning Glassworks). Cells were seeded at a density of 10 5 cells per well in triplicate in the upper chamber of 12-well transwells (8-m pore). After being incubated at 37°C with 5% CO 2 for 24 h, the cells were fixed in 3.7% paraformaldehyde in phosphate-buffered saline for 10 min. The cells on the top surface of the filters were wiped off with cotton swabs. After three washes with phosphate-buffered saline, the filters were stained with 0.4% crystal violet for 10 min, and the dye was detected as the in vitro adhesion assay procedure.
Statistical Analysis-All data are presented as mean Ϯ S.E. of three or four experiments. Analysis was performed using a Student's t test. Values of p Ͻ 0.05 were considered significant.

RESULTS
Osteopontin Expression in 4T1 and 4T07 cells-We first examined differential OPN expression in 4T1 and 4T07 cells. Immunoblot analysis of cell lysate and culture media for cellular and secreted OPN protein, respectively, demonstrated significantly enhanced OPN protein in 4T1 cells (Fig. 1a). Compared with 4T1 cells, 4T07 OPN expression in cell lysate was virtually undetectable (p Ͻ 0.01), whereas that found in culture media was 10-fold less than 4T1 cells (p Ͻ 0.01). Steady state OPN mRNA expression was determined by Northern blot analysis using an 800-bp murine OPN cDNA probe (Fig. 1b). OPN mRNA expression was normalized to that of ␤-actin and was ϳ10-fold greater in 4T1 cells than that found in 4T07 cells (p Ͻ 0.01). OPN mRNA half-life was determined in the presence of actinomycin D (100 g/ml), which was defined as time 0. Northern blots were then performed at 1, 2, 4, and 8 h. Expression of mRNA was normalized to that of the housekeeping gene, ␤-actin, and that of OPN at time 0 and then plotted semilogarithmically as a function of time to determine mRNA half-life. Half-life of OPN mRNA did not differ between the two cell lines. In 4T1 cells, OPN mRNA half-life was 5.0 Ϯ 0.9 h, whereas that of 4T07 cells was 5.8 Ϯ 0.7 h (p ϭ not significant). These data indicate that OPN protein and mRNA are differentially expressed between 4T1 and 4T07 breast cancer cell lines.
OPN Promoter Studies-A 2.1-kb segment of the OPN promoter (OPN-Full), including the transcription start site, from 4T1 and 4T07 cells was amplified and submitted for automated sequencing in the Duke Sequencing Core Facility. The derived sequences were identical between the two cell lines. To further analyze differential OPN promoter activity, deletion-luciferase reporter constructs were transiently transfected into 4T1 and 4T07 cells. Serial deletion constructs demonstrated a significant 8-fold increase in luciferase activity between nt Ϫ69 and nt Ϫ258 in 4T1 cells. In contrast, luciferase activity in 4T07 cells was relatively stable among the various constructs. Sequences upstream of nt Ϫ258 to nt Ϫ2.1 kb did not further significantly increase OPN promoter activity in either 4T1 or 4T07 cells (data not shown). Using further serial deletion constructs between nt Ϫ69 and nt Ϫ258, the area of increased 4T1-associated OPN promoter activity was further localized to the length of the OPN promoter from nt Ϫ69 to nt Ϫ107 (Fig.  2a). Luciferase activity was noted to increase by more than 6-fold in 4T1 cells in the transition between the OPN Ϫ68 and OPN Ϫ107 promoter constructs The 38-bp segment between nt Ϫ69 and nt Ϫ107 was submitted to the TRANSFAC 6.0 data base to determine potential transcription factor binding sites. This analysis indicated the presence of two SP1 and one Yi, Oct 1, REB1, MyoD, D1, and AP-1 binding sites. To determine whether nuclear protein binds to any portion of this segment of OPN promoter, gel shift assays were performed using oligonucleotide sequences that corresponded to overlapping portions of this segment: nt Ϫ124 to nt Ϫ97 (Probe 1), nt Ϫ100 to nt Ϫ80 (Probe 2), and nt Ϫ84 to nt Ϫ64 (Probe 3) (Fig. 2b). Nuclear protein was extracted from 4T1 and 4T07 cells. With Probe 3, a single band was found to shift in the presence of 4T1 nuclear protein; in the presence of a 20-fold molar excess of cold Probe 3, this band was no longer present. No binding was seen with 4T07 nuclear protein. A consensus AP-1 binding sequence, TGACACA, is present in Probe 3 between nt Ϫ69 and nt Ϫ75. To determine whether AP-1 binds at this location in Probe 3 of the OPN promoter, supershift assays were performed with antibody to c-Jun. In this setting, the gel shift band was found to completely shift with c-Jun Ab, suggesting the presence of a c-Jun homodimer in 4T1 nuclear protein. Additional super shift studies were performed with antibody to c-Fos, JunB, and JunD; no band shift was noted (data not shown).
