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Central Role of the Threonine Residue within the p+1 Loop of Receptor Tyrosine Kinase in STAT3 Constitutive Phosphorylation in Metastatic Cancer Cells

Zheng-long Yuan,^1,2, Ying-jie Guan,^1,2, Lijuan Wang,¹ Wenyi Wei,^3, Agnes B. Kane,¹ and Y. Eugene Chin^1,2,3*

Departments of Pathology and Laboratory Medicine,¹ Surgery Science,² Molecular Biology, Cellular Biology, and Biochemistry, Brown University School of Medicine/Rhode Island Hospital, Providence, Rhode Island³

Received 2 February 2004/ Returned for modification 26 May 2004/ Accepted 2 August 2004

	ABSTRACT

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The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met^p+1loopThr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr^p+1loop residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr^p+1loopMet substituted EPH family members constitutively phosphorylated STAT3. In both RTK^Metp+1loop with 2B mutation and wild-type EPH members the Thr^p+1loop residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET^M918T, nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr^p+1loop residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

	INTRODUCTION

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Growth factor receptors are transmembrane protein tyrosine kinases playing critical roles in biological processes, including proliferation, survival, and migration. The core region of the kinase domain, comprising the catalytic loop, the activation loop, and the p+1 loop, is well conserved in receptor tyrosine kinases (RTKs) of different species. RTKs with different types of mutations within these three loops have been associated with developmental disorders and cancer. The point mutation (ATGACG), resulting in replacement of methionine with threonine within the p+1 loop, is associated with aggressive tumors. RET is an RTK that can activate a variety of signaling pathways, including the RAS/ERK, PI3K/AKT, and phospholipase C pathways and plays an important role in neuron survival or differentiation (11). RET with a Met^p+1loopThr substitution (RET^M918T) is associated with the multiple endocrine neoplasia 2B type (MEN-2B) syndrome; this substitution is defined as the 2B mutation (11). In MEN-2B patients, the tumors derived from thyroid C cells are often more aggressive than C-cell tumors that develop in MEN-2A patients who carry mutations in the extracellular domain of RET (11, 22). Similarly, the 2B mutation in HGF receptor MET (Met^M1268T) has been identified in metastatic renal carcinomas (10, 24). Introduction of the 2B mutation in other RTKs, such as RON and epidermal growth factor receptor (EGFR), caused transformation of NIH 3T3 cells with high metastatic potential (20, 23). Although the 2B mutation enhanced kinase activity and such a mutation has been suspected as a gain-of-function mutation (21, 29, 35), the role of the Thr^p+1loop residue in RTK catalytic activity in recruiting specific substrate(s) responsible for the metastatic phenotype has not been clarified.

The importance of autophosphorylation at conserved tyrosine residues within the activation loop on kinase activity, as well as on substrate recruitment, has been well established over recent years. The p+1 loop represents a small motif, residing immediately downstream of the activation loop. It has been implicated to play a role in recognizing the residues next to tyrosine to be phosphorylated in the substrate (36). However, the precise role of the p+1 loop on the catalytic activity resulting in RTK autophosphorylation and substrate selection remains largely unknown. To identify signaling factor(s) preferentially activated by RTK carrying the 2B mutation, a novel antibody array technology was used. We show here that the oncogenic STAT (1), STAT3, was constitutively activated by different RTK^Metp+1loop 2B mutations. The ephrin type receptor (EPH) and ligand ephrin system has been implicated in the regulation of many critical events during developmental patterning processes, including axonal guidance, cell adhesion, and cell migration. Wild-type EPHs are RTK that contain the Thr^p+1loop residue (25). As predicted, wild-type EPH members activated STAT3 in the absence of their ligands, whereas the Thr^p+1loopMet substitution severely impaired this effect. We provide evidence that the Thr^p+1loop residue plays a critical role in kinase tyrosine autophosphorylation and subsequent STAT3 recruitment in a ligand-independent manner. Moreover, STAT3 constitutive activation is associated with expression of the CXCR4 chemokine receptor and multiple mucin isoforms. Temporary depletion of STAT3 by small interfering RNA (siRNA) transiently inhibited expression of these metastasis-related genes and was shown to be invasive in a Matrigel assay.

