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Cancer Research 67, 9869, October 15, 2007. doi: 10.1158/0008-5472.CAN-07-0436
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

TrkC Binds to the Bone Morphogenetic Protein Type II Receptor to Suppress Bone Morphogenetic Protein Signaling

Wook Jin1, Chohee Yun1, Hae-Suk Kim1 and Seong-Jin Kim1,2

1 Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, Maryland and 2 Laboratory of Cell Regulation and Carcinogenesis, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon, Korea

Requests for reprints: Seong-Jin Kim, Laboratory of Cell Regulation and Carcinogenesis, Lee Gil Ya Cancer, and Diabetes Institute, Gachon University of Medicine and Science, 7-45 Songdo-dong, Yeonsu-ku, Incheon, South Korea 406-840. Phone: 82-32-820-4990; Fax: 82-32-820-4991; E-mail: jasonsjkim{at}gachon.ac.kr or sxk396{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TrkC, a member of the tropomyosin-related kinase (Trk) family of neurotrophin receptors, is implicated in the growth and survival of human cancer tissues. TrkC is also a potent oncoprotein expressed in tumors derived from multiple cell lineages, and functions as an active protein tyrosine kinase by neurotrophin-3 (NT-3). We previously reported that TrkC plays an essential role in tumor growth and metastasis in a murine cancer cell line. Here, we report that expression of TrkC suppresses bone morphogenetic protein 2 (BMP-2)–induced Smad1 phosphorylation and transcriptional activation. In the highly metastatic CT26 murine colon cancer cell line, which expresses endogenous TrkC, silencing TrkC expression by small interfering RNA significantly enhanced BMP-2–induced Smad1 phosphorylation and restored BMP-2 growth inhibitory activity. In contrast, expression of TrkC in RIE-1 cells, in which TrkC is not expressed, completely suppressed BMP-2 transcriptional activation. Furthermore, we showed that TrkC directly binds to the BMP type II receptor (BMPRII), thereby preventing it from interacting with the BMPRI. This activity requires a functional TrkC protein tyrosine kinase, and the BMPRII seems to be a direct target of TrkC. Our findings provide evidence for a previously unknown mechanism by which TrkC, a neuronal receptor, can block BMP tumor-suppressor activity. [Cancer Res 2007;67(20):9869–77]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone morphogenetic proteins (BMP) were originally identified as molecules that induce bone and cartilage formation when implanted at ectopic sites in rats (1–3). In accordance with their in vivo effects, BMPs regulate growth and differentiation of chondroblast and osteoblast lineage cells in vitro. However, BMPs have been shown to be multifunctional proteins with a wide range of biological activities on various cell types, including monocytes, epithelial cells, mesenchymal cells, and neuronal cells. BMPs regulate growth, differentiation, chemotaxis, and apoptosis of these cells, and play pivotal roles in the morphogenesis of various tissues and organs (4). The more than 20 types of BMPs, including growth and differentiation factors (GDF), are the largest group of proteins in the transforming growth factor-ß (TGF-ß) family. The R-Smads, including Smad1/5/8, are the major transducers of BMP receptor (BMPR) signals. Phosphorylation of the COOH-terminal of R-Smads via ligand-bound BMPR type I receptor (BMPRI) causes R-Smads to interact with Smad4 and translocate into the nucleus, where they regulate transcription of specific genes (5, 6). Many members in the BMP subfamily are expressed in the prostate, including BMP-2, BMP-3, BMP-4, BMP-6, BMP-7 (6), and GDF-7.

There is growing evidence that BMPs and their receptors may play an important role as tumor suppressors. Treatment of human cancer cells with BMP-4 can completely abrogate their ability to form xenograft tumors in immunodeficient mice (7). A recent study has shown that in vivo inhibition of BMP signaling in murine colonic epithelial cells gives rise to dysplastic and adenomatous lesions (8). BMP-2 is highly expressed in the human stomach and colon (9). However, BMP-2 expression is lost in several cancers, including human familial adenomatous polyposis lesions (10), prostate cancer (11, 12), and human gastric cancers (13), indicating the tumor-suppressor activities of BMP-2.

