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Cancer Research 68, 6224, August 1, 2008. doi: 10.1158/0008-5472.CAN-08-0513
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Transforming Growth Factor-β (TGF-β) and TGF-β–Associated Kinase 1 Are Required for R-Ras–Mediated Transformation of Mammary Epithelial Cells

Mete Erdogan1, Ambra Pozzi1,2,5, Neil Bhowmick1,4, Harold L. Moses1 and Roy Zent1,2,3,5

Departments of 1 Cancer Biology, 2 Medicine (Division of Nephrology), 3 Cell and Developmental Biology, and 4 Urologic Surgery, Vanderbilt University Medical Center; and 5 Department of Medicine, Veterans Affairs Hospital, Nashville, Tennessee

Requests for reprints: Roy Zent, Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Room C3210, MCN, Nashville, TN 37232. Phone: 615-322-4632; Fax: 615-322-4690; E-mail: roy.zent{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Transforming growth factor-β (TGF-β) cooperates with oncogenic members of the Ras superfamily to promote cellular transformation and tumor progression. Apart from the classic (H-, K-, and N-) Ras GTPases, only the R-Ras subfamily (R-Ras, R-Ras2/TC21, and R-Ras3/M-Ras) has significant oncogenic potential. In this study, we show that oncogenic R-Ras transformation of EpH4 cells requires TGF-β signaling. When murine EpH4 cells were stably transfected with a constitutively active R-Ras(G38V) mutant, they were no longer sensitive to TGF-β–mediated growth inhibition and showed increased proliferation and transformation in response to exogenous TGF-β. R-Ras/EpH4 cells require TGF-β signaling for transformation to occur and they produce significantly elevated levels of endogenous TGF-β, which signals in an autocrine fashion. The effects of TGF-β are independent of Smad2/3 activity and require activation of TGF-β–associated kinase 1 (TAK1) and its downstream effectors c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase as well as the phosphoinositide 3-kinase/Akt and mammalian target of rapamycin pathways. Thus, TAK1 is a novel link between TGF-β signaling and oncogenic R-Ras in the promotion of tumorigenesis. [Cancer Res 2008;68(15):6224–31]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The Ras protein family is a group of structurally and functionally conserved small GTP-binding proteins, which cycle between "active" GTP-bound and "inactive" GDP-bound conformations to mediate intracellular signaling events that modulate cell adhesion, migration, invasion, and transformation (1–3). The classic Ras proteins encoded by the H-Ras, K-Ras, and N-Ras genes are the best-studied oncogenes in this family; however, the R-Ras family of Ras-related proteins also has oncogenic potential (4, 5). The R-Ras family consists of three members: R-Ras, R-Ras2/TC21, and R-Ras3/M-Ras. Of these proteins, R-Ras bears the closest resemblance to R-Ras2/TC21 as they share 70% overall amino acid homology and have identical switch I and II domains that form critical interactions with regulatory proteins and downstream effectors (6, 7). R-Ras and R-Ras2/TC21 are regulated by many of the same guanine nucleotide exchange factors and GTPase-activating proteins and show similar subcellular localization at the plasma membrane (7).

Despite their similarities, R-Ras and R-Ras2/TC21 exhibit differential transforming properties in a variety of cell lines. In NIH 3T3 fibroblasts, R-Ras2/TC21 induces foci formation and tumor growth more efficiently than R-Ras (8). R-Ras2/TC21 also potently transforms Rat-1 fibroblasts and various epithelial cell lines, including MCF-10A, RIE-1, and EpH4 (4, 5, 9). By comparison, R-Ras is unable to transform Rat-1 fibroblasts but does promote tumor growth in cervical epithelial cells (3, 10). Phosphoinositide 3-kinase (PI3K) is the predominant effector of R-Ras and R-Ras2/TC21 transforming activity; however, these oncogenes also activate Raf1, Ral-GDS, extracellular signal-regulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) in a cell type–specific manner (4, 5, 11–13). Mutations in R-Ras2/TC21 have been detected in human cancers, and increased R-Ras2/TC21 expression has been correlated with tumor progression (14, 15). Although R-Ras mutations have yet to be identified in human tumors, there is evidence that increased R-Ras activity correlates with carcinogenesis and the promotion of metastasis (1, 16).

