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Cancer Research 67, 1105, February 1, 2007. doi: 10.1158/0008-5472.CAN-06-3867
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

The Tumor Suppressor Activity of the Lysyl Oxidase Propeptide Reverses the Invasive Phenotype of Her-2/neu–Driven Breast Cancer

Chengyin Min1, Kathrin H. Kirsch1, Yingshe Zhao1, Sébastien Jeay1, Amitha H. Palamakumbura2, Philip C. Trackman2 and Gail E. Sonenshein1

1 Department of Biochemistry, Boston University School of Medicine, and 2 Division of Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts

Requests for reprints: Gail E. Sonenshein, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Phone: 617-638-4120; Fax: 617-638-4252; E-mail: gsonensh{at}bu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the lysyl oxidase gene (LOX) was found to inhibit the transforming activity of the ras oncogene in NIH 3T3 fibroblasts and was hence named the ras recision gene (rrg). Lysyl oxidase (LOX) is synthesized and secreted as a 50-kDa inactive proenzyme (Pro-LOX), which is processed by proteolytic cleavage to a functional 32-kDa enzyme and an 18-kDa propeptide (LOX-PP). Recently, the ras recision activity of the LOX gene in NIH 3T3 cells was mapped to its propeptide region. Here, we show for the first time that LOX-PP inhibits transformation of breast cancer cells driven by Her-2/neu, an upstream activator of Ras. LOX-PP expression in Her-2/neu–driven breast cancer cells in culture suppressed Akt, extracellular signal–regulated kinase, and nuclear factor-{kappa}B activation. Her-2/neu–induced epithelial to mesenchymal transition was reverted by LOX-PP, as judged by reduced levels of Snail and vimentin; up-regulation of E-cadherin, {gamma}-catenin, and estrogen receptor {alpha}; and decreased ability to migrate or to form branching colonies in Matrigel. Furthermore, LOX-PP inhibited Her-2/neu tumor formation in a nude mouse xenograft model. Thus, LOX-PP inhibits signaling cascades induced by Her-2/neu that promote a more invasive phenotype and may provide a novel avenue for treatment of Her-2/neu–driven breast carcinomas. [Cancer Res 2007;67(3):1105–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The enzyme lysyl oxidase (LOX) catalyzes oxidative modifications that promote formation of lysine-derived covalent cross-links needed for the normal structural integrity of the extracellular matrix (1, 2). LOX is synthesized and secreted as a 50-kDa inactive proenzyme (Pro-LOX), which is processed by proteolytic cleavage to a functional 32-kDa enzyme (LOX) and an 18-kDa lysyl oxidase propeptide (LOX-PP; ref. 3). Expression of the LOX gene was found to inhibit the transforming activity of the ras oncogene in NIH 3T3 fibroblasts and was hence named the ras recision gene (rrg; refs. 4, 5). Reduced LOX levels were observed in many cancers and derived cell lines (4, 6–11), whereas in spontaneous revertants or upon induced phenotypic reversion, higher normal levels of LOX were again seen (4, 12). Conversely, stable phenotypic revertants of ras-transfected NIH 3T3 cells return to a transformed phenotype upon transfection with an antisense LOX vector (4, 5, 13, 14). Recently, we showed that ectopic expression of Pro-LOX in ras-transformed NIH 3T3 cells inhibits the activities of phosphatidylinositol 3-kinase, Akt, extracellular signal–regulated kinase (Erk) 1/2, and nuclear factor-{kappa}B (NF-{kappa}B; ref. 15). Subsequently, we reported that, unexpectedly, it is LOX-PP, and not the LOX enzyme, that inhibits ras-dependent transformation of NIH 3T3 fibroblasts as determined by effects on proliferation, growth in soft agar, and ras-dependent signaling that induces NF-{kappa}B activity (16). These findings identify the 18-kDa LOX-PP as a novel inhibitor of ras-mediated transformation of fibroblasts.

