Restoration of Transforming Growth Factor-β Receptor II Expression in Colon Cancer Cells with Microsatellite Instability Increases Metastatic Potential in Vivo*

Microsatellite instability (MSI), which occurs in 15% of colorectal cancer, has been shown to have a lower incidence of metastasis and better patient survival rates compared with microsatellite stable colorectal cancer. However, a mechanistic understanding of the basis for this difference is very limited. Here, we show that restoration of TGFβ signaling by re-expression of TGFβ receptor II in MSI colon cancer cells increased PI3K/AKT activation, conferred resistance to growth factor deprivation stress-induced apoptosis, and promoted cell motility in vitro. Treatment with a potent PI3K inhibitor (LY294002) blocked the prosurvival and promotility effects of TGFβ, indicating that TGFβ-mediated promotion of cell survival and motility is dependent upon activation of the PI3K/AKT pathway. Analysis of apoptotic effectors that are affected by TGFβ signaling indicated that Bim is an effector of TGFβ-mediated survival. In addition, TGFβ-induced down-regulation of E-cadherin contributed to the prosurvival effect of TGFβ, and restoration of TGFβ signaling in MSI colon cancer cells increased liver metastasis in an orthotopic model in vivo. Taken together, our results demonstrate that restoration of TGFβ signaling promotes cell survival, motility, and metastatic progression in MSI colon cancer cells and indicate that TGFβ receptor II mutations contribute to the favorable outcomes in colon cancer patients with MSI.

ular basis for the survival advantage in MSI tumors is not well understood.
TGF␤ factors are a group of multifunctional proteins that regulate many cellular processes through binding to TGF␤ receptors. Three major types of TGF␤ receptors, type I (RI), type II (RII), and type III (RIII), have been identified in most cells (8). TGF␤ RI is transphosphorylated and activated by TGF␤ RII after TGF␤ binds to a heteromeric complex of TGF␤ RI and TGF␤ RII. The activated TGF␤ RI kinase then transmits signals through Smad proteins to regulate transcription of target genes (9,10).
TGF␤ acts as a tumor suppressor at early stages of tumorigenesis, but enhances tumor progression, invasion, and metastasis at later stages (11). We and others have demonstrated that autocrine TGF␤ mediates tumor suppressor activity in a variety of cancers, including colon cancers, and that loss of autocrinenegative TGF␤ activity leads to acquisition and progression of malignancy (12)(13)(14)(15)(16)(17)(18)(19). The tumor suppressor function of TGF␤ involves its ability to induce growth arrest and apoptosis, whereas the tumor promoter function of TGF␤ is associated with its ability to induce an epithelial-to-mesenchymal transition (EMT), which facilitates migration and invasion and confers resistance to the apoptotic effects of TGF␤ (20 -22). EMT has been shown to be impaired in MSI colon cancer cells (23). Studies have indicated that TGF␤ RII is commonly mutated in MSI colorectal carcinomas (24) and that TGF␤ RII mutations may be associated with significantly improved survival in MSI colon cancer patients (25). This raises the possibility that inactivation of TGF␤ signaling resulting from TGF␤ RII mutations might contribute to the reduced occurrence of metastasis in MSI colorectal cancer patients.