	MATERIALS AND METHODS

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Antibody array preparation and screening. Three hundred antibodies (Santa Cruz Biotechnology, Inc.) were spotted on nitrocellulose membranes (2.5 by 5 cm) manually at a dose of 40 ng/spot. The antibody-printed nitrocellulose membranes were incubated with 3% bovine serum albumin at room temperature for 2 h prior to incubation for 3 h at room temperature with whole extracts prepared from NIH 3T3 cells (5 x 10⁷) stably transfected with wild-type RET or RET-2B. After three washes with Tris-buffered saline plus Tween, the arrays were blotted with horseradish peroxidase (HRP) conjugated anti-phosphotyrosine (pY20) antibody for 3 h, followed by enhanced chemiluminescence (ECL) analysis.

Matrigel invasion assay. Cell invasion was assayed by using a Boyden chamber assay. In brief, polycarbonate membranes (8.0-µm pore size) were coated with 5% Matrigel in the upper compartment of Transwell culture chambers. Then, 250-µl portions of cells (5 x 10⁵) were suspended in serum-free Dulbecco modified Eagle medium (DMEM) were placed in the upper compartment, and the lower compartment of the chamber was immediately filled with 500 µl of DMEM supplemented with 1% fetal bovine serum. After 16 h of incubation, the membranes were fixed with methanol and stained with hematoxylin and eosin (H&E). Cells located on the upper surface of the filter were completely removed by wiping the filter with a moist cotton swab; cells that had invaded the Matrigel and migrated through the membrane to the lower surface were counted by using a light microscope. Each assay was repeated at least three times.

ChIP experiments. Chromatin preparation and chromatin immunoprecipitation (ChIP) experiments were performed in accordance with the protocol from Upstate Biotechnology. A single-step PCR was used to amplify the MUC1, MUC4, and MUC5B promoters. The PCR conditions were optimized so that amplification was within the linear range for each primer pair. The following primers were used: MUC1-promoter, f-primer (5'-AGAGCAACGGGTGTATCGG-3') and r-primer (5'-GCAGTGTGAGGAGCAGACG-3'); MUC4-promoter, f-primer (5'-AGAGCAACGGGTGTATCGG-3') and r-primer (5'-GCAGTGTGAGGAGCAGACG-3'); and MUC5B-promoter, f-primer (5'-GCTTTGCCATCTAGGACGG-3') and r-primer (5'-CCACGTGTGTTTGCTCTCG-3'). The amplicons were detected by staining with ethidium bromide on a 2% agarose gel.

STAT3 siRNA transfection. A double-stranded siRNA oligonucleotide against STAT3 (5'-AACAUCUGCCUAGAUCGGCUAdTdT-3' and 3'-dTdTGUAGACGGAUCUAGCCGAU-5') was provided by Dharmacon Research, Inc. Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) was used as the transfection reagent according to the manufacturer's directions with 150 nmol of siRNA per well in a six-well dish. A scrambled siRNA was used as the control. siRNA transfected cells were incubated for 48 h in DMEM supplemented with 10% fetal bovine serum.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
STAT3 is constitutively phosphorylated by an RTK^Thrp+1loop but not by an RTK^Metp+1loop. To characterize proteins constitutively phosphorylated by RTK with the 2B mutation, we manufactured antibody arrays by spotting 300 antibodies against different signaling molecules on nitrocellullose membranes (31, 33). Protein extracts were prepared from NIH 3T3 cells transfected with wild-type RET or RET^M918T and incubated with the antibody arrays, followed by anti-phosphotyrosine (pY20-HRP)/ECL immunoblot analysis. Although some signaling proteins, such as ERK1/2 and AKT, were detected in both samples, STAT3, SHP-1, and HDAC2 were only detected in RET-2B transfectants (Fig. 1A). The results obtained with this antibody array were confirmed by immunoprecipitation combined with Western blotting (Fig. 1B). We next examined whether other RTKs with the 2B mutation also constitutively activated STAT3. In 293T cells, constitutive phosphorylation of STAT3 was induced by transfection of MET-2B, RON-2B, or KIT-2B, but not by wild-type RTK constructs (Fig. 1C and D). STAT3 activation upon EGF treatment of cells overexpressing wild-type EGFR has been widely reported (33). Here we show that in the absence of EGF, STAT3 phosphorylation was detected in 293T cells when STAT3 was cotransfected with EGFR containing either the 2B mutation or the inhibitory C-tail truncated (EGFR-Y⁹⁷⁴) mutation (34), but not with wild-type EGFR (Fig. 1E). EGFR-2B protein level in 293T transfectants required for STAT3 constitutive phosphorylation was lower than the endogenous EGFR level in A431 cells in which STAT3 was activated by EGF treatment (Fig. 1E).