To further confirm potential AP-1 c-Jun homodimer binding performed  at this site, DNA affinity chromatography was performed with the biotin-strepavidin technique. The sequence of the biotinlabeled oligonucleotide was: 5Ј-GTG GCA AAA ACC TCA UTGA CAC AUTC ACT CCA CCT-3Ј, corresponding to positions nt Ϫ90 to nt Ϫ58 of the mouse OPN promoter. The putative corresponding transcription factor protein was then purified and isolated from nuclear extract isolated from 4T1 cells. When the eluate was analyzed by Western blot analysis, one major band was identified; this was excised and subjected to automated protein sequencing (Fig. 2c). Analysis of two separate tryptic digests yielded identical matches with only c-Jun. As a control, nuclear extract from 4T07 cells was also subjected to affinity chromatography with the biotin-labeled oligonucleotides. No band was found on Western blotting. FIG. 2. a, differential activity of OPN promoter deletion constructs between 4T1 and 4T07 murine breast cancer cells. The lengths of the osteopontin promoter fragments tested were OPN-69 (Ϫ69 to ϩ79), OPN-107 (Ϫ107 to ϩ79), OPN-174 (Ϫ174 to ϩ79), OPN-209 (Ϫ209 to ϩ79), and OPN-258 (Ϫ258 to ϩ79). Firefly luciferase activity was normalized for transfection efficiency using R. reniformis luciferase activity (pRL-SV40). The data are presented as mean -fold luminescence for three independent experiments performed in triplicate. (*, p Ͻ 0.01 versus OPN Ϫ69). b, gel shift assays in 4T1 and 4T07 cells. To determine whether nuclear protein binds to any portion of this segment of the OPN promoter, a 38-bp segment between nt Ϫ69 and nt Ϫ107, gel shift assays were performed using oligonucleotide sequences that corresponded to overlapping portions of this segment: nt Ϫ124 to nt Ϫ97 (Probe 1), nt Ϫ100 to nt Ϫ80 (Probe 2), and nt Ϫ84 to nt Ϫ64 (Probe 3). Supershift assays were performed with antibody to c-Jun to determine whether AP-1 binds at this location in Probe 3 of the OPN promoter. In competitive binding assays, unlabeled oligonucleotides were added at 20-fold excess. Blot is representative of three experiments. c, isolation of putative transcription factor from 4T1 cells. Western blot of crude nuclear extract and purified nuclear extract from 4T1 cells. Crude nuclear protein and nuclear protein purified using the biotin-streptavidin DNA affinity technique with the identified putative DNA binding sequence were electrophoresed on 8% SDS-PAGE and silver-stained. Blots are representative of four experiments.
These data indicate that a c-Jun homodimer of AP-1 binds to the OPN promoter in 4T1 cells.
Mutation Analysis of the OPN Promoter-To further characterize this AP-1 site, mutation analysis was combined with transient transfection and gel shift assays. A point mutant of the AP-1 binding site was constructed in the full-length OPN promoter-reporter construct (M-OPN-Full) and OPN Ϫ107 (M-OPN Ϫ107). Using two-step PCR, the AP-1 binding site TGA-CACA was mutated to TUCUAUTUAUTUA and confirmed by DNA sequencing. Transient transfection analysis was performed (Fig. 3a). Both M-OPN-107 and M-OPN-Full demonstrated significant depression of OPN promoter activity in 4T1 cells to levels equivalent to that of the OPN-69 construct. In contrast, transfection of mutant constructs into 4T07 cells did not alter OPN promoter activity.