To test the hypothesis that mutated TGF␤ RII contributes to reduced malignancy of colon cancer, we introduced wild-type TGF␤ RII into MSI colon cancer cells bearing mutated TGF␤ RII and examined the phenotypic changes in vitro and in an orthotopic model of colon cancer in vivo (26). Re-expression of TGF␤ RII restored TGF␤ signaling in these cells, which protected them from growth factor deprivation stress (GFDS)-induced apoptosis and promoted their motility in vitro. To determine the molecular mechanisms by which TGF␤ promotes cell survival and motility, we examined the PI3K/AKT pathway because it has been shown to play an important role in cell survival and migration (27). We found that reconstitution of TGF␤ increased AKT phosphorylation under GFDS and that inhibition of PI3K/AKT activation by LY294002 reversed TGF␤-mediated protection from GFDS-induced apoptosis as well as TGF␤-mediated promotion of motility. We have also identified Bim, a pro-apoptotic protein, as a downstream effector of TGF␤ signaling in cell survival. Further studies showed that TGF␤ reduced E-cadherin expression, which contributed to increased cell survival under GFDS in colon cancer cells. Finally, re-expression of TGF␤ RII in MSI colon cancer cells increased metastatic colonization in the liver in an orthotopic model in vivo. Taken together, our results demonstrate that TGF␤ RII is a survival and metastasis promoter, the loss of which provides MSI colon cancer patients a survival advantage.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-The human colon carcinoma cell lines HCT116, HCT116 wt, DLD1, and RKO were cultured at 37°C in a humidified incubator with 5% CO 2 in McCoy's 5A serum-free medium supplemented with 10% fetal bovine serum or 10 ng/ml epidermal growth factor, 20 g/ml insulin, and 4 g/ml transferrin (28). When cells were subjected to growth factor and nutrient deprivation stress, they were cultured in McCoy's 5A serum-free medium in the absence of growth factor or serum supplements.
Apoptosis Assays-Cells were seeded in 96-well plates and then deprived of growth factors by changing to McCoy's 5A serum-free medium for the indicated times (24 -72 h) or treated with 4 ng/ml TGF␤ or 25 mol/liter LY294002 for 24 -48 h. Apoptosis was determined using DNA fragmentation ELISAs (Roche Applied Science) according to the manufacturer's protocol. Statistical analyses were performed using Student's t test.
Transwell Motility Assays-Transwell motility assays were performed utilizing 8-m pore, 6.5-mm polycarbonate Transwell filters (Corning Costar Corp.). After trypsinizing the cells, single cell suspensions were seeded onto the upper surface of the filters in McCoy's 5A serum-free medium in the absence of growth factors and allowed to migrate toward McCoy's 5A serum-free medium with 10% fetal bovine serum. After 18 h of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to the medium. The cells on the upper surface of the filter were removed with a cotton swab, and the cells that had migrated to the underside of the filter were visualized under a microscope, followed by solubilization of the dye in dimethyl sulfoxide and quantification at 570 nm. Statistical analyses were performed using Student's t test.
Plasmids, siRNA Transfection, and Retroviral Infections-Human TGF␤ RII cDNA was cloned into a pBABE-based retroviral expression vector. A shRNA targeting human E-cadherin (5Ј-ACTAGGTATTGTCTACTCTGA-3Ј) was cloned into the pSUPER.retro vector (Oligoengine, Seattle, WA). The 293GP packaging cells (Clontech) were cotransfected with a vesicular stomatitis virus G-expressing vector and retroviral expression constructs using Effectene (Qiagen). The viruses were harvested 48 h later and used to infect HCT116 wt-RII cells. Bim ON-TARGETplus siRNA and negative control siRNA were obtained from Dharmacon (Lafayette, CO). siRNAs were transfected into HCT116 wt-RII cells using Dhar-maFECT 1 reagent (Dharmacon). Transfected cells were either harvested for Western blotting or plated in a 96-well plate for apoptosis assays 48 h later.
In Vivo Orthotopic Model and Immunohistochemistry-Orthotopic implantation was performed as described previously (26). Briefly, exponentially growing GFP-labeled cells (5 ϫ 10 6 cells) were inoculated subcutaneously into BALB/c nude mice. Once xenografts were established, they were excised and minced into 1-mm 3 pieces. Two of these pieces were then subserosally implanted into the cecum of other BALB/c nude mice. 28 days post-implantation, animals were killed . Organs were explanted and imaged. Tissues were then processed and embedded in paraffin. Slides were cut for hematoxylin/eosin and Ki67 staining (Dako Corp.) and for terminal nucleotidyltransferase-mediated nick end labeling (TUNEL) assays (Apotag, Oncor, Gaithersburg, MD). The apoptosis and proliferation were determined quantitatively by counting the number of positively stained cells for TUNEL and Ki67, respectively, per field at ϫ40 magnification. Three histologically similar fields were randomly selected from each slide for analysis. p values were calculated using Student's t test.