View larger version (53K): [in this window] [in a new window]   FIG. 1. STAT3 is constitutively phosphorylated by RTKMetp+1loop with the 2B mutation. (A) Whole extracts prepared from NIH 3T3 cells transfected with wild-type RET or RET-2B (Met918Thr) were incubated with the antibody arrays comprising 300 antibodies immobilized on nitrocellulose membrane. After extensive washes, the antibody arrays were blotted with pY20-HRP, followed by ECL analysis. Positive signals detected only in the RET-2B samples include: STAT3 (L6), HDAC2 (N3), SHP-1 (N11), RET (J10), FAF (C7), and EGFR (F2). (B) Whole extracts, were prepared from 293T cells in 6-well dishes transiently transfected with 1 µg of cDNA of wild-type RET or RET-2B for 48 h. STAT3 immunoprecipitates were blotted with anti-pY705-STAT3 or anti-STAT3 antibody; SHP-1 immunoprecipitates were blotted with pY20 or anti-SHP-1; and phospho-ERKs were detected with anti-pERK1/2 antibody. (C) 293T cells were transiently transfected with wild-type MET, MET-2B (Met1268Thr), wild-type RON, and RON-2B (Met1254Thr). Whole-cell extracts prepared from these transfectants were subjected to Western blotting analysis with different antibodies as indicated. (D) Whole extracts of 293T cells transiently transfected with wild-type KIT or KIT-2B (Met836Thr) were subjected to Western blotting analysis with the antibodies as indicated. (E) Whole extracts of 293T cells transiently cotransfected with cMyc-STAT3 and wild-type EGFR, EGFR-2B (Met881Thr), or C terminus (from Tyr974) truncated EGFR were analyzed with anti-pY705-STAT3, anti-STAT3 and anti-EGFR antibodies, respectively. Also included were whole extracts of A431 cells treated with or without EGF (100 ng/ml) for 30 min.

View larger version (129K): [in this window] [in a new window]   FIG. 2. Wild-type but not Thr/Tyrp+1loopMet substituted EPH or JAK constitutively phosphorylates STAT3. (A) According to the p+1 loop motif, RTKs are divided into two groups: RTKMetp+1loop and RTKThrp+1loop. Secondary structural data were obtained by analyzing the tyrosine kinases with a three-dimensional structural program (http://www.sbg.bio.ic.ac.uk/3dpssm). Yellow shading indicates ß-sheets, and blue shading indicates -helix. The amino acids (in red) are the core of the p+1 loop motif, and the autophorphorylated tyrosine residues in activation loop are blue. (B) Wild-type EphA1, EphA2, EphB3, EphB4, EphA5, and EphB2, as well as the EphA5T845M and EphB2T793M mutants, were cotransfected with c-Myc-STAT3 in 293T cells. Whole-cell extracts prepared from these transfectants were subjected to Western blotting analysis with anti-pY705-STAT3 or anti-STAT3 antibody as indicated. (C) 293T cells were transfected with empty vector, EphB2, or EphB2 and ephrinB1. Cell lysates were analyzed in Western blotting with anti-pY705-STAT3, anti-EphB2, and anti-ephrinB2 antibodies, respectively. (D) In 293T cells cMyc-STAT3 was cotransfected with empty vector, wild-type JAK1, JAK1Y1033S, JAK1Y1033 M, or JAK1Y1033T, followed by alpha interferon (2,000 U/ml) treatment for 30 min. Whole lysates prepared from these transfectants were immunoblotted with anti-pY705-STAT3, anti-STAT3, and anti-JAK1 antibodies.