The multifunctional cytokine, transforming growth factor-β (TGF-β), regulates numerous cell processes, including transformation, by binding the type II TGF-β receptor (TBRII), which subsequently recruits the type I receptor (TBRI) into a heterotetrameric signaling complex; the type II receptor phosphorylates and activates the type I receptor (17). Once active, TBRI can initiate signaling by phosphorylating Smad2 and Smad3, which translocate along with Smad4 to the nucleus and initiate gene transcription resulting in G1-S cell cycle arrest. In addition, TGF-β signaling can activate PI3K/Akt (18), the mammalian target of rapamycin (mTOR; ref. 19), and the MAPK cascade (20). The MAPK kinase kinase (MKK), TGF-β–associated kinase 1 (TAK1), plays a key role in activating its downstream kinases JNK and p38 MAPK (21, 22).

Ras proteins promote cellular transformation either alone or in conjunction with TGF-β (23, 24). The EpH4 line of nontumorigenic cells derived from spontaneously immortalized mouse mammary gland epithelial cells is well characterized for the effects of Ras and TGF-β signaling (24, 25). These fully polarized cells form organotypic structures when grown in collagen gels (26) and undergo G1-S cell cycle arrest in response to exogenous TGF-β (27). It was previously shown that H-Ras cooperates with TGF-β to induce proliferation, invasion, and metastasis of EpH4 cells (3, 4). In contrast, we recently reported that expressing an activated mutant of R-Ras2/TC21 is sufficient to highly transform EpH4 cells and that exogenous TGF-β only marginally increased its oncogenic potential (9).

In this study, we determined the effect of TGF-β signaling on EpH4 cells transfected with constitutively activated R-Ras. We show that similar to H-Ras, but in contrast to R-Ras2/TC21, R-Ras transformation of EpH4 cells is dependent on TGF-β signaling and requires autocrine TGF-βproduction. Furthermore, R-Ras/EpH4 cells are extremely susceptible to the transforming ability of exogenous TGF-β. Consistent with these findings, TGF-β stimulation activates TAK1, JNK, p38 MAPK, Akt, and mTOR signaling in R-Ras/EpH4 cells. Finally, we show that TAK1 is required for TGF-β–dependent R-Ras/EpH4 cell transformation, suggesting that TAK1 plays a critical role in the TGF-β–dependent transformation of EpH4 mammary epithelial cells by oncogenic R-Ras.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. Phoenix 293 cells were provided by Dr. Gary Nolan (Stanford University, Stanford, CA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Murine EpH4 cells were obtained from Dr. Carlos Arteaga (Vanderbilt University, Nashville, TN) and maintained in DMEM with 10% FBS. PAI/L cells were obtained from Dr. Dan Rifkin (New York University, New York, NY) and maintained in 10% FBS.

Plasmids and cell lines. R-Ras(G38V) was subcloned into the LZRS-green fluorescent protein (GFP) vector (28) modified for bicistronic expression of GFP and the protein of interest. Vectors were transfected into Phoenix 293 packaging cells using Lipofectamine (Invitrogen), and EpH4 cells were subsequently infected daily with retrovirus for 10 d. Stable populations of cells expressing mutant R-Ras or empty vector were isolated by GFP using a FACStar Plus cell sorter (BD Biosciences). Pooled small interfering RNA (siRNA) for Akt (targeting Akt1, Akt2, and Akt3) was obtained from Ambion and transfected using DharmaFECT reagent 2 (Dharmacon). Pooled siRNA for JNK (targeting JNK1, JNK2, and JNK3) and specific siRNA for p38 MAPK, mTOR (FRAP), TBRII, TAK1, PI3K (targeting the p110β subunit), Smad2/3, and scrambled control siRNAs were obtained from Santa Cruz Biotechnology, Inc. and transfected using the manufacturer's reagents and protocol. Silencing of target gene expression was evaluated 3 d following transfection and did not decrease significantly by 9 d after transfection as determined by Western blot analysis.