The loss of expression of BMPRs has been reported in several human cancers. BMPRs are preferentially expressed by epithelial cells in the human prostate, and human prostate cancer cells frequently have reduced levels of expression of BMPRI-A, BMPRI-B, and BMPRII (14). Human prostate cancer, human renal cancer, and human bladder transitional cell carcinoma tissues frequently display a loss of expression of BMPRII (15–17). The most compelling evidence for the role of BMP signaling in colon cancer is the recent discovery that mutations in BMPRI-A cause a rare inherited cancer predisposition syndrome known as familial juvenile polyposis (18, 19).

Neurotrophins were first identified as promoters of neuronal survival; however, it is now appreciated that they regulate many aspects of neuronal development and function, including synapse formation and synaptic plasticity (20–24). In mammals, the neurotrophin family consists of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and NT-4/5 (22–24). The neurotrophins are ligands for receptor protein tyrosine kinases of the tropomyosin-related kinase (Trk) family.

Trk was initially discovered from a colon cancer–derived oncogene in which tropomyosin was fused to a novel tyrosine kinase domain (25, 26). The normal cellular counterpart of this Trk is a single-pass transmembrane molecule that is highly expressed in the developing nervous system. The extracellular domain of Trk, which is not present in the oncogene, encodes a leucine-rich motif flanked by two cysteine-rich domains and an immunoglobulin-like domain, which is required for ligand binding (27, 28). The intracellular domain of Trk is much smaller than that of many other receptor tyrosine kinases and contains only 70 amino acids before and 15 amino acids after the tyrosine kinase domain. NGF binds p140trkA, brain-derived neurotrophic factor and NT-4/5 bind to p145trkB, whereas NT-3 is the ligand for p145trkC (26, 29, 30). Neurotrophins and their corresponding receptors have been shown to induce a variety of pleiotropic responses in malignant cells, including enhanced tumor invasiveness and chemotaxis (31–35). Also, neurotrophins and their receptors are important in the regulation of angiogenesis and mitogenic signals that facilitate tumor growth, the prevention of apoptosis, and the spreading of cells and metastasis (36–39).

In the present study, we investigated whether TrkC modulates BMP signaling in CT26 cells, a highly metastatic murine colon cancer cell line that expresses TrkC. Overexpression of TrkC in normal rat intestinal epithelial (RIE) cells inhibits anoikis. Also, knockdown of TrkC in CT26, a highly metastatic murine colon cancer cell line, not only suppressed cell proliferation but also significantly reduced the ability of cells to colonize in the lungs of BALB/c mice. We also show that TrkC suppresses BMP signaling by directly binding to the BMPRII, thereby preventing it from recruiting the BMPRI and activating downstream BMP effector cascades.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, antibodies, and reagents. RIE cells, colon cancer (CT26) cells, and 293T cells were maintained in DMEM high glucose (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humidified 5% CO2 incubator. Antibodies were obtained from the following companies: anti-TrkC (C-14, 798), anti-BMPRII (T-18), anti-Smad1 (A-4), anti-hemagglutinin (HA; Y-11), and anti-Myc (9E10) from Santa Cruz Biotechnology; anti-V5 from Invitrogen; anti–phosphorylated Smad1/3 from Cell Signaling Technology; and anti–ß-actin from Sigma-Aldrich. Pharmacologic inhibition was done using the protein kinase inhibitor, K252a, from Calbiochem.

Plasmids. Each of two small interfering RNA (siRNA)–coding oligonucleotides against mouse TrkC was designed and verified to be specific to TrkC by Blast search against the mouse genomes, respectively. To construct hairpin-type single RNA interference vectors, 5 µL (100 mmol/L) of the synthesized sense and antisense oligonucleotides (Supplementary Table S1) were combined with 1 µL of 1 mol/L NaCl and annealed by incubation at 95°C for 2 min, followed by rapid cooling to 72°C, and ramp cooling to 4°C over a period of 2 h. The mouse TrkC-siRNA inserts were subcloned into the XbaI/XhoI sites of the pFG12 lentivirus vector. An siRNA that does not match any known mouse coding cDNA was used as control.