Thr^p+1loop is critical for RTK constitutive autophosphorylation. To further characterize the role of Thr^p+1loop in kinase activity, we assayed for tyrosine autophosphorylation of RTK. In 293T cells, RET tyrosine autophosphorylation was only detected in cells transfected with RET-2B but not with wild-type RET, RET-2A, and RET-HS constructs (Fig. 3A). RET-2A carries a Cys⁶³⁴Arg mutation in the extracellular domain and causes MEN-2A, a less aggressive carcinoma predisposition syndrome than MEN-2B, whereas RET-HS with the Arg⁸⁹⁷Glu mutation is responsible for Hirschsprung disease, a gut motility defect caused by an absence of ganglion cells in the nerve plexuses of the lower digestive tract (9, 18). Similarly, EGFR autophosphorylation was switched from EGF-dependent into EGF-independent with introduction of the 2B mutation or with the negative C-tail truncation (Fig. 3B). Under the same conditions, different wild-type EPH family members all displayed constitutive tyrosine autophosphorylation following transfection in 293T cells (Fig. 3C). However, the Thr^p+1loopMet substitution markedly reduced tyrosine autophosphorylation in both EphA5 and EphB2 (Fig. 3C).

View larger version (37K): [in this window] [in a new window]   FIG. 3. Thrp+1loop residue is critical for tyrosine autophosphorylation. (A) 293T cells were transiently transfected with different forms of RET as indicated. Whole extracts prepared from these 293T transfectants were subjected to Western blotting analysis with anti-pTyr and anti-RET antibodies. (B) 293T cells were transiently transfected with different forms of EGFR. Whole extracts prepared from these 293T transfectants were subjected to Western blotting analysis with anti-pTyr (pY20) or anti-EGFR antibody. (C) 293T cells were transiently transfected with wild-type EphA1, EphA3, EphB3, EphB2, EphB2T793M, EphA5, and EphA5T845M. Whole extracts prepared from these transfectants were subjected to Western blotting analysis with pY20 or anti-EphA5 antibody. (D) In 293T cells, wild-type EGFR and EGFR-2B were transiently transfected, followed by treatment with or without EGF as indicated. Whole-cell extracts prepared from these cells were Western blotted with anti-pERK1/2, anti-ERK1/2, anti-pAKT (catalog no. 9271S; Cell Signaling) antibodies (left panel). Same Western blotting analysis was performed with 293T cells transfected with empty vector, wild-type EphB2, EphB2T793M, wild-type EphA5, and EphA5T845M as indicated (right panel).

The 2B mutation was found to change the substrate specificity of RET from an RTK to a cytoplasmic tyrosine kinase by using peptides with defined sequences as substrates (20, 27). We compared the ability of mutant RTK-2B with wild-type RTK to recruit STAT3 in vivo. Although RET-2A was previously reported to activate STAT3 (28), among the four forms of RET tested here, RET-2B was most efficient in recruiting STAT3 proteins, a finding consistent with strong tyrosine autophosphorylation associated with this mutation (Fig. 4A). As for EGFR, constitutive STAT3 association was detected with both EGFR-2B and EGFR-Y974 but not with wild-type EGFR (Fig. 4B). In contrast, wild-type EphA5 or EphB2, but not Thr^p+1loopMet substituted constructs were coimmunoprecipitated with STAT3 in 293T transfectants (Fig. 4C). Therefore, the Thr^p+1loop residue is required for an RTK to become constitutively autophosphorylated, which in turn is required for a constitutive complex formation between RTK and STAT3, regardless of the absence or presence of the ligand.