Antibodies and other reagents. TGF-β1 was obtained from R&D Systems. The 2G7 TGF-β–neutralizing antibody was kindly provided by Dr. Carlos Arteaga. Antibodies to R-Ras, PI3Kβ, TBRII, and actin were from Santa Cruz Biotechnology. Antibodies to phosphorylated and total ERK1/2, JNK, p38 MAPK, Akt (total and phosphorylated Ser473), mTOR (total and phosphorylated Ser2448), and Smad2 were from Cell Signaling Technology, as were antibodies to phosphorylated MKK3/6 and total TAK1. Total Smad3 antibodies were from Zymed Laboratories and antibodies to phosphorylated Smad3 were kindly provided by Dr. Ed Leof (Mayo Clinic, Rochester, MN). LY294002, U0126, PD98059, SB203580, Akt inhibitor II, Akt inhibitor III, and rapamycin were from Calbiochem (EMD Biosciences). SP600125 was purchased from Biomol. Recombinant MKK6 was purchased from Sigma-Aldrich.

Tumor formation. Five-week-old female BALB/c athymic mice were obtained from Harlan Laboratories. Cells were trypsinized and resuspended in PBS, and then injected s.c. on either side of the back (1.0 x 106/100 µL PBS per injection). Tumor size was measured after 3 wk using a dial caliper and volumes were calculated as (length) x (width) x (height).

Colony formation. Cells (1 x 104) in suspension (DMEM/10% FBS/0.3% agar) with TGF-β (5 ng/mL) or inhibitors (10 µmol/L) were overlaid onto a solidified layer of agar (DMEM/10% FBS/0.7% agar) in 35-mm dishes. Cells were incubated at 37°C for 9 d. Colonies were scored counting multiple fields using an inverted microscope.

Cell proliferation. Cells (3 x 103) were plated per well in 24-well plates and maintained in DMEM (2% FBS) for 70 h and then pulsed for 2 h with 4 µCi/well [3H]thymidine (Perkin-Elmer Life Sciences). Cells were washed with 10% trichloroacetic acid and solubilized with 0.2 N NaOH, and radioactivity was measured using a scintillation counter. Cell counting assays were performed by plating 3 x 103 cells per 35-mm dish and counting cell number over 5 d using a hemocytometer.

Immunoblotting. Cells were serum starved overnight and stimulated with TGF-β (5 ng/mL) for the times indicated. Cells were lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mmol/L EDTA] supplemented with protease and phosphatase inhibitors. Total cell lysates were run onto 10% SDS gels and then transferred to nitrocellulose membranes and blocked with 5% milk in TBS with Tween 20 [TBS-T; 150 mmol/L NaCl, 100 mmol/L Tris (pH 7.5), 0.1% Tween 20]. Immunoblotting was performed with primary (1:1,000) and secondary (1:5,000) antibodies in TBS-T with 5% milk and visualized using the enhanced chemiluminescence Western blotting detection system (Perkin-Elmer Biosystems).

Reporter assays. Transcriptional activation of TGF-β–responsive genes was evaluated by performing reporter assays. Using Lipofectamine, cells were transiently transfected with either 3TP-Lux luciferase or CAGA reporter constructs in conjunction with a cytomegalovirus-driven Renilla luciferase plasmid. Subsequently, cells were treated with TGF-β (5 ng/mL) for 24 h and then lysed and dual-luciferase assays were performed to determine relative levels of TGF-β–induced transcription as indicated by the manufacturer (Promega) and measured on a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Ratios of firefly and Renilla luciferase were calculated in normalizing data to relative luminescent units. The PAI/L reporter assay for TGF-β production was performed as previously described (29). Briefly, PAI/L cells were incubated for 24 h in serum-free medium with or without TGF-β (5 ng/mL) or in conditioned serum-free medium collected from R-Ras/EpH4 or LZRS/EpH4 cells. Cells were then lysed and luciferase assays were performed as described above.

