Novel Function of Transcription Factor Nrf2 as an Inhibitor of RON Tyrosine Kinase Receptor-mediated Cancer Cell Invasion*

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a “repressor” and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.

Recepteur d' origine nantais (RON) 2 , a tyrosine kinase receptor, for macrophage-stimulating protein (MSP) was reported to be overexpressed in various cancers of epithelial origin (1,2). Activation of the RON receptor in breast cancer cells is linked to tamoxifen resistance (3). Further, gene expression analyses suggested that increases in RON expression are associated with metastatic disease. Transgenic mice that overexpress a wild type or constitutively active RON receptor in the mammary epithelium induced mammary transformation and associated with a high degree of metastasis (4). Aberrant RON expression in human breast cancer is associated with an aggressive cancer phenotype with decreased disease-free survival time in patients and an increase in breast cancer metastasis (5). These studies demonstrated that RON overexpression can be a causative factor for metastatic breast cancer. Nrf2 (NF-E2-related factor 2) belongs to the cap n collar subfamily of basic leucine zipper family of transcription factors (6). Its role in chemoprevention as the inducer of several hundred cytoprotective genes, which contain one or more antioxidant response elements (ARE: 5Ј-TGACnnnGC-3Ј) is well documented (7). Nrf2-null mice are susceptible to carcinogen-induced cancer development (8 -12). Nrf2 activity is regulated by keap1 (kelch-like ECHassociated protein 1). Keap1 serves as an adapter protein for interaction of cul3-based E3-ubiquitin ligase complex with Nrf2 leading to continuous ubiquitination of Nrf2 and its proteasomal degradation (13). Activators of Nrf2 like sulforaphane (SFN), an isothiocyanate, have been shown to modify the keap1 protein leading to the dissociation of Nrf2-keap1 complex resulting in the escape of Nrf2 from proteasomal degradation (14). Nrf2 was reported to be depleted in breast cancer cell lines (15). The data presented in this report indicate depletion of Nrf2 is prevalent in breast tumors and is associated with overexpression of RON. We also report that Nrf2 binds a closely related cis-element (5Ј-TGA(C/G)TCA-3Ј) to ARE but displays a novel function as an inhibitor of oncogene RON and invasion of carcinoma cells. SFN induced Nrf2 and blocked RON expression in invasive carcinoma cells from various tumor types. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" of an oncogene and RON kinase as one of the potential molecular targets of SFN, which mediates the antitumor effects of SFN.

EXPERIMENTAL PROCEDURES
Cell Culture-Cell lines were obtained from American Type Culture Collection (ATCC). Sulforaphane was purchased from LKT Laboratories.
Immunohistochemical Analysis-Breast cancer metastasis tumor microarray (60 samples) was purchased from IMGENEX (catalogue number: IMH-364). Immunohistochemical analysis using RON and Nrf2 antibodies was performed by our institution pathology core facility.
Generation of Stable Cell Lines-Scramble shRNA and shRNA keap1 and Nrf2 expression plasmids were purchased from Oregene. We have generated keap1 knock-down clones for characterization. Control Neo or Nrf2 cDNA was tran-siently expressed in MDA MB 231 cells to analyze the ectopic Nrf2 effect on RON expression.
Western Blot Analysis-All antibodies were purchased from Santa Cruz Biotechnology. To analyze the effect of sulforaphane, cells were treated for 16 h with varying concentrations of SFN prior to Western analysis.
Invasion Assay-The invasive behavior of cancer cells was analyzed as described previously (16). Quantification data represent the mean Ϯ S.D. of three separate invasion assays.
Chromatin Immunoprecipitation Assays-The ChIP assay was performed as described previously (16). RON promoter primers covering the Nrf2 sites were used to carry out PCR on DNA isolated from chromatin immunoprecipitation using control IgG or Nrf2 antibodies. Primers used to generate a 293-bp fragment covering the Nrf2 binding sites on the RON promoter. Forward: 5Ј-CTC CAA GGG CCG GAA GAG TCG GAT GG-3Ј; Reverse: 5Ј-TTA AGC AGC GGT CCC GAC AGC CCC AA-3Ј.
Luciferase Assay-Luciferase assays were performed as described previously (16). To determine the effect of SFN on wild type and the Nrf2 mutant RON promoter activity, cells transfected with Ϫ400-bp wild type or the Nrf2 mutant RON promoter were treated for 16 h with varying concentrations of SFN prior to the luciferase assay.