View larger version (24K): [in this window] [in a new window]   FIG. 4. The Thrp+1loop residue is required for RTK to recruit STAT3 constitutively. (A) Different forms of RET were cotransfected with cMyc-STAT3 in 293T cells. Anti-RET immunoprecipitates were subjected to Western blotting analysis with anti-c-Myc or anti-RET antibody as indicated. (B) Different forms of EGFR were cotransfected with c-Myc-STAT3 in 293T cells. Anti-EGFR immunoprecipitates were subjected to Western blotting with anti-c-Myc or anti-EGFR antibody. (C) Wild-type EphA5 or EphA52B and wild-type EphB2 or Eph2B were cotransfected with c-Myc-STAT3 in 293T cells. Anti-EphA5 or anti-STAT3 immunoprecipitates were analyzed by Western blotting with anti-c-Myc and anti-EphA5 or anti-c-Myc and anti-EphB2 as indicated.

p+1loop

View larger version (12K): [in this window] [in a new window]   FIG. 5. RTK-Thrp+1loop is highly active in STAT3-dependent transcriptional activation. (A) In 293T cells, different RTKs (1 µg) as indicated were cotransfected with Luc reporter construct (0.5 µg) containing 2x STAT3 binding SIE fragment of the promoter region of mouse IRF1 gene. At 48 h after transfection, cell extracts were prepared and subjected to luciferase activity assay. The averages and standard deviations (error bars) of the values of luciferase activities from triplicates of a representative experiment are shown. Luciferase activities in the cell extracts were measured by using a luminometer to estimate transcriptional activity. (B) In 293T cells, the Luc reporter construct (0.5 µg) described above was cotransfected with 1 µg of cDNA of wild-type EGFR or EGFR-2B. As indicated, in some cases, wild-type STAT3 or STAT3Y705F was included. Luciferase activity was measured with the whole-cell lysates prepared from these transfectants. (C) In 293T cells, wild-type RET and the 2B-mutant RET of different doses (0.25 µg and 0.5 µg) were cotransfected with the above Luc reporter construct (0.5 µg) and pcDNA3 (empty vector). Luciferase activity was assayed 48 h after transfection.

View larger version (46K): [in this window] [in a new window]   FIG. 6. STAT3, constitutively activated by RTKMetp+1loop with 2B mutation or wild-type EPH, is enriched in cancer cell nuclei. (A) Medullary thyroid C-cell carcinoma tumor samples from thyroid gland, lymph nodes, and liver from of a 14-year-old male MEN-2B patient were stained with H&E, anti-calcitonin antibody, or anti-STAT3 antibody as indicated. (B) In the left panel, whole extracts prepared from A431 cells treated with or without EGF were analyzed by Western blotting with anti-pY705-STAT3, anti-STAT3, and anti-EGFR antibodies. In the right panel, whole extracts of T47D, MCF-7, and MB-468 cells were analyzed in a Western blot with antibodies to pY705-STAT3, STAT3, and different EPH family members as indicated. (C) Whole extracts of EGF treated MCF-7, MD-468, and T47D cells were blotted with anti-pERK1/2 and anti-ERK1/2 antibodies in Western blotting analysis. (D) Confluent MB-468 and T47D cells in six-well dishes received treatment of genistein (30 µM), AG-490 (10 µM), PP1 (10 µM), PD153035 (20 µM), or a combination of these drugs for 2 h prior to harvest. Whole-cell lysates were analyzed by Western blotting with anti-pY705-STAT3 or anti-STAT3 antibody.