The epidermal growth factor receptors (EGFR) are a family of tyrosine kinases, which are generally activated by peptide ligand binding and subsequent receptor dimerization and tyrosine phosphorylation on the cytoplasmic tail (17). The family consists of the EGFR gene ERBB1 (HER1), ERBB2/HER2/neu, ERBB3/HER3, and ERBB4/HER4, with EGFR and Her-2/neu being overexpressed in a wide variety of tumors. Overexpression of the ERBB2/HER2/neu receptor has been seen in ~30% of breast cancers (18, 19) and is sufficient to activate receptor signaling. Her-2/neu receptor activation, via dimerization with other EGFR family members or itself, signals via Ras to activate the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase (MAPK)/Erk kinase pathways that lead to induction of NF-{kappa}B (20–23). Breast cancers expressing high levels of Her-2/neu usually express undetectable or very low levels of estrogen receptor (ER){alpha} and typically display resistance to antiestrogens (24). Many clinical and laboratory investigations have shown that overexpression of Her-2/neu in breast cancer is associated with increased mesenchymal properties [or epithelial to mesenchymal transition (EMT)], metastasis and resistance to chemotherapeutic agents (25), and poor prognosis and overall survival (26, 27). EMTs occur as key steps during embryonic morphogenesis and are now implicated in the progression of primary tumors towards metastasis. During EMT, cancer cells lose expression of proteins that promote cell-cell contact, such as E-cadherin and {gamma}-catenin, and acquire mesenchymal markers, such as Snail, a repressor of E-cadherin gene transcription, and vimentin, which leads to a more migratory and invasive phenotype (28). Furthermore, the inhibition of ER{alpha}, seen in Her-2/neu breast cancer, leads to a decrease in transcription of MTA3, a component of the histone deacetylase complex NURD, which represses Snail gene transcription, and thereby reduces E-cadherin expression. Here we have asked whether the rrg activity of LOX-PP can be extended to breast cancer cells driven by the Her-2/neu receptor, which signals via Ras. We report that LOX-PP, which is underexpressed in many breast cancer cell lines, is a potent inhibitor of EMT of Her-2/neu–driven breast cancer cells in vitro and tumor formation in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and treatment conditions. The mouse mammary tumor virus (MMTV)-Her-2/neu cell line NF639, MMTV-v-H-ras cell line SH1-1, and MMTV-c-myc cell line M158 (kindly provided by P. Leder, Harvard Medical School, Boston, MA) were derived from mammary gland tumors and cultured as described. The ras-transformed cell line 3T3-Ras (kindly provided by D. Faller, Boston University Medical School, Boston, MA; ref. 15) and BOSC 23 (29) were cultured as described. For NF639 stable lines, 2 µg/mL doxycycline (Sigma, St. Louis, MO) was added to the medium to induce transgene expression. Where indicated, cells were pretreated with doxycycline in DMEM-10% fetal bovine serum (FBS) for 4 h. The medium was then changed to DMEM-0.5% FBS with doxycycline and the cultures were incubated for another 48 h and then stimulated with addition of FBS to 10% for either 90 min or 15 h.

Plasmids. Wild-type murine Pro-LOX and LOX enzyme containing COOH-terminal V5/His tag were excised from pcDNA4-V5/His vector (kindly provided by P. Sommer, Institut de Biologie et Chimie des Protéines, Lyon, France; ref. 30) and cloned into retroviral vector pC4bsrR(TO) containing a doxycycline-inducible promoter or vector pCXbsr under the control of constitutive cytomegalovirus immediate-early promoter (generously provided by T. Akagi, OBI, Osaka, Japan). LOX-PP cDNA was generated by PCR and inserted into pcDNA4-V5/His vector in frame with the V5/His tag. The tagged LOX-PP fragment was then subcloned into pC4bsrR(TO) and pCXbsr vectors.

Retroviral infection and expression. Retrovirus stocks were generated using the ecotropic packaging cell line BOSC 23 (29). pCLEco vector (Imgenex, San Diego, CA) was cotransfected with either empty effector vector pC4bsrR(TO) or vector bearing the indicated cDNA fragment with COOH-terminal V5/His tag, or the regulator vector pCXneoTR2. After 48 h, NF639 cells were dually infected with filtered culture supernatant from BOSC 23 cells containing viruses that carry the regulator vector and effector vectors supplemented with 6 µg/mL polybrene. Infected cells were selected with 10 µg/mL blasticidin (Invitrogen, Carlsbad, CA) and 1.4 mg/mL active geneticin (Invitrogen) to generate separate pools of stable infectants of EV-, Pro-LOX–, LOX-, and LOX-PP–expressing NF639 cells. Expression of the LOX constructs in whole-cell extracts and conditioned medium was confirmed by immunoblotting.