Restored TGF␤ Signaling Protects Colon Cancer Cells from
GFDS-induced Apoptosis-HCT116 cells have inactivated TGF␤ RII due to MSI-associated mutations (24). The cell model we chose to use in the study is HCT116 wild-type PIK3CA cells (designated HCT116 wt), which have only the wild-type PIK3CA allele as a result of asymmetrical knock-out of the mutant PIK3CA allele (29). The reason to choose HCT116 wt cells is that they are more sensitive to GFDS-induced apoptosis in vitro and less metastatic in vivo than HCT116 cells bearing only the mutant PIK3CA allele (designated HCT116 mut) and parental HCT116 cells (heterozygous for PIK3CA mutation) (30,31), which offers a bigger window to observe reduced malignancy if our hypothesis is correct. To ensure that results obtained are not specific to haploid HCT116 wt cells, parental diploid HCT116 cells were included for in vitro experiments. Wild-type TGF␤ RII was ectopically expressed in HCT116 wt and parental HCT116 cells. Consequently, TGF␤ RII was re-expressed at a level comparable with that in microsatellite stable colon cancer cells (Fig. 1A, left panels), and TGF␤ signaling was restored as reflected by increased phosphorylation of Smad2 by TGF␤ treatment (right panels). However, TGF␤-mediated growth inhibition was not restored (data not shown). Nevertheless, when subjected to GFDS, TGF␤ protected TGF␤ RII-expressing cells from GFDS-induced apoptosis as shown by decreased caspase-3 cleavage, whereas little effect was observed in vector-expressing cells (Fig. 1B). This observation was further confirmed by DNA fragmentation ELISAs. In both cell types, there was significantly decreased apoptosis (*, p Ͻ 0.006) in TGF␤ RII-expressing cells when treated with TGF␤, whereas TGF␤ had no effect on vector-expressing cells (Fig. 1C). Of note, in the absence of exogenous TGF␤, HCT116 wt cells expressing TGF␤ RII (designated HCT116 wt-RII) showed markedly reduced apoptosis (**, p Ͻ 0.01) compared with vector-expressing cells (designated HCT116 wt-V) due to the effect of endogenous TGF␤ (Fig. 1C). In addition, SB525334, a potent inhibitor of TGF␤ RI kinase, was used to confirm the effect of TGF␤. Treatment of HCT116 wt-RII cells with the TGF␤ RI inhibitor reversed the protective effect of TGF␤ under GFDS (**, p Ͻ 0.008) (Fig. 1D). These results indicate that restoration of TGF␤ signaling protects both parental HCT116 and HCT116 wt cells from GFDS-induced apoptosis.
To ensure that TGF␤-mediated cell survival is not specific to HCT116 cells and their derivative cells, TGF␤ RII was re-expressed in two other colon cancer cell lines with MSI, DLD1 and RKO (24,32). Restoration of TGF␤ RII expression in DLD1 cells protected them from GFDS-induced apoptosis as reflected by significantly reduced caspase-3 cleavage (Fig. 2A). The addition of exogenous TGF␤ did not further reduce caspase-3 cleavage in these cells ( Fig. 2A), suggesting that DLD1 cells have very strong endogenous TGF␤ signaling. Similar to HCT116 and HCT116 wt cells, TGF␤ protected TGF␤ RII-expressing RKO cells from GFDS-induced apoptosis as shown by decreased caspase-3 cleavage, whereas little effect was observed in vector-expressing cells (Fig. 2B).