View larger version (32K): [in this window] [in a new window]   FIG. 7. STAT3 regulates CXCR4 and multiple mucin genes. (A) STAT3-binding SIE motifs present in 5' upstream promoter regions of CXCR4, MUC1, MUC4, and MUC5B genes. (B) Nuclear extracts prepared from EGF-treated A431 cells were incubated with p32-labeled probes with the SIE sequences of CXCR4, MUC1, MUC4, and MUC5B promoters and separated in 5% polyacrylamide gel. For supershift assays, anti-STAT3 antibody was included in the binding reactions. (C) A431 cells were transfected with the indicated CXCR4-Luc reporter constructs or the control vector pcDNA3. After EGF treatment for 6 h, luciferase activity was measured with the whole lysates as described above. (D) From different cells as indicated, DNA fragments coimmunoprecipitated with anti-STAT3 antibody were amplified with the primers for CXCR4, MUC1, MUC4, and MUC5B gene promoters. (E) MCF-7, MB-468, and T47D cells were transiently delivered with STAT3 siRNA. After incubation for 48 h, whole-cell extracts were prepared and subjected to Western blotting for STAT3, MUC1, MUC4, MUC5B, and ß-actin. (F) Medullary thyroid carcinoma metastasized to liver within the blood vessel (left) and liver tissue (right) in MEN-2B patients: anti-CXCR4 immunoperoxidase staining (magnification, x400).

Since increased cell motility and invasion are correlated with increased metastatic potential, we investigated the invasive ability of these human breast carcinoma cell lines in a transwell chamber assay. MCF-7, MB-468, and T47D all showed 45 to 50% reduction in migration following down regulation of STAT3 with siRNA (Fig. 8A). NIH 3T3 cells transfected with RET-2B or wild-type EphB2 constructs showed 50% more invasion than cells transfected with wild-type RET or EphB2^T703M (Fig. 8B and C). Cotransfection of STAT3^Y705F restrained this effect of RET-2B and EphB2^T703M on cell invasion (Fig. 8B). Together, these results indicate that constitutive phosphorylation of STAT3 can activate expression of various genes involved in invasion and metastasis in different types of tumor cells.

View larger version (61K): [in this window] [in a new window]   FIG. 8. STAT3 constitutive activation leads to increased invasion. (A) MCF-7, MB-468, and T47D cells were regulated with siRNA (+) or scrambled siRNA (Ctl) against STAT3. Equal numbers of cells (2 x 105) was seeded in the upper chamber. The cells that invaded the low chamber 10 h later were fixed, stained with H&E, and counted. (B) Stable NIH 3T3 (empty vector), NIH 3T3-RETWT, or NIH 3T3-RET2B cell lines were transformed with c-Myc-tagged STAT3Y705F or wild-type STAT3. Similarly, these two forms of STAT3 were introduced into NIH 3T3 cells expressing EphB2 and EphB2T793M. Equal numbers of cells (2 x 105) were seeded in the upper chamber, and the cells that invaded the lower chamber 10 h later were fixed, stained with H&E, and counted. (C) Cells that migrated through Matrigel and the other side of the membrane in the chambers in panel B were stained with H&E and examined by using light microscopy.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The p+1 loop was so named for its role in contacting the residue immediately COOH terminal to the phosphorylated tyrosine (the p+1 position of the residue) in the substrate (8). EGFR with the 2B mutation was associated with a decrease in the selectivity of the kinase for Phe and an increase in the selectivity for acidic residues (Glu or Asp) at the p+1 position compared to wild-type EGFR (20). The motif of pTyr⁷⁰⁵ residue in STAT3 is "YLK," which does not obey this rule. If the side chain of Arg⁴⁰⁹ site inserts into a pocket near Thr⁴²⁹ site of the p+1 loop in Src, as suggested by X-ray crystallography (36), Thr⁴²⁹ site is most likely phosphorylated in order to form a salt bridge with residue Arg⁴⁰⁹. The substitution of the Arg residue at the same position of the activation loop of RET (Arg⁸⁹⁷Glu) in Hirschsprung disease is a loss-of-function mutation. However, in this case, the Met^p+1loop site may not form a bridge with Arg⁸⁹⁷. The results from previous studies and the present study clearly show that RET tyrosine autophosphorylation and STAT3 activation were not detected in RET-HS, providing additional evidence that the p+1 loop forms a specific conformation with the Arg residue in the activation loop required for Tyr autophosphorylation and catalytic activity of the kinase (35, 36).