In vitro kinase assay. Cell lysates (300 µg) were immunoprecipitated using anti-TAK1 antibodies. Immunoprecipitates were incubated with 1 µg of bacterially expressed MKK6 in 10 µL of kinase buffer containing 10 mmol/L HEPES (pH 7.4), 1 mmol/L DTT, 5 mmol/L MgCl2, and 5 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol) at 25°C for 2 min. The reactions were terminated by adding SDS sample buffer and boiling for 5 min. Samples were then fractionated by 10% SDS-PAGE followed by Western blotting with antibodies to phosphorylated MKK3/6 or TAK1.

Statistical analysis. The Student's t test was used to compare two groups. Values with P ≤ 0.05 were considered significant. Results from colony formation, proliferation, reporter, and kinase assays are representative of three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Oncogenic R-Ras requires Smad-independent TGF-β signaling for transformation. EpH4 cells were infected with the LZRS-GFP retroviral vector carrying either an activated mutant of R-Ras(G38V) or an empty construct, and cell populations expressing comparable levels of GFP were isolated by fluorescence-activated cell sorting (data not shown). Overexpression of mutant R-Ras was verified by Western blot analysis (data not shown). Tumorigenicity in vivo was measured by injecting R-Ras–expressing cells s.c. into nude mice. As shown in Fig. 1A , R-Ras/EpH4 cells formed small-sized tumors by 21 days after injection, whereas LZRS/EpH4 cells were nontumorigenic. The soft agar assay was used as an in vitro assay to correlate the tumorigenic activity observed in vivo. Similar to in vivo results, R-Ras/EpH4 cells formed colonies in soft agar 9 days after plating, whereas LZRS/EpH4 cells did not (Fig. 1B). To determine the effect of oncogenic R-Ras on cell proliferation, we performed [3H]thymidine incorporation and sequential cell counting assays. R-Ras/EpH4 cells proliferated significantly faster than control cells (Fig. 1C) and continued to proliferate after reaching confluence, whereas control cells were contact inhibited (Fig. 1D). Thus, oncogenic R-Ras transforms EpH4 cells both in vitro and in vivo. To determine the effects of TGF-β on transformation in R-Ras/EpH4 cells, colony formation assays were performed in the presence of exogenous TGF-β. As shown in Fig. 2A , TGF-β stimulation dramatically increased R-Ras/EpH4 colony formation. To test whether TGF-β signaling was required for transformation of R-Ras/EpH4 cells, TBRII receptor expression was down-regulated using specific siRNA (Fig. 2B) or cells were treated with a TGF-β–neutralizing antibody (2G7). As seen in Fig. 2A, these interventions effectively inhibited the transforming effects of exogenous TGF-β and reduced basal colony formation of R-Ras/EpH4 cells. When proliferation was measured by [3H]thymidine incorporation assays, R-Ras/EpH4 cells proliferated significantly more than LZRS/EpH4 (Fig. 2C). On stimulation with exogenous TGF-β, there was a 50% increase in R-Ras/EpH4 cell proliferation but a 30% decrease in LZRS/EpH4 cell proliferation. Blocking TGF-β signaling decreased the basal and TGF-β–induced proliferation rate of R-Ras/EpH4 cells by 50% with no change in LZRS/EpH4 cells (Fig. 2C). As the basal levels of TBRII were similar in both cell populations (Fig. 2B), we hypothesized that R-Ras/EpH4 cells might be producing more activated TGF-β than control cells. To test this possibility, PAI/L reporter cells were incubated with conditioned medium from each cell population and luciferase activity was measured. As shown in Fig. 2D, R-Ras/EpH4 cells produced significantly higher levels of TGF-β than LZRS/EpH4 cells. Thus, R-Ras/EpH4 cells are markedly transformed by exogenous TGF-β and require autocrine TGF-β production for basal transformation.