Focus formation assay. Cells were plated in triplicate at 104/mL in top plugs consisting of complete F-12 nutrient mixture (Ham) medium and 0.4% SeaPlaque agarose. Where indicated, cells were incubated in the presence of 2.5 µg purified bovine aorta LOX enzyme (31), 2.5 µg recombinant rat LOX-PP (32), or the same volume of vehicle potassium phosphate (16 mmol/L, pH 7.8). Following incubation for 14 days, colonies were stained with 0.0005% crystal violet solution for 1 h and counted using a dissecting microscope (x50 magnification). Three random fields were counted from each triplicate sample and values presented as average ± SD.

Immunoblotting. Whole-cell extracts were prepared in radioimmunoprecipitation assay (RIPA) lysis buffer with phosphatase and protease inhibitors (1 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 10 mmol/L p-nitrophenylphosphate, 1 mmol/L Na3VO4, 10 mmol/L NaF, 10 mmol/L ß-glycerol phosphate) and samples subjected to immunoblotting, as described (15). Antibodies against phospho-Akt (Ser473), Akt, phospho-Erk1/2, and Erk1/2 were obtained from Cell Signaling (Danvers, MA). ER{alpha} and vimentin antibodies were from NeoMarker (Fremont, CA) and E-cadherin and {gamma}-catenin antibodies from BD Transduction Laboratories (Franklin Lakes, NJ). Antibodies against cyclin D1, ß-actin, and the V5 epitope were from Santa Cruz Biotechnology (Santa Cruz, CA), Sigma, and Invitrogen, respectively. A mouse monoclonal antibody against Snail protein (33) was kindly provided by D. Schendel (GSF-Institut für Molekulare Immunologie, Munich, Germany). To detect expression of recombinant proteins in cell culture medium, 1 mL of the 10-mL culture medium was subjected to immunoprecipitation with a V5 antibody and protein A-Sepharose. Immunoblot analysis was done with anti-V5 antibody followed by incubation with protein A conjugated to horseradish peroxidase.

Electrophoretic mobility shift assay. Nuclear extracts were prepared as described (15). The sequence of the oligonucleotide containing NF-{kappa}B element upstream of the c-myc gene is as follows: 5'-GATCCAAGTCCGGGTTTTCCCCAACC-3'. Electrophoretic mobility shift assay was done using 5 µg of nuclear extracts with either the NF-{kappa}B or Oct-1 oligonucleotide as probe, as we previously described (15).

Reverse transcription-PCR analysis. Total RNA was extracted and purified using TRIzol reagent. Samples (5 µg) were reverse transcribed using SuperScript II Reverse Transcriptase with random primers (Invitrogen). Amplification of Snail cDNA was done with the following primers: forward, 5'-ACATCCGAAGCCACACGCTG-3'; reverse, 5'-AGTGAGGAGGAGGGTGAGCT-3'. PCR was done in a Thermal Cycler for 19 cycles as follows: 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s. PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Migration assay. NF639 stable cells were treated with 2 µg/mL doxycycline in DMEM-0.5% FBS for 48 h. Subsequently, suspensions of 1 x 105 cells were layered in the upper compartments of Transwells (Costar, Cambridge, MA) on an 8-mm diameter polycarbonate filter (8-µm pore size) and incubated at 37°C for 6 h. Migration of the cells to the lower side of the filter was evaluated with the acid phosphatase enzymatic assay using p-nitrophenylphosphate (Sigma) and A410 nm determination.

Matrigel outgrowth assay. Following induction with 2 µg/mL doxycycline for 24 h, 1 x 104 cells were resuspended in 10 µL of serum-free DMEM and mixed with 0.2 mL of ice-cold Matrigel (6.3 mg/mL; BD Biosciences, Bedford, MA) as we have described (34). The mixtures were put onto a preset Matrigel layer in 24-well plates, solidified, and overlaid with 500-µL DMEM containing 10% FBS and 2 µg/mL doxycycline. After 5 days, structures of the cells were analyzed with a Zeiss Axiovert 200M microscope.