TGF␤ Activates the PI3K/AKT Pathway and Promotes Cell Survival in a Smad2/3-dependent Manner-Because PI3K/ AKT is a major survival pathway in colon cancer cells, we next determined the effect of TGF␤ on AKT activation. Phosphorylation of AKT at Thr 308 and Ser 473 leads to its kinase activation (33). As shown in Fig. 3A, TGF␤ treatment increased the levels of phosphorylation of AKT at Ser 473 in HCT116 wt-RII cells under GFDS, whereas it has little effect on AKT phosphorylation in HCT116 wt-V control cells. Functionally, targeting PI3K/AKT with a potent PI3K inhibitor (LY294002) reversed the protective effect of TGF␤ in HCT116 wt-RII cells (**, p Ͻ 0.002) (Fig. 3B), indicating that TGF␤-mediated protection from GFDS-induced apoptosis is PI3K/AKT-dependent.
TGF␤ signals through Smad proteins. However, Smad-independent TGF␤ signaling has been reported in many different cell types (34,35). To determine whether TGF␤-mediated cell survival is dependent on Smad signaling, expression of Smad2 and Smad3 was knocked down individually or simultaneously in HCT116 wt-RII cells by shRNA targeting Smad2 or Smad3. Expression of Smad2 and/or Smad3 was reduced efficiently and specifically in Smad2, Smad3, or Smad2 and Smad3 knockdown cells (Fig. 3C). Consequently, phosphorylation of Smad2 and/or Smad3 was inhibited in the knockdown cells after TGF␤ treatment (Fig. 3C). DNA fragmentation assays showed that protection from GFDS-induced apoptosis by TGF␤ was abrogated in Smad2, Smad3, or Smad2 and Smad3 knockdown cells (Fig.  3D). These results indicate that TGF␤ mediates cell survival in a Smad2/3-dependent manner.
TGF␤ Down-regulates Pro-apoptotic Protein Bim-Bim is a BH3-only member of the Bcl-2-related protein family that has been implicated in initiating apoptosis by engaging anti-apoptotic members of the Bcl-2 family (36,37). We found that expression of Bim was reduced by TGF␤ in HCT116 wt-RII cells but not in HCT116 wt-V control cells (Fig. 4A). In addition, down-regulation of Bim expression by TGF␤ was abrogated in Smad2 and Smad3 knockdown cells (Fig. 4B). These results indicate that TGF␤ regulates Bim expression in a Smad2/3-dependent manner. Furthermore, treatment of HCT116 wt-RII cells with LY294002 inhibited reduction of Bim expression by TGF␤ (Fig. 4C), indicating that TGF␤-mediated down-regulation of Bim expression is PI3K-dependent. Therefore, we hypothesized that TGF␤ increases cell survival through down-regulation of Bim expression. To test this hypothesis, we knocked down Bim expression in HCT116 wt-RII cells using a siRNA pool. As a result, Bim expression was significantly reduced in Bim siRNA-transfected cells compared with cells transfected with a nonspecific control siRNA (Fig. 4D). When subjected to GFDS, Bim knockdown cells were more resistant to GFDS-induced apoptosis than the control cells as reflected by reduced caspase-3 cleavage (Fig. 4E). DNA fragmentation assays showed that apoptosis under GFDS was reduced by 40% in Bim siRNA-transfected cells compared with control siRNA-transfected cells (*, p Ͻ 0.02) (Fig. 4F). These results indicate that reduction of Bim expression increases the resistance of HCT116 wt-RII cells to GFDS-induced apoptosis. Taken together, our results demonstrate that TGF␤ signaling down-regulates expression of Bim, which leads to increased cell survival under stress conditions.