Gelshift assays using 4T1 and 4T07 nuclear extracts were then performed using a labeled oligonucleotide with a mutated AP-1 site; 5Ј-AAA ACC TCA UTGA CAC AUTC AC-3Ј was mutated to 5Ј-AAA ACC TCA UTCA TAT AUTC AC-3Ј (Fig.  3b). In 4T1 cells, the typical AP-1 band was noted with the wild-type probe; in contrast, no binding was noted with the mutant probe. In addition, 20ϫ excess unlabeled mutant probe did not compete with labeled wild-type probe for binding. No specific binding was found using nuclear protein from 4T07 cells. These mutation studies suggest that this AP-1 binding site is necessary for OPN promoter activity in 4T1 cells.
Expression of c-Jun and JNK in 4T1 and 4T07 Cells-Cell and nuclear protein from 4T1 and 4T07 cells were assayed to determine relative c-Jun protein levels (Fig. 4a). No differences in total or nuclear c-Jun protein levels were found between the two cell types. Because JNK activity is a well characterized activation mechanism for c-Jun, immunoblot analysis was also performed to determine c-Jun phosphorylated at Ser-63 and Ser-73 in 4T1 and 4T07 cells. Nuclear protein levels of constitutively phosphorylated c-Jun were noted to be significantly higher in 4T1 cells. ␤-Actin protein levels did not differ between 4T1 and 4T07 cells (data not shown). A specific pharmacologic inhibitor of JNK activity, SP600125, and an AS oligonucleotide inhibitor of JNK 1/2 (5Ј-TTC CAC TGA TCA ATA TAG TCC CTT-3Ј) were used in further studies. A dose-response relationship was determined for SP600125, and 100 nM SP600125 was found to ablate phosphorylation of c-Jun in 4T1 cells (Fig. 4b).
No alteration in c-Jun and ␤-actin protein expression and no change in cell viability, as determined by trypan blue exclusion, were noted in the presence of this inhibitor. Transient transfection studies of the OPN promoter, gel shift assays, and immunoblots of OPN protein were then performed with SP600125 and/or AS-JNK (Fig. 4, c-e). Inhibition of JNK activity with SP600125 and/or treatment with AS-JNK 1/2 resulted in levels of OPN promoter activation in OPN-107 and OPN-Full that were not statistically different from that of OPN Ϫ69. In a parallel fashion, using gel shift assays, AP-1 binding in 4T1 cells at nt Ϫ69 to nt Ϫ75 of the OPN promoter was ablated in the presence of SP600125 and/or treatment with AS-JNK 1/2. Finally, immunoblot analysis demonstrates dramatically diminished OPN protein levels in 4T1 cell lysates in the presence of SP600125 and/or treatment with AS-JNK 1/2. These results demonstrate that inhibition of JNK 1/2 activity significantly decreases AP-1 DNA binding, OPN promoter activity, and OPN protein expression in 4T1 cells.
Total and phosphorylated JNK 1/2 protein levels in the nuclear fraction and whole-cell lysates were measured in the 4T1 and 4T07 cell lines (Fig. 5). Monoclonal antibody to phosphorylated Thr-183 and Thr-185 on JNK-1/2 was used. Total cell lysate levels of JNK-1 and JNK-2 were similar between the two cell types; however, there was a significant difference in phos-phorylated JNK-1 and JNK-2 between the two cell lines. This indicates that JNK-1/2 is constitutively phosphorylated in 4T1 cells.