In vivo xenograft mouse model. NCrnu/nu nude mice were purchased from Taconic Laboratories (Albany, NY) at 7 to 9 weeks of age. Mice were housed in a two-way barrier at the Boston University School of Medicine Transgenic mouse facility in accordance with the regulations of the American Association for the Accreditation of Laboratory Animal Care. NF639 cells infected with empty pCXbsr vector (EV) or pCXbsr carrying LOX-PP were injected s.c. (4 x 106 per injection) in both flanks (EV, left; LOX-PP, right) of nude mice (n = 6) 16 h postinfection. Tumor size was measured with calipers and tumor volumes were calculated using the following formula: (length x width2)/2. Mice were sacrificed when tumors of at least one mouse in the set had reached a size of ~1.0 cm in diameter (day 23). Tumors were dissected out and weighed and snap frozen in liquid nitrogen. Protein extracts were prepared by homogenizing frozen tumor specimens in RIPA lysis buffer containing a cocktail of protease and phosphatase inhibitors, as above. Equal amounts of protein were subjected to immunoblotting, as above. Tumor weights between groups EV and LOX-PP were compared using two-tailed, paired Student's t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LOX-PP, but not the LOX enzyme, inhibits anchorage-independent growth of breast cancer cells transformed by Ras and Her-2/neu. The SH1-1 v-H-ras– and NF639 Her-2/neu–transformed mammary tumor cells have the ability to grow in soft agar, a hallmark of transformed phenotype. We compared the effects of purified LOX enzyme and LOX-PP versus vehicle control on anchorage-independent growth of these cells. LOX-PP was strongly inhibitory, whereas the LOX enzyme was unable to inhibit soft agar colony formation of SH1-1 or NF639 cells (Fig. 1A ). As a positive control, we similarly tested the effects of LOX-PP and LOX on NIH 3T3 cells transformed by H-Ras (3T3-Ras). Consistent with our previous work (16), growth in soft agar of NIH 3T3-Ras cells was potently (~80%) inhibited by LOX-PP but not by LOX enzyme (Fig. 1B). As a negative control for specificity of action, the c-myc–transformed M158 fibroblasts were similarly analyzed. No effect of LOX-PP was seen (Fig. 1), indicating that LOX-PP–mediated reversion is not universal and seems to be selective for cells transformed by Ras or Her-2/neu. Thus, the 18-kDa LOX-PP inhibits anchorage-independent growth induced via aberrant Ras signaling in breast cancer cells.


Figure 1
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  		Figure 1. LOX-PP inhibits colony formation of Ras- and Her-2/neu–induced breast cancer cells. Ras-transformed NIH 3T3 cells (3T3-Ras) and breast cancer cells transformed by either v-H-ras (SH1-1), Her-2/neu (NF639), or c-myc (M158) were plated in 0.4% soft agar, in the absence (Control) or the presence of 2.5 µg/mL of either 32-kDa LOX active enzyme (+LOX) or 18-kDa LOX-PP (+LOX-PP). A, after 2 wks, the colonies were stained with 0.0005% crystal violet and photographed by using a digital camera coupled to a dissecting microscope. B, top, schematic representation of Pro-LOX. SP, signal peptide; PP, propeptide domain. Bottom, to determine the number of foci in (A), three random fields were counted from each triplicate sample. Columns, mean colony-forming units per square centimeter from two separate experiments; bars, SD.

 
LOX-PP inhibits signaling pathways induced by Her-2/neu. To directly test the effects on Her-2/neu signaling, NF639 mammary tumor cells were infected with replication-defective retrovirus carrying either doxycycline-inducible Pro-LOX, LOX, LOX-PP, or empty vector (EV) constructs (Fig. 2A ) and stable cell populations were isolated (termed NF639-Pro-LOX, NF639-LOX, NF639-LOX-PP, and NF639-EV, respectively). Western blot analysis with V5 antibody of cell extracts and conditioned medium 48 h postinduction with 2 µg/mL doxycycline showed that all of the constructs were expressed (Fig. 2B). The Pro-LOX (50 kDa) was efficiently secreted into the medium and processed to the mature enzyme and the propeptide, which is not detected due to the loss of the tag. LOX-PP (18 kDa) was detected as a doublet of ~25 kDa, possibly due to N-glycosylation. Although the signal peptide of Pro-LOX was fused to the coding region for the mature enzyme, the enzyme from the LOX construct was only detected in cell extracts and not in the medium (Fig. 2B), consistent with the reported requirement of the propeptide region for normal secretion of Pro-LOX (30).