Immunoblotting and immunoprecipitation. 293T cells were used for the detection of protein-protein interaction in vivo. 293T cells were transiently transfected with the indicated plasmids. After 24-h transfection, cells were switched to 0.2% serum overnight. Cells were lysed in a buffer containing 25 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EDTA, and protease inhibitor mixture (Complete, Roche). Extracts were separated by SDS-PAGE followed by electrotransfer to polyvinylidene difluoride membranes and probed with polyclonal or monoclonal antisera, followed by horseradish peroxidase–conjugated anti-rabbit and anti-mouse IgG, respectively, and visualized by chemiluminescence according to the manufacturer's instructions (Pierce). For immunoprecipitation, the cell lysates were incubated with the appropriate antibody for 1 h, followed by incubation with Gamma-bind beads (Amersham Pharmacia Biosciences) for 1 h at 4°C. Beads were washed four times with the buffer used for cell solubilization. Immunocomplexes were then eluted by boiling for 3 min in 2x Laemmli buffer (pH 6.8), and then extracts were analyzed by immunoblotting as described above.

Reverse transcription-PCR reactions. Total RNA was extracted using Quizol reagent (Qiagen, Inc.). Reverse transcription was done using a one-step reverse transcription-PCR (RT-PCR) kit at the suggested conditions (Qiagen). The primers used for RT-PCR are shown in Supplementary Table S1. PCR products were separated on 1% agarose gels and visualized by the ethidium bromide technique.

Transfection and reporter assays. Normal RIE-1 cells, RIE-1 cells expressing TrkC, CT26 cells, and CT26 cells carrying trkC-siRNAs were transiently transfected with BRE-luc and the internal control pCMV-ß-gal in six-well plates using Lipofectin (Invitrogen) according to the manufacturer's instructions. After 24-h transfection, cells were treated with 20 ng/mL BMP-2 or K252a for 24 h in medium. Luciferase activity was quantified using the Enhanced Luciferase Assay Kit (BD Biosciences). Values were normalized with ß-galactosidase activity. All assays were done in triplicate and represented as the mean (±SE) of three independent transfections.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TrkC is expressed in CT26 cell line. We compared the expression of neurotrophin receptors in the CT26 cell line, which is an undifferentiated adenocarcinoma of the colon that is characterized to be highly metastatic, with nonmalignant RIE cell line (RIE-1 cell). Results obtained from RT-PCR using the specific primers for each neutrophin receptor revealed that although RIE-1 cells had no or negligible trk expression, CT26 had mRNA expression of trkB and trkC (Fig. 1A ). TrkA mRNA expression was low to undetectable in both cell lines. Expression of TrkC protein in CT26 cells was also observed, but not in RIE cells (Fig. 1A). In addition, TrkC protein in CT26 cells were found to be tyrosine phosphorylated, indicating an activated state (Supplementary Fig. S1).


Figure 1
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  		Figure 1. Inhibition of TrkC restores BMP signaling. A, expression of TrkA, TrkB, and TrkC in mouse colorectal cancer cell line (CT26) and in RIE cell line (RIE-1) was examined by RT-PCR. The endogenous Gapdh mRNA level and ß-actin level were measured as an internal control. Expression of TrkC protein was examined by immunoblotting. B, the relative levels of BMPRI, and BMPRII mRNA measured by real-time RT-PCR in the CT26 cells compared with RIE-1 cells. Columns, mean of the PCRs in triplicate; bars, SE. *, P < 0.05. C, inhibition of TrkC expression by K252a restores BMP-2–induced growth inhibition. CT26 cells were treated with varying concentrations of BMP-2 with or without K252a (20 nmol/L) as indicated. After 24 h of BMP-2 treatment for the cells, the cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated thrice. Points, averages of means from three determinations; bars, SD. *, P < 0.01; #, P < 0.05. D, CT26 cells or RIE-1 cells were pretreated with K252a (20 nmol/L) for 1 h, and Smad1 phosphorylation was examined 1 h after stimulation with BMP-2 (20 ng/mL). Phosphorylated Smad1 and total Smad1 were blotted with anti–phosphorylated Smad1/3 Ab or anti-Smad1 antibody. E, the BMP-2–responsive reporter BRE-luc was transfected into RIE-1 or CT26 cells. Luciferase activity was measured 24 h after treatment with BMP-2 or K252a. Data are representative of at least three independent experiments. *, P < 0.05.