Figure 1
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  		Figure 1. R-Ras induces tumor formation in vivo and soft agar growth in vitro. A, EpH4 cells were infected with retrovirus carrying activated R-Ras(G38V) or empty vector (LZRS) as described in Materials and Methods. Tumorigenicity in vivo was determined by s.c. injecting athymic BALB/c mice on either side of the back with 1 x 106 cells expressing R-Ras or empty LZRS vector. Tumor volumes were measured 3 wk later using a dial caliper. The circles represent individual tumors (n = 6) and the bars represent the mean. Tumor volumes were significantly higher in R-Ras/EpH4 cells. B, soft agar colony formation assays were performed as described in Materials and Methods, and colonies were scored after 9 d. Colony number was significantly higher in R-Ras/EpH4 compared with control cells. *, P < 0.001. C, cell proliferation was measured by performing 72-h [3H]thymidine incorporation assays as described in Materials and Methods. R-Ras/EpH4 cells proliferated significantly faster than LZRS/EpH4 cells. *, P < 0.01. D, cell proliferation was determined by plating 3 x 103 cells in 35-mm dishes and sequential cell counting. R-Ras/EpH4 cells proliferated significantly faster than LZRS/EpH4 cells. *, P < 0.01. Points, mean values from transformation and proliferation assays from triplicate wells of a single representative experiment; bars, SD.

 

Figure 2
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  		Figure 2. TGF-β signaling is required for R-Ras–induced transformation. A, colony formation in soft agar by R-Ras/EpH4 or LZRS/EpH4 cells alone (–), cells treated with the TGF-β–neutralizing antibody 2G7 (2G7Ab), or cells with TBRII expression knocked down by siRNA (TBRII siRNA). Colonies were grown in the presence or absence of TGF-β (5 ng/mL) and scored after 9 d. Differences in colony formation of cells treated with TGF-β were significant. **, P < 0.01. Knockdown of TBRII expression or treatment with 2G7 significantly reduced colony formation in R-Ras/EpH4 cells. *, P < 0.01. B, gene silencing was performed as described in Materials and Methods. Shown are cell populations either not transfected (NT), transfected with target siRNA for TBRII (TBRII), or transfected with a scrambled control (Scram). Immunoblotting was performed on 20 µg of total cell lysate from transfected cells to determine levels of target gene expression using the antibodies indicated. Blotting for actin was performed as a loading control. C, 72-h cell proliferation assays were performed on untreated cells (–), cells treated with 2G7 (2G7Ab), or cells transfected with TBRII siRNA (TBRII siRNA) in the presence or absence of TGF-β (5 ng/mL). Stimulation with TGF-β significantly increased R-Ras/EpH4 cell proliferation, whereas the proliferation of LZRS/EpH4 cells was significantly decreased. **, P < 0.05. Inhibition of TGF-β signaling significantly reduced R-Ras/EpH4 cell proliferation. *, P < 0.01. Columns, mean values from transformation and proliferation assays from triplicate wells of a representative experiment; bars, SD. D, production of autocrine TGF-β was determined using the PAI/L assay as described in Materials and Methods. PAI/L cells were incubated for 24 h in serum-free medium (SF) with or without TGF-β (5 ng/mL) or in conditioned serum-free medium (CM) collected from R-Ras/EpH4 or LZRS/EpH4 cells. PAI/L cells were then harvested and assayed for luciferase activity. Differences in relative luciferase activity were significant. *, P < 0.01. Columns, mean values from triplicate wells of a representative experiment; bars, SD.