Ras-, PI3K-, and JNK-dependent OPN Expression in 4T1 and 4T07 Cells-Because enhanced Ras actvity can be a potential mechanism for constitutive activation of JNK 1/2, a dominant-negative form of Ras protein (pCMV-RasN17; BD Biosciences Clontech) that contains a serine-to-asparagine mutation at residue 17 was transfected into 4T1. Expression of this mutant Ras variant will "knock out" endogenous Ras ex-  OPN-Full). b, mutation analysis of the AP-1 binding site in OPN promoter in 4T1 cells. Gel shift competition studies were performed using nuclear extract prepared from 4T1 cells. Gel shift assays using 4T1 and 4T07 nuclear extracts were then performed using a labeled oligonucleotide with a mutated AP-1 site; 5Ј-AAA ACC TCA UTGA CAC AUTC AC-3Ј was mutated to 5Ј-AAA ACC TCA UTCA TAT AUTC AC-3Ј. In competitive binding assays, unlabeled mutant oligonucleotides were added at 20-fold excess. Blot is representative of three experiments. pression in mammalian cells. Western blot analysis demonstrates dramatically enhanced Ras protein expression in 4T1 cells after transfection (Fig. 6a). In the presence of dominant-negative Ras protein, 4T1 levels of phosphorylated c-Jun, phosphorylated JNK1 and JNK2, and total OPN protein were then determined. Inhibition of Ras activity by dominant-negative Ras protein did not alter levels of phosphorylated c-Jun, phosphorylated JNK, or total OPN, suggesting that alternative pathways for JNK activation are functioning in 4T1 cells to augment OPN transcription and protein expression.
PI3K is an additional signal transduction pathway that may activate JNK. To determine the role of PI3K, we used ⌬p85 (Gift from Dr. Julian Downward, Imperial Cancer Research Fund, London, UK), a dominant-negative form of the regulatory subunit of PI3K that is unable to bind the catalytic p110 subunit. ⌬p85 is thought to inhibit endogenous PI3K activation by sequestering upstream stimulatory proteins that bind to p85. When 4T1 cells are transfected with ⌬p85, levels of phosphorylated JNK-1 and JNK-2 protein are significantly decreased in association with decreased levels of phosphorylated c-Jun (Fig. 6b). OPN protein expression in the presence of PI3K inhibition is minimally detectable. These data suggest that 4T1 cells possess constitutively active PI3K, which ultimately increases OPN protein expression. To corroborate the potential role of PI3K in OPN expression in 4T1 cells, transient transfection studies were performed in 4T1, 4T1 ϩ ⌬p85, and 4T1 ϩ LY294002, a pharmacologic inhibitor of PI3K activity, using the OPN Ϫ107 and OPN-Full constructs (Fig. 6c). In the presence of ⌬p85 and LY294002, OPN promoter activation is significantly decreased by 6 -8-fold in both the OPN Ϫ107 promoter and the full-length OPN promoter constructs. In association with the previous Western blot data in Fig. 6b, these results suggest that PI3K-dependent JNK-and c-Jun activation constitutively up-regulate OPN promoter activity to increase OPN protein expression.
OPN-dependent in Vitro Adhesion, Migration, and Invasion of 4T1 and 4T07 Cells-The functional correlate of altered OPN expression in 4T1 and 4T07 cells was determined using in vitro adhesion, migration, and invasion assays (Fig. 7). OPN protein expression in 4T1 cells was inhibited using AS-JNK, AS-OPN, and SP600125. Relative to unstimulated 4T1 cells, 4T1 ϩ AS-JNK, 4T1 ϩ AS-OPN, and 4T1 ϩ SP600125 cells exhibited significant 3-4-fold decreases in adhesion, migration, and invasion. The level of activity was not statistically different from that of 4T07 cells. This suggests that inhibition of OPN expression, JNK expression, or JNK activity is associated with significantly decreased in vitro correlates of metastatic behavior in 4T1 cells. DISCUSSION In this study, we use the murine mammary epithelial cell lines 4T1 and 4T07 to examine the transcriptional mechanisms underlying differential expression of OPN and OPN-dependent function between these two cell lines. Our data indicate that consti- tutive JNK activation in 4T1 cells phosphorylates c-Jun to ultimately promote OPN transcription in 4T1 cells. This activation of JNK occurs via phosphorylation and is PI3K-dependent but also Ras-independent. These results indicate a potential mechanism by which these cells, from a common lineage, develop divergent metastatic phenotypes that are OPN-dependent.