 
Because previous studies suggested that both the Ras/extracellular signal-regulated kinase (ERK) pathway and the phosphatidylinositol 3-kinase/Akt pathway play an important role in TrkC-mediated oncogenesis, we examined the activation status of both of these pathways in RIE-1 and CT26 cells. We first examined the phosphorylation (activation) status of mitogen-activated protein/ERK kinase 1/2 (MEK1/2) and Akt, along with level of total cyclin D1 expression in RIE-1 and CT26 cells. Levels of phosphorylated (activated) MEK1/2, phosphorylated Akt, and total cyclin D1 were markedly increased in TrkC-expressing CT26 cells compared with RIE-1 cells (Supplementary Fig. S2).

Ectopic expression of TrkC has a protective effect from anoikis in RIE-1 cells. Anoikis, a form of apoptosis, is induced by detachment from the surrounding extracellular matrix of anchorage-dependent cells. Because metastatic tumor cells must be able to escape from anoikis to invade other organs, comprehending the mechanism of anoikis is crucial to better understand the tumor progression closely associated with cell invasion and migration. To determine whether TrkC plays a role in anoikis suppression in colorectal tumors, we asked whether ectopic expression of TrkC could protect cells from anoikis. We infected RIE-1 cells with retroviral vector transducing a DNA segment specifying a full-length mouse trkC sequence and selected neomycin-resistant cells (Supplementary Fig. S3A). Anoikis was tested in vitro using cell culture dishes to which RIE-1 cells cannot attach. RIE-1 cells stably overexpressing TrkC proliferated large spheroid aggregates in suspension, whereas the normal counterpart RIE cells showed reduced survival (Supplementary Fig. S3B). These data suggest that ectopic expression of TrkC alone is sufficient to protect RIE-1 cells from anoikis.

TrkC suppresses BMP signaling. We next examined expression levels of BMPRI and BMPRII in RIE-1 and CT26 cell lines. Both RIE-1 and CT26 cells expressed BMPRI and BMPRII mRNA, although expression levels were lower in CT26 cells (Fig. 1B). We then examined whether inhibition of Trk signaling enhances BMP-2–induced inhibition of DNA synthesis by measuring thymidine incorporation. To do so, we treated CT26 cells with BMP-2 in the presence or absence of K252a, an inhibitor of the Trk tyrosine kinases (40). CT26 cells were resistant to BMP-2–induced growth-inhibitory activity, and BMP-2 treatment enhanced thymidine incorporation. However, to our surprise, inhibition of Trk kinase activity by K252a restored BMP-2–induced growth-inhibitory activity in CT26 cells (Fig. 1C). The inhibition of Trk kinase activity by K252a reduced thymidine incorporation of CT26 cells and BMP-2 cotreatment slightly decreased the thymidine incorporation (Supplementary Fig. S4). On the other hand, epidermal growth factor (EGF) treatment slightly induced thymidine incorporation of CT26 cells. However, EGF had no effect on the growth of CT26 cells in the presence of K252a (Supplementary Fig. S4). We also examined whether K252a treatment enhances BMP-2–induced Smad1 phosphorylation. BMP-2 treatment induced Smad1 phosphorylation in RIE-1 cells, and the addition of K252a further enhanced BMP-2–induced Smad1 phosphorylation (Fig. 1D). BMP-2 treatment did not induce Smad1 phosphorylation in CT26 cells; yet, in the presence of K252a, BMP-2 markedly induced Smad1 phosphorylation (Fig. 1D). We also assessed the effects of K252a on BMP transcriptional activity using a BMP reporter construct (BRE-luc) in RIE-1 and CT26 cells. K252a had no effect on BMP-2–induced transcriptional activity in RIE-1 cells, whereas K252a treatment in CT26 cells enhanced BMP-2–induced transcriptional activity (Fig. 1E). The intensity of the bands in the Western blots for phosphorylated Smad1 and Smad3 was stronger in RIE-1 cells treated with BMP-2 in the presence of K252a, but K252a had no effect on BMP-2–induced transcriptional activity in RIE-1 cells. This may be due to the saturation of BMP reporter activity.