TGF␤ Signaling Increases Cell Survival under GFDS by Inhibition of E-cadherin Expression-TGF␤ signaling has been
shown to induce EMT in many different cell types (38,39). To determine whether TGF␤ has a similar effect in HCT116 wt-RII cells, expression of EMT markers (E-cadherin, vimentin, and Slug) was examined in the presence or absence of TGF␤. Fig. 5A showed that TGF␤ treatment decreased expression of E-cadherin, whereas it increased expression of vimentin and Slug in HCT116 wt-RII cells, which is characteristic of EMT phenotypes. No changes were observed in expression of other EMTrelated transcription factors examined (data not shown). This indicates that TGF␤ signaling induced at least partial EMT in HCT116 wt-RII cells. EMT has been associated largely with invasion/motility (40). However, its role in cell survival is not very clear. To determine whether TGF␤-induced EMT contributes to resistance to GFDS-induced apoptosis, E-cadherin was knocked down in HCT116 wt-RII cells by transfecting a shRNA construct into the cells. Expression of E-cadherin was significantly reduced in E-cadherin shRNA-transfected cells compared with control shRNA-transfected cells (Fig. 5B). When subjected to GFDS, E-cadherin knockdown cells were more resistant to GFDS-induced apoptosis than the control cells as reflected by reduced caspase-3 cleavage (Fig. 4C). In addition, treatment with TGF␤ under GFDS decreased caspase-3 cleavage significantly in the control cells, whereas it caused a slight decrease in caspase-3 cleavage in E-cadherin knockdown cells (Fig. 4C). These results were further confirmed by DNA fragmentation assays, which showed that E-cadherin knockdown cells displayed 35% reduction of apoptosis under GFDS compared with control shRNA-transfected cells (**, p Ͻ 0.003) and that TGF␤ treatment did not further decrease apoptosis in these cells (Fig. 5D). Furthermore, overexpression of E-cadherin in HCT116 wt-RII cells abolished the protective effect of TGF␤ against GFDS-induced apoptosis (Fig. 5, E and F). These results indicate that TGF␤ protects HCT116 wt-RII cells from GFDS-induced apoptosis through down-regulation of E-cadherin and suggest that EMT plays an important role in aberrant cell survival of cancer cells under stress conditions.

FIGURE 2. TGF␤ promotes cell survival under GFDS in DLD1 and RKO cells. TGF␤ RII was stably transduced into DLD1 (A) and RKO (B) cells. Expression of exogenous TGF␤ RII was determined by Western blot analyses (left panels). The levels of cleaved caspase-3 were determined in cells under GFDS for 4 days in the presence or absence of TGF␤1 (right panels). Vec, vector.
TGF␤ Promotes Cell Motility through the PI3K/AKT Pathway-In addition to cell survival, TGF␤ has been shown to promote cell motility in many cancer cell types (41). To determine the effect of TGF␤ on cell motility in colon cancer cells, Transwell assays were performed in the presence or absence of TGF␤. As shown in Fig. 6A (left panel), HCT116 wt-RII cells  displayed an increase in the motility of Ͼ2-fold compared with HCT116 wt-V control cells (*, p Ͻ 0.006). The addition of exogenous TGF␤ increased the cell motility of HCT116 wt-RII cells by 2-fold (**, p Ͻ 0.01) (Fig. 6A, left panel). Although parental HCT116 cells expressing TGF␤ RII (designated HCT116-RII) showed similar motility compared with vector control cells (designated HCT116-V), exogenous TGF␤ treatment did increase the motility of HCT116-RII cells by Ͼ50% (**, p Ͻ 0.01) (Fig. 6A, right panel). To confirm the effect of TGF␤, a TGF␤ RI kinase inhibitor was used in the Transwell assays. Treatment of HCT116 wt-RII cells with the TGF␤ RI inhibitor abolished the promoting effect of TGF␤ on cell motility (*, p Ͻ 0.01) (Fig. 6B). Of note, the addition of the TGF␤ RI inhibitor alone reduced the motility of HCT116 wt-RII cells, confirming the effect of endogenous TGF␤ in these cells. These results indicate that restoration of TGF␤ signaling in HCT116 and HCT116 wt cells increases their motility. To determine whether TGF␤ promotes motility through activation of the PI3K pathway, cells were treated with LY294002. LY294002 abrogated promotion of motility by TGF␤ (*, p Ͻ 0.007) (Fig. 6C), indicating that TGF␤ signals through PI3K to increase cell motility.