OPN is a secreted glycoprotein that is rich in aspartate and sialic acid residues and contains functional domains for calcium-binding, phosphorylation, glycosylation, and extra-cellular matrix adhesion (14). OPN seems to mediate cell-matrix interactions and cellular signaling through binding with integrin, primarily ␣V␤3, and CD44 receptors. OPN is expressed in multiple species, including humans and rodents (15). Cells that express OPN include osteoclasts, osteoblasts, kidney, breast and skin epithelial cells, nerve cells, vascular smooth muscle cells, and endothelial cells (14, 16 -19). Activated immune cells such as T cells, natural killer cells, macrophages, and Kupffer cells also express OPN. The secreted OPN protein is widely distributed in plasma, urine, milk, and bile (20 -22). Constitutive expression of OPN exists in several cell types, but induced expression has been detected in T lymphocytes, epidermal cells, bone cells, macrophages, and tumor cells in remodeling processes such as inflammation, ischemia-reperfusion, bone resorption, and tumor progression (14,18,19). A variety of stimuli, including phorbol 12-myristate 13-acetate, 1,25-dihydroxyvitamin D, basic fibroblast growth factor, tumor necrosis factor-␣, interleukin-1, interferon-␥, and lipopolysaccharide, seemed to up-regulate OPN expression (14,18,19,23). OPN has multiple molecular functions that mediate cell adhesion, chemotaxis, macrophage-directed interleukin-10 suppression, stress-dependent angiogenesis, prevention of apoptosis, and anchorage-independent growth of tumor cells (14,18,19,23). A substantial body of data has recently linked OPN with the regulation of metastatic spread by tumor cells. However, the molecular mechanisms that define the role of OPN expression in tumor metastasis are incompletely understood.
With regard to breast cancer, OPN expression has been correlated with breast cancer metastasis. Fedarko and colleagues (9) demonstrated significantly increased serum levels of OPN in 20 breast cancer patients compared with those of normal control subjects. When breast cancer primary tumors were compared with bone metastases, elevated cellular OPN expression, as detected by immunohistochemistry, was noted to be significantly higher with bone metastasis (24). In addition, using expression microarray analysis, Korkola and colleagues (25) found that OPN is differentially expressed between lobular and ductal breast carcinomas. In contradistinction to the bulk of the correlative clinical data, Coppola et al. (7) recently surveyed immunohistochemical expression of OPN in a variety human tumors. These investigators found little or no OPN expression in 23 of 26 breast cancers, although increased expression of OPN was noted in a number of other cancer types. Although speculative, these divergent pieces of data suggest a differential role for OPN in metastasis in contrast to tumorigenesis. As a result, OPN is the subject of a great deal of investigative interest as the result of its presumed primary role in mediating metastasis.
A number of studies have addressed the potential mechanisms by which exogenous OPN stimulates cell motility in breast cancer. However, little is known of the regulation of OPN expression. Several studies have addressed transcriptional regulation of OPN in the context of breast malignancy. When small 1-kb fragments of genomic DNA obtained from human malignant breast cancer lines are transfected into a benign rat mammary cell line, OPN gene expression is upregulated with subsequent metastatic behavior when implanted into syngeneic rats (26). Subsequent work indicated that the endogenous inhibitory Tcf-4 is sequestered and as a result, OPN transcription is increased. Work by this same group (27) has also shown that Ets, PEA3, ␤-catenin-Lef-1, and c-Jun synergize to increase OPN transcription in a rat mammary cell line. It is interesting that although c-Jun was found to be essential for OPN transcription, these authors identified an AP-1 binding site at nt Ϫ1872 and did not examine the nt Ϫ107 region, which is integral to our observations. Finally, Liu and colleagues (28) investigated the transcriptional activation of the OPN promoter in the human metastatic cancer cell line A2058. They found that the region between Ϫ170 and Ϫ127 in the human OPN promoter acts as an enhancer element. This region was found to contain overlapped AML-1 and CCAAT/ enhancer-binding protein binding site motifs. Functional analysis showed that the CCAAT/enhancer-binding protein ␣ was more potent than the complex of AML-1 and its cofactor CCAAT-binding transcription factor ␤ to up-regulate the OPN promoter. The authors conclude that AML-1 and CCAAT/enhancer-binding protein ␣ play an important role in the upregulation of the OPN gene in metastatic tumor cells. When our results are examined against this background, the relevant role of c-Jun corroborates the findings of El-Tanani and coworkers (27). Indeed, other activators, co-activators, or repressors may contribute to OPN expressional programming.