Knockdown of TrkC in CT26 cells inhibits metastasis in vivo. Because CT26 cells express TrkC, we next examined whether TrkC is responsible for the resistance of CT26 cells to BMP-2–induced transcriptional activity and growth-inhibitory activity. To address this, we used two CT26 cell lines expressing trkC-specific siRNA, which we had generated. Expression of the trkC-specific siRNAs significantly reduced the expression of TrkC protein in CT26 cells (Supplementary Fig. S5A). CT26 cells carrying trkC-siRNAs grew slower compared with those carrying control luciferase siRNA (Supplementary Fig. S5B). To investigate the functional role of TrkC in tumor metastasis, we examined if inhibition of TrkC expression in the highly metastatic CT26 cells would affect their metastatic ability in vivo. Pools of CT26 cells containing either control-siRNA or trkC-siRNAs were injected into the tail veins of BALB/c mice, and mice were sacrificed 3 weeks later for gross examination of lungs for metastatic lesions. The number of metastatic lung nodules was strikingly lower in the mice that were injected with trkC-siRNA–transfected cells, compared with those in the control counterparts (Supplementary Fig. S5C). Histologic analyses showed that the size and the number of micrometastatic lesions were also markedly diminished in the lungs of mice inoculated with cells carrying trkC-siRNAs (Supplementary Fig. S5D). These data suggest that TrkC might play a role in one or more steps in the metastatic process, which makes colonization in distant organs more efficient.

Knockdown of TrkC enhances BMP signaling. We next tested if knockdown of TrkC expression in CT26 cells had any effect on Smad1 phosphorylation, transcriptional activity, and thymidine incorporation in response to BMP-2. Knockdown of TrkC expression in CT26 cells markedly enhanced BMP-2–stimulated Smad1 phosphorylation (Fig. 2A ) and BMP-2–induced BRE-luciferase activity (Fig. 2B). We next examined whether knockdown of TrkC expression affected the ability of CT26 cells to proliferate in vitro. Indeed, BMP-2 inhibited thymidine incorporation in CT26 cells carrying trkC-siRNAs, whereas BMP-2 slightly enhanced thymidine incorporation in CT26 cells carrying control-siRNA (Fig. 2C). These results suggest that TrkC expression attenuates BMP-2 tumor-suppressor activity.


Figure 2
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  		Figure 2. Suppression of TrkC expression by stable TrkC-siRNA enhances BMP-2–induced Smad1 phosphorylation, transcriptional activation, and growth inhibition. A, CT26 cells expressing TrkC-siRNAs restored Smad1 phosphorylation induced by 1-h stimulation with BMP-2 (20 ng/mL). B, BMP-2–responsive reporter BRE-luc was transfected into CT26 cells expressing control siRNA and CT26 cells expressing TrkC siRNAs. Luciferase activity was measured 24 h after BMP-2 stimulation. Data are representative of at least three independent experiments. *, P < 0.05. C, suppression of TrkC expression restores BMP-2–induced growth inhibition. CT26 cells expressing control siRNA and CT26 cells expressing TrkC siRNAs were treated with varying concentrations of BMP-2 as indicated. After 24 h of BMP-2 treatment, cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated thrice. Points, averages of means from three determinations; bars, SD. *, P < 0.05.

 
Overexpression of TrkC suppresses BMP signaling. To confirm whether TrkC expression suppresses BMP signaling, we examined BMP-2 responses in three different colonies of RIE-1 cells expressing TrkC (Supplementary Fig. S3). We first examined whether TrkC regulates BMP-2–stimulated Smad1 phosphorylation. TrkC expression significantly suppressed BMP-2–stimulated endogenous Smad1 phosphorylation in RIE-1 cells compared with control cells (Fig. 3A ). Expression of TrkC also markedly repressed the activity of a BMP-2–responsive BRE-luc reporter (Fig. 3B and C). Consistent with this suppressive activity of TrkC, we observed a reproducible reduction of the basal, uninduced transcription level of the BMP-2–responsive reporter. Then, we examined whether expression of TrkC renders resistance of RIE-1 cells to BMP-2–mediated growth-inhibitory activity. RIE-1 cells carrying TrkC became resistant to BMP-2 growth-inhibitory activity compared with control cells (Fig. 3D). Taken together, these results suggest that TrkC suppresses BMP signaling.