Immunofluorescence-Cells were fixed with paraformaldehyde and incubated with RON or Nrf2 primary antibodies overnight. After washing, the cells were incubated with secondary antibody for 2 h and RON. Nrf2 expression was analyzed using a Carl Zeiss inverted fluorescence microscope using Axio Vision Version 4.8 software.

Inverse Correlation between Nrf2 and RON Expression in
Invasive Carcinoma Cells-RON tyrosine kinase was reported to be overexpressed in various cancers of epithelial origin (1). It was recently shown that Nrf2 was present in the normal human mammary epithelial cells (HMEC) but depleted in breast tumor samples and breast cancer cell lines (15). Genomatix search identified the putative Nrf2 binding sites on the RON promoter. Immunohistochemical analysis on breast tumors with normal matching controls indicated an inverse correlation between Nrf2 and RON expression i.e. presence of high Nrf2 and low RON expression in the normal mammary tissue and lack/or low Nrf2 and high RON expression in breast tumors (Fig. 1A). We carried out Western analysis using RON (Fig. 1C). As previously reported, Nrf2 message is detected because Nrf2 expression is regulated through a post-transcriptional mechanism (15).

SFN-mediated Nrf2 Stabilization Blocks RON Expression and Invasion of Carcinoma Cells-Chemoprevention agent SFN
has been documented to modify the keap1 protein leading to the dissociation of Nrf2-keap1 complex and thus stabilizing the Nrf2 protein (14). To determine if SFN-mediated Nrf2 stabilization affects RON expression, we have treated MDA MB 231 and MDA MB 468 cells for 16 h with varying concentrations of SFN and analyzed RON and Nrf2 expression. SFN treatment stabilized Nrf2 expression as previously reported (15). However, Nrf2 expression was associated with the inhibition of RON expression ( Fig. 2A, whole extract; Fig. 2B, cytosolic and nuclear fractions) contrary to the earlier reports of Nrf2 association with gene induction. Immunofluorescence analysis confirmed the inverse correlation between Nrf2 induction and RON inhibition in the SFN-treated breast cancer cells (Fig. 2C). We carried out in vitro matrigel assay as previously described (16) to determine if Nrf2-mediated RON inhibition blocks invasion of breast cancer cells. The RON ligand, MSP-mediated invasion is abrogated in the SFN-treated carcinoma cells where RON expression was inhibited (Fig. 2D).
Nrf2 Directly Regulates the RON Promoter Activity-To determine if the decrease in RON protein expression in the SFN-treated cells was caused by a decrease in the RON mRNA expression, we have analyzed RON and Nrf2 message by RT-PCR with actin as a control. A reduction in RON but not Nrf2 message was observed in the SFN-treated cells (Fig.  3A). We have next analyzed the RON promoter activity to determine if a decrease in the RON transcription is contributing to the decreased RON message levels in the SFNtreated cells. SFN treatment inhibited the RON promoter (Ϫ400 bp wt) activity in the breast cancer cells analyzed (Fig.  3B). We have previously characterized the RON promoter and demonstrated the requirement of Sp1 binding sites at Ϫ94 and Ϫ113 bp for the basal activity (17). There are two overlapping Nrf2 binding sites at Ϫ232 bp and another Nrf2 binding site at Ϫ269 bp relative to the transcription start site (Fig. 3C). We have performed in vivo ChIP analysis as previously described (17) to identify if SFN-mediated Nrf2 binding to the RON promoter is contributing to the inhibition of RON gene expression. The binding of Nrf2 to the RON promoter fragment was observed only in the SFN-treated cells (Fig. 3C). To further confirm the contribution of Nrf2 binding sites in the inhibition of RON promoter activity, we have mutated the overlapping Nrf2 site at Ϫ232 (Nrf2 1m) and another Nrf2 site at Ϫ269 (Nrf2 2m) and analyzed the activities of the wild type (Ϫ400 bp wt) and these mutant constructs either in the absence or presence of SFN. While SFN inhibited the activity of Nrf2 sites intact wild type RON promoter the Nrf2 mutant constructs were resistant to SFNmediated inhibition (Fig. 3D).