 
To determine whether altered signaling through the Smad pathway caused R-Ras/EpH4 cell insensitivity to TGF-β–mediated growth inhibition, we performed 3TP-Lux and CAGA reporter assays for Smad-dependent transcriptional activation. As shown in Fig. 3A , TGF-β–induced transcriptional activation was intact and comparable in R-Ras/EpH4 and LZRS/EpH4 cells. Furthermore, each of these cell populations showed similar levels of Smad2/3 phosphorylation, both basally and in response to TGF-β (Fig. 3B). To determine whether Smad2/3 activity was required for transformation of R-Ras/EpH4 cells, we silenced expression of Smad2/3 using siRNA (Fig. 3C) and performed soft agar colony formation assays. As shown in Fig. 3C, inhibiting expression of Smad2 and Smad3 did not affect transformation of R-Ras/EpH4 cells at either the basal level or in response to exogenous TGF-β. In addition, inhibiting Smad2/3 expression did not alter proliferation, as measured by thymidine incorporation assays, in either R-Ras/EpH4 or control cells (Fig. 3D). These data suggest that the differences in response of LZRS/EpH4 and R-Ras/EpH4 cells to TGF-β are mediated through Smad-independent signaling pathways.


Figure 3
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  		Figure 3. Smad signaling is unaffected by transformation with R-Ras. A, reporter assays for transcriptional activation of TGF-β–induced genes were performed as described in Materials and Methods. Cells were cotransfected with either 3TP-Lux or CAGA luciferase constructs in conjunction with a Renilla construct, treated with TGF-β (5 ng/mL) for 24 h, and then harvested to measure luciferase activity. Columns, mean values from triplicate wells after being normalized to Renilla activity; bars, SD. B, cell populations were analyzed for levels of activated and total Smad2 and Smad3 by Western blot analysis as described in Materials and Methods. Serum-starved cells were stimulated with TGF-β (5 ng/mL) for the times indicated, and immunoblots were performed on 20 µg of total cell lysate using the antibodies indicated. A representative of three experiments performed is shown. C, gene silencing was performed on cell populations using siRNA. Shown are cell populations either nontransfected (NT), transfected with siRNA targeting both Smad2 and Smad3 (Smad2/3), or transfected with a scrambled control (Scram). Immunoblots were performed on 20 µg of total cell lysate to determine levels of Smad2 and Smad3 expression using the antibodies indicated. Soft agar colony formation assays were performed using knockdown R-Ras/EpH4 cells and colony formation was scored after 9 d. Knockdown of Smad2 and Smad3 expression did not affect colony formation in R-Ras/EpH4 cells. D, 72-h cell proliferation assays were performed using knockdown cells. Silencing Smad2 or Smad3 expression did not affect R-Ras/EpH4 cell proliferation. Columns, mean values from transformation and proliferation assays from triplicate wells of a representative experiment; bars, SD.

 
Signaling pathways mediating R-Ras transformation and the effects of TGF-β on R-Ras/EpH4 cells. Because the Smad pathway remained intact in R-Ras-EpH4 cells, we investigated other well-described TGF-β effectors (19, 23, 30). Surprisingly, there was no difference in ERK1/2 phosphorylation between LZRS/EpH4 and R-Ras/EpH4 cells either basally or in response to TGF-β stimulation (data not shown). Furthermore, the MAPK/ERK kinase inhibitors U0126 and PD98059 did not decrease R-Ras-EpH4 cell proliferation as measured by [3H]thymidine incorporation or soft agar colony formation (data not shown). In contrast, when JNK and p38 MAPK were evaluated, increased basal activation of both kinases was evident in R-Ras/EpH4 cells when compared with LZRS/EpH4 cells (Fig. 4A ). On treatment with TGF-β, R-Ras/EpH4 cells showed a dramatic increase in JNK and p38 MAPK activity, whereas control cells showed an increase in activation of p38 MAPK but not JNK. To test whether these MAPKs were involved in R-Ras/EpH4 cell transformation, expression of JNK or p38 MAPK was silenced using siRNA (Fig. 4B) and colony formation and proliferation assays were performed. Loss of p38 MAPK expression reduced R-Ras/EpH4 colony formation by 50%, whereas silencing JNK expression reduced colony formation by >90% (Fig. 4C). Furthermore, down-regulating either JNK or p38 MAPK completely blocked TGF-β–induced colony formation in R-Ras/EpH4 cells. However, R-Ras/EpH4 knockdown cells treated with TGF-β still showed greater colony formation and proliferation than untreated knockdown cells, suggesting that additional pathways are involved in R-Ras–induced transformation. The proliferation rate of R-Ras/EpH4 cells was also significantly reduced by decreasing either JNK or p38 MAPK expression, in both the presence and absence of TGF-β (Fig. 4D). Similar results for transformation and cell proliferation were obtained using specific inhibitors of JNK (SP600125) or p38 MAPK (SB203580; data not shown). Thus, JNK and p38 MAPK are critical in mediating the transforming effects of TGF-β on R-Ras/EpH4 cells.