Figure 2
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  		Figure 2. LOX-PP reduces Her-2/neu–mediated activation of Akt and Erk. A, schematic representation of the V5/His-tagged Pro-LOX, LOX enzyme, and LOX-PP cDNA fragments. These fragments were cloned into the doxycycline-inducible retroviral vector pC4bsrR(TO) and transduced into NF639 cells. EV DNA was used as control. B, expression of the recombinant proteins was induced in stable polyclonal pools of NF639-EV, NF639-Pro-LOX, NF639-LOX, or NF639-LOX-PP cells by treatment with 2 µg/mL doxycycline for 48 h. Whole-cell protein extract (WCE) and cell culture medium (MED) were collected. One milliliter of the 10-mL medium was subjected to immunoprecipitation as described in Materials and Methods. Immunoblot analysis was done on whole-cell extracts (20 µg) and immunoprecipitated medium samples with anti-V5 antibody followed by protein A conjugated to horseradish peroxidase. Representative of two independent experiments with similar results. The positions of Pro-LOX, LOX, LOX-PP, immunoglobulin G heavy chain, and marker polypeptides (50, 37, and 25 kDa, respectively) are indicated. *, position of Pro-LOX above the heavy chain band. C and D, left, NF639-EV, NF639-Pro-LOX, NF639-LOX, and NF639-LOX-PP cells were treated with doxycycline for 4 h. The medium was changed to DMEM-0.5% FBS with doxycycline and the cultures were incubated for another 48 h and then stimulated with addition of FBS to 10% for 90 min. Whole-cell extracts were prepared and samples (50 µg protein) subjected to immunoblotting with antibodies against Ser473 phosphorylated Akt and total Akt or phosphorylated Erk1/2 and total Erk1/2, as indicated. C and D, right, cultures were treated with doxycycline for 24 h and whole-cell extracts analyzed by immunoblotting as above. Representative of two independent experiments with similar results.

 
Oncogenic Her-2/neu, working via Ras, activates the phosphatidylinositol 3-kinase/Akt and MAPK/Erk signal transduction pathways (15, 23). The effects of Pro-LOX, LOX, and LOX-PP on Akt and Erk activation were compared following FBS stimulation of serum-deprived cells or 24 h postinduction with doxycycline (Fig. 2C and D, left and right, respectively). In both cases, Pro-LOX suppressed phosphorylation of both Akt and Erk1/2, as was seen previously with Ras-transformed NIH 3T3 fibroblasts (15). Importantly, LOX-PP was even more potent than Pro-LOX in suppressing phosphorylation of Akt and Erk1/2 in NF639 breast cancer cells (Fig. 2C and D). Surprisingly, expression of the LOX enzyme resulted in a modest increase in activation of Akt as well as Erk1/2. Thus, LOX-PP effectively reduces Her-2/neu–mediated activation of Akt and Erk in NF639 breast cancer cells.

The transforming activity of Her-2/neu and Ras is mediated, in part, via induction of NF-{kappa}B by the phosphatidylinositol 3-kinase/Akt and MAPK/Erk signaling pathways (23, 35). Thus, we examined the effects of Pro-LOX, LOX, and LOX-PP on NF-{kappa}B activity in NF639 cells. A substantial reduction in NF-{kappa}B binding was observed in response to expression of LOX-PP and, to a lesser extent, of Pro-LOX (Fig. 3A ). In contrast, expression of LOX enzyme led to an increase in NF-{kappa}B binding activity. Cyclin D1, a target gene of NF-{kappa}B and ß-catenin, is expressed at an elevated level in >50% of human breast tumors of all histologic types (36). Only expression of LOX-PP substantially blocked the induction of cyclin D1 levels seen after serum stimulation for 15 h (Fig. 3B). Taken together, our data indicate that LOX-PP suppresses downstream targets mediating transformation by oncogenic Her-2/neu more potently than Pro-LOX, consistent with the tumor suppressor activity residing within this domain. Furthermore, expression of the LOX enzyme seems to have opposing effects on NF-{kappa}B, Akt, and Erk activities.