Although OPN transcription has received some attention, even less is known of the upstream signal transduction pathways that regulate endogenous OPN expression in the setting of malignancy. Denhardt and coworkers (29) have studied the potential role of Ras in OPN regulation. Ras signaling in fibroblasts and epithelial cells stimulates binding of the Ras-response factor to a Ras-activated enhancer in the OPN promoter to enhance OPN transcription. In a further study, Wu and Denhardt (30) used 3T3 cells derived from wild-type and OPNdeficient mice and transformed by transfection with oncogenic Ras to assess the role of OPN in transformation in vitro and in tumorigenesis in vivo. Ras-transformed cell lines from both wild-type and OPN-deficient mice could form colonies in soft agar, indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced compared with wild-type cell lines. Their results indicate that maximal transformation by Ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the Ras oncogene. However, in 4T1 cells, our results suggest that OPN expression is not dependent upon Ras activity.
The contribution of PI3K to a metastatic phenotype in breast cancer cells has been studied. Verbeek and colleagues (31) have demonstrated a critical role for PI3K in the migration characteristics of the ZR75-1 human breast cancer cell line. In a similar fashion, Tan and colleagues (32) found that heregulin-␤1 activation of PI3K enhances breast cancer cell aggregation in MCF-7 and SKBR3 breast cancer cells. Finally, van Golen et al. (33) examined the role of PI3K in the SUM149 inflammatory breast cancer cell line and demonstrated a critical role for PI3K in anchorage-independent growth and survival. Very little is known with respect to the participation of PI3K in the expression of OPN. Zhang and colleagues (34) used benign breast epithelial cell lines to demonstrate that PI3K-Akt kinase activity can regulate accelerated cell division and increase OPN expression. Otherwise, in HepG2 cells, inhibition of the upstream activator of Akt, PI3K, using wortmannin significantly inhibited epidermal growth factor induced OPN expression (35). The underlying transcriptional regulatory mechanisms have not been previously addressed in the context of OPN-associated metastatic phenotypes.
In this study, we demonstrate that 4T1 breast cancer cells overexpress OPN compare with a sister subclone line, 4T07 cells. Enhanced expression of OPN is associated with significantly enhanced properties of adhesion, migration, and invasion. On the other hand, inhibition of OPN expression in these 4T1 cells ablates this more aggressive metastatic phenotype. Elevated OPN production is transcriptionally mediated via a Ras-independent, PI3K-dependent, and JNK-dependent pathway that activates c-Jun with a subsequent increase in binding of an AP-1 c-Jun homodimer to a region of the OPN promoter. The mechanism by which PI3K is constitutively activated is presently unknown, but the potential contribution of growth factors and/or phosphatase and tensin homolog is the subject of ongoing studies. Investigators have previously demonstrated a correlation between decreased PTEN expression in breast cancer. Garcia and colleagues (36) found that 40% of breast cancers show a decrease or absence of PTEN, with hypermethylation of the PTEN promoter in 48%. Likewise, Chung and coworkers (37) also found that loss of PTEN expression was common in the setting of breast cancer, and this loss correlates with tumor progression and lymph node metastasis. These data suggest that PTEN/PI3K and their relationship with OPN may come to be a fruitful area of investigation.