Manipulation of the Nrf2 Expression Confirmed Its Role as a Repressor-We have increased the Nrf2 levels in the breast carcinoma cells either through ectopic Nrf2 expression or knockdown of endogenous keap1, which stabilizes Nrf2 to comple-ment the pharmacological data that SFN-mediated Nrf2 stabilization inhibits RON expression. RON expression was inhibited in the ectopic Nrf2 as well as keap1 knock-down MDA MB 231 cells (Fig. 4A). Immunofluorescence data confirmed that knock-down of keap1 increases Nrf2 and decreases RON expression (Fig. 4B). We have carried out the matrigel invasion assay to confirm if Nrf2-mediated RON inhibition in the keap1 knock-down cells abrogates RON ligand, MSP induced invasion. MSP-mediated invasion is reduced in the keap1 knock-down clones (Fig. 4C).

DISCUSSION
Numerous studies documented the clinical significance between elevated RON tyrosine kinase expression and the pathological features of breast tumors. RON receptor activation also contributes to the resistance of breast cancer cells to tamoxifen treatment (3). Hence, development of agents that can specifically inhibit RON expression/activity has the potential to impact breast cancer treatment and benefit patients. The published literature to-date documented the prominent role of Nrf2 as an inducer of gene expression including phase I and phase II cytoprotective genes that are involved in the chemo-prevention of tumorigenesis (6,7). We now report a novel functional role for Nrf2 as a repressor of an oncogene RON that was reported to be aberrantly expressed in various cancers of epithelial origin and one of the players in tumor cell invasion and metastasis (1,2). Nrf2 was reported to be present in the normal HMEC but depleted in the breast tumors and established breast tumor cell lines (15). Our studies indicated depletion of Nrf2 is widespread in carcinoma cells from various tumor types and is associated with overexpression of RON (Fig. 1B). Several reports indicated the susceptibility of Nrf2 knock-out mice to the development of carcinogen-induced tumorigenesis demon-  Nrf2 regulates RON promoter activity. A, total RNA from control or SFN-treated cells was reverse transcribed into cDNA, and PCR analysis was performed using primers for RON, Nrf2, and actin. B, cells were transfected with pGL3 control vector or Ϫ400 bp wild type (wt) RON promoter. 24 h following transfection, cells were treated with SFN for 16 h, and luciferase activity was measured following normalization to protein levels. C, ChIP assay on the chromatin fragments from control or SFN-treated cells using control IgG or Nrf2 antibodies was performed. D, cells were transfected with the Ϫ400-bp wild type RON promoter or Nrf2 binding site mutant RON promoter-luciferase reporter constructs. 24 h following transfection, cells were treated with SFN for 16 h, and luciferase activity was measured following normalization to protein levels. Mutations at the Nrf2 binding site had no significant effect on the basal RON promoter activity. Each construct is normalized to the untreated sample.
strating the importance of Nrf2 in cancer chemoprevention (8 -12). Nrf2 protein levels are regulated through an ubiquitinmediated proteasomal degradation involving adapter protein keap1 and cul3 based E3-ubiqitin ligase complex (13). The activators of Nrf2 including sulforaphane, an isothiocyanate, used in this study have been reported to modify the thiol groups of cysteine residues in keap1 protein leading to the dissociation of Nrf2 and keap1 complex thus blocking ubiquitin-mediated proteasomal degradation resulting in increased Nrf2 levels (14). SFN treatment increased Nrf2 expression in the MDA MB 231 and MDA MB 468 breast cancer cells ( Fig. 2A) and prostate, ovarian, pancreatic, and colon cancer cells (supplemental Figs. S1-S3). There are contradictory reports in the literature about the requirement of Nrf2-keap1 dissociation and stabilization of Nrf2 for the nuclear translocation of Nrf2. According to one hypothesis, Nrf2 is sequestered in the cytoplasm by keap1, and endogenous as well as exogenous activators induce dissociation of Nrf2 and keap1 complex leading to the stabilization of Nrf2 and nuclear translocation. The alternative hypothesis is that Nrf2 is continuously expressed and trans-located to the nucleus under basal conditions and keap1 shuttles between cytoplasm and nucleus and targets Nrf2 to degradation in the nucleus following trans-activation of the genes (18). The data presented in this report indicated the presence of steady state Nrf2 in both the cytoplasm and nuclear fractions in the SFN-treated breast cancer cells (Fig. 2B) thus suggesting a third alternative