Figure 3
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  		Figure 3. LOX-PP reduces Her-2/neu–mediated activation of NF-{kappa}B and cyclin D1. A, cultures were serum starved and stimulated as in Fig. 2C (left) and nuclear extracts subjected to electrophoretic mobility shift assay for binding of NF-{kappa}B (top) or Oct-1 (bottom). Essentially equal loading was verified by analysis of Oct-1 binding levels; the slightly lower level of Oct-1 seen with the LOX enzyme suggests that the increase in binding may be somewhat underestimated. Representative of two independent experiments with similar results. B, cultures were serum starved as in Fig. 2C (left) and stimulated with 10% FBS for 15 h. Whole-cell extracts were analyzed by immunoblotting for cyclin D1 and ß-actin, which confirmed essentially equal loading. Representative of three independent experiments with similar results.

 
LOX-PP reverses EMT. Her-2/neu and Ras downstream signaling has been implicated in promoting a mesenchymal, invasive phenotype (34, 37–39). Her-2/neu–transformed NF639 cells have a fibroblastoid, invasive phenotype (Fig. 4A ) and express low levels of ER{alpha} (40) and of the epithelial marker E-cadherin and high levels of the mesenchymal markers Snail and vimentin (41). The cultures were examined for changes in morphology upon induction of LOX-PP, and a marked increase in cell-cell adhesion was noted (Fig. 4A). Consistent with more cell-cell contacts, induction of LOX-PP substantially increased the level of E-cadherin, whereas the effects of Pro-LOX were much less pronounced (Fig. 4B). Furthermore, LOX-PP expression resulted in a substantial increase in ER{alpha} (Fig. 4C). The protein {gamma}-catenin is another adherens junction protein that is frequently associated with epithelial phenotype. LOX-PP induced a moderate increase in {gamma}-catenin expression (Fig. 4D). Consistent with the induction of ER{alpha}, LOX-PP expression was found to substantially reduce levels of Snail mRNA and protein (Fig. 5A ). Furthermore, LOX-PP inhibited the expression of the mesenchymal marker vimentin more efficiently than Pro-LOX (Fig. 5B). Conversely, the LOX enzyme caused a substantial decrease in levels of ER{alpha} and {gamma}-catenin and a modest increase in vimentin (Figs. 4C and D and 5B), which has been shown to be an NF-{kappa}B target gene, in agreement with its effects on signaling observed above (Fig. 3).


Figure 4
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  		Figure 4. LOX-PP induces a more epithelial phenotype. A, after induction with doxycycline for 48 h, NF639-EV and NF639-LOX-PP cells were photographed using an Orca-ER camera attached to an Axiovert 200M microscope. B to D, NF639 cells were induced with doxycycline and serum starved and stimulated with FBS as in Fig. 3B and whole-cell extracts prepared. Samples (50 µg) were subjected to immunoblot analysis for E-cadherin (E-cad; B), ER{alpha} (C), or {gamma}-catenin (D), or ß-actin, which confirmed equal loading. B and C, representative of two to four independent experiments with similar results.

 

Figure 5
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  		Figure 5. LOX-PP inhibits invasive phenotype. NF639 cells were induced with doxycycline and serum starved and stimulated with FBS as in Fig. 3B and either RNA or whole-cell extracts prepared. A, Snail and GAPDH mRNA levels were determined by reverse transcription-PCR (top). Representative of two independent experiments with similar results. Alternatively, whole-cell extracts were subjected to immunoblot analysis for Snail protein or ß-actin as loading control (bottom). B, samples (50 µg) were subjected to immunoblot analysis for vimentin or ß-actin, which confirmed equal loading. Representative of three independent experiments with similar results. C, following a 48-h period of induction with doxycycline, NF639 cells were subjected to a migration assay for 6 h, in triplicate, and cells that migrated to the lower side of the filter were quantified by spectrometric determination at A410 nm. Columns, mean; bars, SD. D, after induction with doxycycline for 24 h, NF639-EV, NF639-LOX-PP, and NF639-LOX cells were subjected to Matrigel outgrowth assay. After 5 d, cultures were photographed using a Zeiss Axiovert 200M microscope and an Orca-ER camera at a magnification of x50 (top) or x100 (bottom); representative images are shown from one of two independent experiments with similar results.