Figure 3
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  		Figure 3. Overexpression of TrkC represses BMP signaling in RIE-1 cells. A, BMP-2–induced Smad1 phosphorylation of three different colonies of RIE-1 cells infected with retroviruses containing control vector alone (pLNCX) or TrkC. Cells were treated with BMP-2 (20 ng/mL) for 1 h and then BMP-2–induced Smad1 phosphorylation was examined. B, BMP-2–responsive reporter BRE-luc was transfected into RIE-1 cells stably expressing TrkC as well as control cells (pLNCX). Luciferase activity was measured after cells were treated with BMP-2 for 24 h. Data are representative of at least three independent experiments. C, either pcDNA or TrkC was cotransfected into RIE-1 cells with BRE-luc, and luciferase activity was measured 24 h after BMP-2 stimulation. D, control RIE-1 cells and RIE-1 cells stably expressing TrkC were treated with varying concentrations of BMP-2 as indicated. After 24 h of BMP-2 treatment, cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated thrice. Points, averages of means from three determinations; bars, SD. *, P < 0.01, **, P < 0.05.

 
TrkC directly interacts with the BMPRII. Because our results suggested that the actions of TrkC are positioned upstream of Smad1 phosphorylation, we examined the possibility that TrkC may interact directly with the surface receptors for BMP-2 in vivo. We examined the endogenous interaction of TrkC and BMPRII in CT26 cells that express TrkC. Extracts prepared from RIE-1 cells that do not express TrkC were used as negative controls. Endogenous TrkC was able to interact with BMPRII (Fig. 4A ). Similarly, overexpressed TrkC was able to interact with endogenous BMPRII in RIE-1 cells expressing TrkC (Fig. 4B). We also examined the interaction between TrkC and BMPRII in 293T cells by transiently transfecting Myc-tagged BMPRII or HA-tagged BMPRI and TrkC. TrkC did not interact with BMPRI, whereas TrkC interacted strongly with BMPRII (Fig. 4C–E).


Figure 4
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  		Figure 4. TrkC interacts with BMPRII. A, identification of TrkC-BMPRII complexes in CT26 cells. Cell lysates were subjected to immunoprecipitation (IP) using anti-TrkC antibody, followed by immunoblotting (IB) with anti-BMPRII antibody. Whole-cell lysates were also probed for TrkC, BMPRII, and ß-actin as a loading control. B, identification of TrkC-BMPRII complexes in RIE-1 cells stably expressing TrkC. The cell lysates were subjected to immunoprecipitation using anti-V5 antibody, followed by immunoblotting with anti-BMPRII antibody. C to E, TrkC interacts with BMPRII, but not with BMPRI. V5-tagged TrkC was cotransfected into 293T cells with HA-tagged BMPRI or Myc-tagged BMPRII constructs. Cell extracts were subjected to immunoprecipitation using anti-V5, Myc, or HA antibody followed by immunoblotting with anti-Myc or V5 antibody. F, BMPRII interacts with TrkC but not with EN. Myc-tagged BMPRII was cotransfected into 293T cells with either V5-tagged TrkC or V5-tagged EN. Cell extracts were subjected to immunoprecipitation using anti-V5 antibody followed by immunoblotting with anti-Myc antibody.

 
The trkC gene is also known to be a partner of the ETV6-NTRK3 (Tel-TrkC) fusion gene. In a previous report, we have shown that ETV6-NTRK3 (EN) binds to TGF-ß type II receptor, but not BMPRII (41). To verify this finding, we transfected either V5-tagged EN or V5-tagged TrkC with Myc-tagged BMPRII and examined the interaction between TrkC and BMPRII or EN and BMPRII. As shown in Fig. 4F, TrkC strongly interacted with BMPRII, whereas EN did not.

Characterization of the BMPRII/TrkC interaction. BMPRII has a relatively short extracellular domain with some conserved cysteine residues, a single transmembrane domain, and a cytoplasmic domain containing a serine/threonine kinase region (Fig. 5A ). To identify the functional domain of BMPRII responsible for the interaction with TrkC, we used a series of deletion constructs. The BMPRII mutant lacking the cytoplasmic tail still interacted with TrkC (Fig. 5B), whereas TrkC did not interact with the cytoplasmic tail of BMPRII (hLAP16/22, hLAP41, h LAP 15s; Fig. 5C).