Restoration of TGF␤ Signaling in HCT116 wt Cells Increases Metastasis in an Orthotopic Model-Because cell survival and motility are two important determinants of metastasis (42,43), we next used an orthotopic model to determine the effect of restoring TGF␤ signaling on metastasis of MSI colon cancer  cells. HCT116 wt-V and HCT116 wt-RII cells were stably transfected with GFP and characterized in the orthotopic model as described previously (26).
In vivo studies showed that animals implanted with xenografts formed by HCT116 wt-V and HCT116 wt-RII cells demonstrated 100% primary growth at the site of implantation with clear invasion of the bowel upon histological evaluation (Fig.  7A, upper panels). However, compared with HCT116 wt-V cells, orthotopic implantation of HCT116 wt-RII cells gave rise to a significantly increased incidence of metastatic localization to the liver (Table 1). HCT116 wt-RII orthotopic tumors generated liver metastases in ϳ94% of the animals compared with 50% for HCT116 wt-V orthotopic tumors. Moreover, fluorescence imaging of explanted liver showed a remarkable increase in the numbers of liver metastases in the animals implanted with HCT116 wt-RII cells relative to control animals (Fig. 7B,  upper panels). Quantitation of liver metastatic loci indicated a Ͼ10-fold difference in the numbers of liver metastases between these two groups of animals (*, p Ͻ 0.0005) (Fig. 7B, lower  panel). The presence of metastatic disease was confirmed by microscopic histological analysis (Fig. 7A, lower panels). These results indicate that restoration of TGF␤ signaling by re-expression of TGF␤ RII increases metastasis of HCT116 wt cells in vivo. To determine whether TGF␤-mediated cell survival is associated with metastatic potential in vivo, TUNEL assays  were performed in primary tumors. TUNEL staining of primary tumors showed that there were significantly fewer apoptotic cells in the tumors of HCT116 wt-RII cells (ϳ1% positive cells) than in those of HCT116 wt-V cells (Ͼ50% positive cells) (Fig. 7, C and D, upper panels). Meanwhile, Ki67 staining showed that tumors of HCT116 wt-RII cells had fewer proliferative cells than those of HCT116 wt-V cells (30% versus 65%) (Fig. 7, C and D, lower panels). Although restoration of TGF␤ signaling in HCT116 wt cells decreased cell proliferation in primary tumors by ϳ2-fold, it increased cell survival by almost 50-fold. These data are fully consistent with cell survival as a key factor in determining metastasis. Taken together, these in vivo results demonstrate an important role for TGF␤ signaling in distant metastatic colonization of MSI colon cancer cells, providing a molecular mechanism of the favorable outcome in MSI colorectal cancer patients.

DISCUSSION
TGF␤ signaling plays a dual role in tumorigenesis. It elicits tumor-suppressive functions in tumor initiation, whereas it enhances tumor progression and metastasis at later stages, attributed to its ability to protect cancer cells from stress-induced apoptosis, induce EMT, and promote cell migration and invasion (11). TGF␤ RII is mutated in up to 90% of colon cancer with MSI (24). Mutations of TGF␤ RII has been implicated to be associated with favorable prognosis and better survival in patients with MSI colon cancers (25). We have shown in this study that introduction of wild-type TGF␤ RII into MSI colon cancer cells provided a survival advantage to these cells, which contributed to increased metastasis to the liver in an orthotopic model. Our studies indicate that mutated TGF␤ RII is at least partially responsible for significantly reduced incidence of metastatic disease and improved survival in patients with MSI colon cancers compared with microsatellite stable colon cancer patients.