 
To determine whether LOX-PP expression can reduce the invasive properties of Her-2/neu breast cancer cells, NF639-EV and NF639-LOX-PP cells were subjected to migration assay. Expression of LOX-PP reduced migration of NF639 cells (Fig. 5C). Finally, we examined the effects of LOX-PP on the ability of highly malignant NF639 cells to form invasive, branching colonies in Matrigel (Fig. 5D). NF639-EV cells formed the typical branching, invasive colonies seen with NF639 cells after 5 days in culture in Matrigel. The expression of LOX-PP prevented the formation of these structures and only small round colonies were observed with NF639-LOX-PP cells (Fig. 5D, top). In contrast, expression of the LOX enzyme seemed to facilitate more rapid formation of branching structures, which were thus larger, than those seen with the control NF639-EV cells (Fig. 5D, bottom). Only a marginal suppression of invasive colony formation in Matrigel was observed by expression of Pro-LOX (data not shown), consistent with the effects on E-cadherin. Thus, LOX-PP promotes a less invasive phenotype and reverses EMT in Her-2/neu breast cancer cells.

LOX-PP suppresses Her-2–driven tumor formation in vivo. Next, we sought to determine whether LOX-PP can reduce tumor formation in vivo. The effects of LOX-PP on tumorigenicity in nude mice were examined by s.c. injection of newly infected populations of NF639 cells with either the EV or LOX-PP. Specifically, nude mice (n = 6) were injected in the left or right flanks with 4 x 106 NF639 cells infected with retroviruses carrying EV or LOX-PP constructs, respectively. Tumors developed only slowly in both populations; however, by day 17, the tumors resulting from the EV-infected NF639 cells began to grow at a faster rate and were notably larger than those expressing LOX-PP (Fig. 6A ). The differences in tumor volume continued to increase until the experiment was halted on day 23 (Fig. 6B). A substantially lower weight of tumors formed by cell populations expressing LOX-PP versus the EV was noted (Fig. 6C, inset). The average tumor weight for LOX-PP xenografts was 38.5% of that for the EV group on day 23 (P = 0.037; Fig. 6C). Analysis of protein extracts from tumor tissues revealed substantially lower cyclin D1 levels in tumors derived from cells infected with LOX-PP (Fig. 6D). Furthermore, consistent with the in vitro data on expression of EMT genes, the tumors formed with cells expressing LOX-PP displayed an induction of epithelial markers {gamma}-catenin, ER{alpha}, and E-cadherin versus EV (Fig. 6D). Similar data were obtained with extracts from other mice (data not shown). Thus, LOX-PP suppresses Her-2/neu–driven tumor formation in vivo.


Figure 6
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  		Figure 6. LOX-PP reduced in vivo tumor growth of NF639 cells. NF639 cells infected with retroviruses carrying empty pCXbsr vector (EV) or LOX-PP were injected s.c. (4 x 106 cells per injection) at two sites in the flanks (EV, left; LOX-PP, right) of NCrnu/nu nude mice 16 h postinfection. Six mice were analyzed for tumor growth for 23 d posttransplantation. A, tumor volumes were measured with calipers and tumor growth curves plotted. Bars, SE. B, image of a representative mouse sacrificed on day 23. C, total tumor weights were determined on day 23. Columns, average tumor weights; bars, SE. Statistical significance of the effects of LOX-PP relative to EV on tumor weights was confirmed by Student's t test (P = 0.037). Inset, representative tumors derived from the same animals by injection of NF639 cells infected with EV or LOX-PP on the left and right flanks, respectively. D, protein extracts from tumor tissue of one representative mouse were analyzed by immunoblotting for cyclin D1, {gamma}-catenin, ER{alpha}, E-cadherin, and ß-actin, which confirmed equal loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we show for the first time that LOX-PP functions as a tumor suppressor in breast cancer cells both in vitro and in vivo, and reverts the invasive phenotype of Her-2/neu–driven cancer cells. LOX-PP reduced transformed phenotype, signaling via the phosphatidylinositol 3-kinase/Akt and MAPK/Erk pathways, and downstream NF-{kappa}B activity and cyclin D1 levels in Her-2/neu–driven cells in culture. Expression of LOX-PP induced an epithelial profile of gene expression (i.e., induced E-cadherin and {gamma}-catenin and reduced Snail and vimentin). LOX-PP also inhibited formation of branching structures in Matrigel and migration of the cancer cells. Anchorage-independent growth of Ras-transformed breast cancer cells was also inhibited by LOX-PP (Fig. 1) or Pro-LOX (data not shown). Importantly, LOX-PP suppressed the growth of Her-2/neu–driven tumors in a xenograft model, with a >60% reduction in tumor burden measured. Decreased levels of LOX gene expression have been seen to typify many breast and other cancer cell lines (4, 6–8) and primary human cancers, such as colorectal, prostate, and gastric cancers (9–11). Furthermore, ectopic Her-2/neu expression in NIH 3T3 cells was recently shown to lead to a profound reduction in LOX mRNA levels (42). Our data indicate that reduced LOX-PP expression, as mediated by Her-2/neu, likely leads to increased susceptibility to invasive breast cancer.