Figure 5
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  		Figure 5. Immunoprecipitation analyses of overexpressed TrkC and its interaction with BMPRII proteins in 293T cells. A, schematic representation of full-length and truncated BMPRII proteins. Immunoprecipitation and immunoblot analyses of Flag-tagged or Myc-tagged BMPRII proteins (B) or GFP-tagged BMPRII proteins (C) interacting with V5-tagged TrkC. TrkC interacts with the extracellular domain and kinase domain of the BMPRII but cannot associate with the cytoplasmic tail of BMPRII.

 
Because the interaction between BMPRII and TrkC potentially represents a novel mechanism of cross-talk between two distinct signaling pathways, we investigated how these proteins associate. To identify the region(s) of TrkC that interact with BMPRII, several truncated versions of TrkC were generated (Supplementary Fig. S6A). To facilitate analysis of these mutants, all the constructs contained an epitope tag of V5 at the amino terminus. Transfection of these constructs into 293T cells confirmed that all the mutant versions of TrkC were expressed, as evidenced by Western blot analysis (Supplementary Fig. S6B). Immunoprecipitation with anti-V5 showed that the V5-TrkC 609, V5-TrkC 682, and V5-TrkC 747 coprecipitated with Myc-BMPRII, whereas V5-TrkC 435, V5-TrkC 530, and V5-TrkC 590 showed no interaction with BMPRII (Supplementary Fig. S6B). These results suggest that BMPRII interacts with residues 591 to 609 of the intracellular domain of TrkC. Because BMPRII did not associate with EN, which contains the TrkC kinase domain, conformation of EN may prevent the interaction between EN and BMPRII.

TrkC/BMPRII interaction blocks the ability of BMPRII to recruit and activate BMPRI. Ligand binding leads the BMPRII to phosphorylate and activate the type I receptors, BMPRI-A (also known as ALK3) and BMPRI-B (ALK6). BMPRI, in turn, phosphorylates Smad proteins, causing their activation and translocation to the nucleus where they regulate the transcription of BMP-responsive genes (5). Thus, BMPRII phosphorylation results in activation of BMPRI with subsequent initiation of downstream signaling pathways. Because TrkC interacts with BMPRII, but not with BMPRI, we hypothesized that TrkC may exert its effect by blocking the ability of BMPRII to recruit and activate BMPRI. To examine whether TrkC suppresses the ability of BMPRII kinase to activate BMPRI by blocking BMPRI-BMPRII complex formation, Myc-tagged BMPRII was coexpressed with V5-tagged TrkC along with HA-tagged Alk3 in 293T cells. As expected, BMPRI bound to BMPRII only after stimulation with BMP-2. However, coexpression of TrkC significantly reduced the level of BMPRI associated with BMPRII, and the serine phosphorylation level of BMPRI (Alk3) was significantly reduced in the presence of TrkC (Fig. 6A ). These findings strongly indicate that TrkC inhibits BMP signaling through its interaction with BMPRII, and this association inhibits BMPRI-BMPRII complex formation (Fig. 6B).


Figure 6
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  		Figure 6. TrkC blocks BMPRI and BMPRII complex formation. A, V5-tagged TrkC was cotransfected into 293T cells with both HA-tagged BMPRI (Alk3) and Myc-tagged BMPRII. Twenty-four hours after transfection, cells were treated with or without BMP-2 (20 ng/mL) for 1 h. Cell extracts were immunoprecipitated using anti-HA or anti-Myc antibodies and Gamma-bind beads, followed by immunoblotting with anti-phosphoserine, anti-HA, and anti-V5 antibodies. The expression of TrkC, BMPRI, and BMPRII was monitored as indicated. B, a model for suppression of BMP signaling by TrkC.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results of the present study show a suppression of BMP signaling by TrkC receptor tyrosine kinase in colon cancer cells, and the binding of TrkC with BMPRII to inhibit BMPRI-BMPRII complex formation. Furthermore, there was a correlation between the resistance of colon cancer cells to BMP-2 and the expression of TrkC in the murine highly metastatic colon cancer cell line, CT26. When CT26 cells were transfected with trkC-specific siRNA, BMP-2 sensitivity was restored. Overexpression of TrkC in RIE-1 cells, which do not normally express TrkC, renders resistance of these cells to BMP-2–induced transcriptional activity and growth-inhibitory activity. Taken together, these results provide a valuable insight concerning the role of TrkC in BMP signaling in colon cancer cells.