i.e. keap1 mediated Nrf2 degradation in both the cytosolic and nuclear compartments. However, interestingly, Nrf2 stabilization in the SFN-treated breast cancer cells is associated with inhibition of gene (RON) expression and not gene induction as previously reported (Fig. 2, A and B). The immunofluorescence data complemented the Western analysis confirming the inverse correlation between Nrf2 stabilization and RON inhibition in the SFN-treated breast cancer cells (Fig. 2C). Nrf2 is widely reported to induce target gene expression by binding to the antioxidant response element (ARE: 5Ј-TGACnnnGC-3Ј). However, RON promoter does not contain typical ARE but a closely related DNA binding sequence of Nrf2 (5Ј-TGA(C/G)TCA-3Ј) (19). The common feature in both the cis-elements is the presence of TGAC core sequence and both reportedly induce gene expression. Hence, it was surprising that our data in contrast showed that SFN treatment inhibited the RON promoter activity in breast carcinoma cells (Fig. 3B). RON promoter contains one overlapping Nrf2 (Ϫ232 bp) and another Nrf2 (Ϫ269 bp) binding sites relative to the transcription start site (Fig. 3C). We have previously identified the HIF-1␣ binding site at Ϫ285 bp (16). Chromatin immunoprecipitation analysis demonstrated the binding of Nrf2 protein to the RON promoter only in the SFN-treated breast cancer cells (Fig.  3C) and site-directed mutagenesis of the core TGAC Nrf2 binding sequence on the RON promoter further confirmed the requirement of Nrf2 binding to inhibition of the RON promoter activity (Fig. 3D). We have expected the RON promoter constructs with one wild type and one mutant Nrf2 binding sites to exhibit some inhibition in the presence of Nrf2 inducer, SFN. However, mutation at either one of the Nrf2 binding sites led to the complete loss of Nrf2 inducer, SFN mediated repression of the RON promoter. One possible explanation could be that mutation at one of the Nrf2 binding sites on the RON promoter may slightly alter the conformation of the DNA at the second Nrf2 binding site, thus preventing the binding of Nrf2 to the wild type Nrf2 binding site on the RON promoter. This will be further explored in our future studies. Previous reports suggested that small Maf proteins serve as dual function transcription factors i.e. activator or repressor-based on the heterodimerization partners (20). Consequently, it is plausible that Nrf2 binds the same cis-element but displays diverse function based on its hetero-dimerization partner. The RON promoter lacks the TATA box, is GC-rich, and depends on Sp1 transcription factor for basal activity (18). Consequently, it is possible that Sp1 in association with other positive regulators of RON expression such as NFB p65, HIF-1␣ along with histone acetyltransferases may favor the RON gene expression in the carcinoma cells. However, Nrf2 stabilization and binding to RON promoter may aid in the recruitment of histone deacetylases or other co-repressors and may prevent the binding of Sp1 activator complex thus blocking RON expression in SFN-treated cells. We will test this hypothesis in the future studies. Although, the mechanism(s) of Nrf2-mediated RON inhibition is not completely clear, the role of Nrf2 as a repressor is unequivocally demonstrated in this report because increased Nrf2 expression either through transient ectopic Nrf2 expression or stable knock-down of endogenous keap1, which stabilizes Nrf2 expression, inhibited RON tyrosine kinase expression in the invasive breast carcinoma cells (Fig. 4, A and B). Significantly, RON inhibition through SFN-mediated Nrf2 stabilization or keap1 knock-down mediated Nrf2 stabilization blocked RON ligand, MSP-mediated invasion of breast carcinoma cells (Fig. 2, D and C). Nrf2 role as a repressor of RON is not limited to breast cancer cells because SFN-induced Nrf2 expression and inhibited RON expression in carcinoma cells from prostate, ovarian, colon, and pancreas (supplemental Figs. S1-S3). Nrf2 role as a "repressor" of RON gene expression was further validated by our studies where ectopic expression of the Nrf2 negative regulator, keap1 led to a decrease in the Nrf2 levels with a concomitant increase in the RON expression in the normal human mammary epithelial cells (supplemental Fig. S4). As illustrated in Fig. 4D, we have shown that increasing Nrf2 expression through Nrf2 activator SFN or ectopic Nrf2 expression or keap1 knock-down inhibits RON expression and consequently blocks RON ligand MSP-induced invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" of an oncogene and RON kinase as one of the potential molecular targets of SFN that mediates the anti-tumor effects of SFN.