In contrast to the effects of LOX-PP, we observed that intracellular expression of the LOX enzyme enhanced Akt, Erk, and NF-{kappa}B activities, suggesting that it promotes a more invasive phenotype. Interestingly, intracellular localization of the LOX enzyme was noted in metastatic breast disease, whereas in normal tissue it resided predominantly in the stromal tissue (43). Furthermore, the active enzyme was recently found to promote breast cancer cell migration and metastasis (43–45). In a recent study, Payne et al. (43) observed enhanced migration only in MCF-7 cells expressing LOX, whereas expression of Pro-LOX resulted in reduced migration. Consistent with these observations, Kaneda et al. (11) showed the ability of Pro-LOX to inhibit tumor formation by gastric cancer cells in a nude mouse model. In our studies, expression of Pro-LOX also inhibited activation of Akt and Erk, but to a lesser extent than LOX-PP. Taken together, these findings indicate that the propeptide region and mature enzyme encoded by the LOX gene exert opposing effects on tumor progression and invasion. These findings suggest that the propeptide region of Pro-LOX may be the natural regulator of the growth-promoting and motility-enhancing activities of the enzyme LOX, which is required for elastin and collagen cross-linking essential for formation of a functional cardiovascular system (46). This likely explains the more potent inhibition of Her-2/neu–mediated transformation by LOX-PP compared with Pro-LOX, and further indicates the rrg activity of the LOX gene resides within the propeptide domain. As discussed above, decreased levels of LOX gene expression typify many primary human cancers, including those frequently associated with ras mutations, such as lung and pancreatic cancers. We have begun testing the hypothesis that the loss of LOX-PP activity plays an important role in the etiology of these cancers, and have shown the ability of LOX-PP to inhibit the transformed phenotype of human lung and pancreatic cancer cell lines with mutant ras genes.3 Overall, our findings suggest the potential use of this peptide (or of subdomains) in treatment modalities of human cancers driven by Ras or Her-2/neu.

The precise mechanism by which LOX-PP inhibits Her-2/neu– or Ras-mediated transformation remains to be determined. Structure prediction studies using DISOPRED (47), GlobPlot (48), and DisProt (49) indicate that the propeptide region assembles as an intrinsically disordered protein. Intrinsically disordered protein structures have atomic coordinates and Ramachandran angles that vary significantly over time (50). When intrinsically disordered proteins bind to other proteins, the bound protein confers structure and biological activity to the "disordered" protein. Purified LOX-PP has aberrant mobility in SDS-gel electrophoresis, which is a characteristic of proteins that contain a disordered structure (51). Yeast two-hybrid screening is in progress to identify interacting proteins that could affect propeptide folding. LOX-PP has not yet been crystallized, and more structural studies are needed to understand its mechanism of action and to design more effective inhibitors in cancer therapeutics.


   Acknowledgments
 
Grant support: NIH grants CA82742 and ES11624 and Department of the Army grants DAMD17-03-1-0452 and DAMD 05-1-0286.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank E. Fajardo (Albert Einstein College of Medicine, New York, NY) for expert help with structure prediction analysis, P. Sommer and D. Schendel for generously providing LOX expression vectors and Snail antibody, D. Faller and P. Leder for cell lines, and T. Akagi for retroviral vectors.


   Footnotes
 
3 M. Wu, C.M., K.H.K., P.C.T. and G.E.S., in preparation. Back

Received 10/18/06. Revised 11/15/06. Accepted 11/21/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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