The RTK^Metp+1loop-2B is a gain-of-function mutation that is associated with activation of STAT3, SHP-1, and HDAC2 (Fig. 1); however, this 2B mutation is rare. The large EPH family represents a naturally occurring "2B" form of RTK. Although EPH receptors play roles during vertebrate cranial development and neural crest cell migration from hindbrain segments to specific branchial arches, many EPH members have been overexpressed in cancer cells with high metastatic potential (14, 39). Our results strongly indicate that ligand binding and receptor dimerization are apparently not required for EPH receptor autophosphorylation and subsequent substrate recruitment and activation. This conclusion is supported by the crystallographic analysis of EphB2 revealing that the overall structure of the ligand-binding domain of EphB2 in the complex is similar to that of the unbound EphB2 (6). It has been reported that wild-type EPH transformed cells, whereas the stimulation with its ligand actually reversed the oncogenic phenotype (17). Thus, ligand-free activation of specific signaling pathways is sufficient for EPH to fulfill its specific functions in vivo under conditions when the ligand is not available.

Studies of STAT3 conditional knockout mice indicate that STAT3 plays a role in migration rather than in proliferation of keratinocytes (13). In the ovary of Drosophila, the JAK-STAT pathway is required to convert the stationary epithelial cell into a migratory or invasive cell (26). Recently, dominant-negative STAT3 was shown to block human endothelial cell migration (37). However, downregulation of STAT3 protein by 90% did not completely block invasion (Fig. 5), suggesting that STAT3 is not the sole factor responsible for metastasis. Chemokine receptor CXCR4 and mucin isoforms have been implicated in metastasis in different types of cancer, but the mechanisms involved may be quite different. SIE (TTCxxxGAA) sequence presents within the region from –500 to –100 of all three mucin (MUC1, MUC4, and MUC5B) promoters or within the region –1010 to –1000 of the CXCR4 promoter. All of these SIE sequences bound to STAT3 in vitro. Constitutively activated STAT3 preferentially bound to the mucin promoters in the breast cancer cells, whereas CXCR4 stained positively in C-cell carcinoma. Hence, STAT3 may differentially activate these genes in breast cancer cell lines and neuroendocrine C-cell carcinoma. It provides additional evidence that STAT3 requires organ- or cell-specific factors for differential regulation of these genes. Extracellular signal-activated NF-B was reported to bind to the promoters of CXCR4, MUC1, and MUC2 genes (5, 15, 16). Thus, constitutively activated STAT3 and NF-B may have a synergistic effect in the regulation of these genes for constant expression. Taken together, our findings indicate that targeting the p+1 loop of RTK may provide a novel therapeutic intervention to curb metastasis in both adenocarcinoma and neuroendocrine cancer cells.

.

	ACKNOWLEDGMENTS

 
We thank S. M. Jhiang for wild-type and mutant RET constructs, P. Accornero and K. Furge for mouse Met constructs, A. Danilkovitch-Miagkova for human RON construct, R. Arceci for mouse KIT construct, and E. B. Pasquale, B. Wang, and R. Zhou for various EPH and ephrin constructs. Patient samples were obtained from Rhode Island Hospital and Mayo Clinic Foundation.

This study was partially supported by NIH RO1 grant (CA82549) to Y.E.C. and NIH COBRE grant (RR-15578) to Brown University.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Surgery Science, Brown University School of Medicine/Rhode Island Hospital, 593 Eddy St., Providence, RI 02903. Phone: (401) 444-0172. Fax: (401) 444-3278. E-mail: y_eugene_chin{at}brown.edu.

Z.-L.Y. and Y.-J.G. contributed equally to this study.

Present address: Department of Medical Oncology, Dana-Farber Cancer Center, Boston, MA 02115.

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