There is great interest in understanding how oncogenic tyrosine kinases function in malignant transformation, because these proteins are attractive targets for therapeutic intervention (42). Trks seem to be activated by overexpression of the full-length protein in a number of human tumors (43–47). Trks have a high capacity for ligand-independent activation, presumably via spontaneous interactions. Thus, high levels of Trk expression elicit autophosphorylation in the absence of neurotrophins. Because CT26 cell line expresses not only TrkB and TrkC but also ligands for receptor protein tyrosine kinases of the Trk family, we cannot exclude the possibility that at least brain-derived neutrophic factor and NT-3 could exert autocrine effect in CT26 cells.

In agreement with the paradigm of promotion of tumorigenesis by the ETV6-NTRK3 chimeric oncoprotein, we have shown a significant decrease in tumorigenic potential in CT26 cells when the expression of TrkC was silenced by siRNA technology. Specifically, CT26 cells carrying trkC-siRNAs grew slower compared with those carrying control luciferase siRNA, and the number of metastatic lung nodules was strikingly lower in the mice that were injected with trkC-siRNA–transfected cells, compared with those in the control counterparts. These results show that TrkC is a promoter of tumorigenicity in the CT26 colon cancer cell line.

BMPs regulate cell proliferation, apoptosis, and differentiation, and participate in the mesenchymal development of most tissues and organs in vertebrates. However, the role of BMPs in epithelial growth regulation is not well understood. Multiple studies have suggested that BMPs may be important regulators of neoplastic cells. Hallahan et al. (48) reported that BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells, whereas Kawamura et al. (49) showed that BMP-2 induces apoptosis in human myeloma cells. We have previously shown a correlation between the Gleason score and BMPR expression status in prostate cancer cells, as well as a loss of BMPRII expression in bladder transitional cell carcinoma tissues and renal cancer tissues (15–17). It is of note that a recent study by Nishianian et al. (7) has reported that colon cancer cells are resistant to the differentiative and antiproliferative effects of BMP4. However, the mechanism that confers this resistance is not known. In this regard, the results of the present study suggest that overexpression of TrkC may be responsible for resistance to BMP.

In a previous study (41), we showed a remarkable specificity in the regulation of the TGF-ß family type II receptors by EN. This oncoprotein did not interact with activin or with BMPRII; EN only interacted with TßRII. This seems to be mediated at least in part by a five amino acid (TSEQF) sequence within the intracellular domain of TßRII, which is not present in related TGF-ß family type II receptors, including activin and BMPRII (41). Because both TßRII and BMPRII bind to wild-type NTRK3 (TrkC; ref. 41), the TrkC binding region in the BMPRII may be different from that in TßRII.

TrkC is frequently overexpressed in human cancers, including pancreatic and prostate carcinoma, neuroblastoma, and medulloblastoma (48–50). Moreover, recent mutational analysis of the tyrosine kinome in colorectal cancers has shown somatic mutations within the kinase domain of TrkC (50). Overexpression of TrkC confers constitutive activation of its tyrosine kinase activity to induce continuous proliferation of the cell. There is to date no report of cross-talk between the Trk receptor tyrosine kinase and the BMPRII serine-threonine kinase. The biological significance of the potential cross-talk between Trk receptor signaling and BMP signaling therefore remains to be determined.

In summary, we have identified that TrkC suppresses BMP signaling specifically through interaction with BMPRII and inhibition of BMPRI-BMPRII complex formation. The identification of BMPRII as a potential target for TrkC suggests that the loss of BMP signaling by TrkC may be an important step in colon carcinogenesis.


   Acknowledgments
 
Grant support: Intramural Research Program of the National Cancer Institute, NIH, Bethesda, MD.

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 O. Bernard for providing the BMPRII constructs and A. Hobbie for the critical reading of the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

W. Jin and C. Yun contributed equally to this work.

Received 2/ 2/07. Revised 7/11/07. Accepted 8/17/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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