EMT plays an important role in cancer development. It has been implicated in progression to distant metastasis, acquisition of therapeutic resistance, and generation of cancer-initiating cells (44). Reduction of E-cadherin expression is a hallmark of EMT. In colon cancer, loss of E-cadherin correlates with increased metastasis and decreased patient survival (45), which indicates the clinical relevance of EMT in colon cancer patients. EMT has been implicated to contribute mainly to migration and invasion. However, its role in cell survival under stress conditions has not been well studied. We have shown that restoration of TGF␤ signaling by re-expression of wild-type TGF␤ RII in MSI colon cancer cells promoted cell survival under GFDS through decreasing E-cadherin and inducing EMT. Therefore, TGF␤ RII mutations commonly observed in colon cancers with MSI block TGF␤-induced EMT and, consequently, not only reduce cell migration and invasion but also prevent these cells from obtaining aberrant survival capabilities under stress. Our studies suggest that TGF␤ RII mutations may be a key determinant of reduced incidence of metastasis in patients with MSI colon cancer, providing a novel mechanistic link between TGF␤ signaling, EMT, cell survival, and metastasis.
We have shown previously that TGF␤ acts as a suppressor of cell survival and metastasis in a subgroup of colon cancer cells (46). 4 However, TGF␤ becomes a promoter of survival and metastasis in MSI colon cancer cells as demonstrated in this study. Little is known about how the switch of TGF␤ functions occurs. Our cell models with TGF␤ acting as either a tumor suppressor or promoter provide a very useful tool to study the molecular determinants of the dual functions of TGF␤. Smad4 mutation has been proposed to be a molecular switch for TGF␤ signaling because a high frequency of Smad4 mutation and inactivation is closely associated with increased metastases and poor prognosis in colon cancer (47,48), and Smad4-independent TGF␤ signaling has been shown to promote colon cancer metastasis (49). However, parental HCT116 and HCT116 wt cells express wild-type Smad4 (data not shown). Therefore, other molecules are involved in the metastasis-promoting function of TGF␤ in these cells. More studies are under way to identify TGF␤ "switches" in our cell models.
Bim is one of the downstream effectors of TGF␤ in cell survival identified in this study. A major mechanism of the functional regulation of Bim-dependent apoptosis is the regulation of Bim expression by transcription factors such as FOXO and AP-1 family members (50,51). Another regulatory mechanism implicated in the control of Bim-dependent apoptosis is phosphorylation of Bim (52), which may regulate Bim protein stability (53). In our study, TGF␤ activated the PI3K/AKT pathway in HCT116 wt-RII cells (Fig. 3A) and inhibited Bim expression in a PI3K-dependent manner (Fig. 4C). Activation of PI3K/AKT has been shown to phosphorylate FOXO3a, which leads to down-regulation of Bim (54). Our data suggest that TGF␤ regulates Bim expression through AKT-FOXO3a signaling. Recent studies by Hoshino et al. (55) indicated that TGF␤ protects breast cancer cells from apoptosis through the TGF␤/ Foxc1/Bim pathway. In their studies, TGF␤ repressed expression of Foxc1. Foxc1 activates transcription of Bim in certain cell types. Therefore, we do not exclude the possibility that TGF␤ may affect Foxc1 expression and down-regulate Bim expression. More studies are needed to determine the mechanisms by which TGF␤ regulates Bim expression in colon cancer cells.
In summary, we have shown that re-expression of TGF␤ RII in MSI colon cancer cells increases their aberrant cell survival and motility in vitro and enhances their metastatic potential in vivo. This demonstrates that TGF␤ RII is at least one of the determinants of metastasis in MSI colon cancer. Our study provides a molecular explanation for the favorable outcomes observed in MSI tumors, thereby enabling us to better understand the mechanisms of colon cancer progression and metastasis.