Figure 4
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  		Figure 4. Signaling through JNK and p38 MAPK in R-Ras–transformed cells. A, cell populations were analyzed for levels of activated and total JNK and p38 MAPK by Western blot analysis. Serum-starved cells were stimulated with TGF-β (5 ng/mL) for the times indicated, and immunoblots were performed on 20 µg of total cell lysate using the antibodies indicated. A representative of three experiments performed is shown. B, gene silencing was performed on cell populations using siRNA. Shown are cell populations either nontransfected (NT), transfected with siRNA targeting either JNK or p38 MAPK (Target), or transfected with a scrambled control (Scram). Immunoblots were performed on 20 µg of total cell lysate to determine levels of target gene expression using the antibodies indicated. C, soft agar colony formation assays were performed using knockdown R-Ras/EpH4 cells and colony formation was scored after 9 d. Knockdown of JNK or p38 MAPK expression significantly reduced colony formation in R-Ras/EpH4 cells. *, P < 0.001. D, 72-h cell proliferation assays were performed using knockdown cells. Silencing JNK or p38 MAPK expression significantly reduced R-Ras/EpH4 cell proliferation (*, P < 0.01), whereas p38 MAPK knockdown significantly reduced proliferation in control cells (**, P < 0.05). Columns, mean values from transformation and proliferation assays from triplicate wells of a representative experiment; bars, SD.

 
Because TGF-β is known to require TAK1 to activate JNK and p38 MAPK (21, 22), TAK1 activity was determined by performing in vitro kinase assays. As shown in Fig. 5A , R-Ras/EpH4 cells have significantly greater levels of TAK1 activation both basally and in response to TGF-β stimulation when compared with LZRS/EpH4 cells. To test whether TAK1 was required for cell growth and transformation, we down-regulated TAK1 expression using siRNA (Fig. 5B) and performed colony formation and proliferation assays. As shown in Fig. 5C, silencing TAK1 in R-Ras/EpH4 cells decreased colony formation in soft agar by 60% under basal conditions and by 75% following TGF-β treatment. Similarly, down-regulation of TAK1 decreased the proliferation rate of R-Ras/EpH4 cells both basally and in the presence of TGF-β, whereas no effect was observed in LZRS/EpH4 knockdown cells (Fig. 5D). Taken together, these results show that TAK1 is a mediator of the transforming effects of TGF-β in R-Ras/EpH4 cells.


Figure 5
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  		Figure 5. TAK1 is required for transformation in R-Ras/EpH4 cells. A, TAK1 activation was determined by an in vitro kinase assay as described in Materials and Methods. Immunoprecipitates were incubated with 1 µg of bacterially expressed MKK6 in 10 µL of kinase buffer containing [{gamma}-32P]ATP at 25°C for 2 min. The samples were then fractionated by 10% SDS-PAGE and immunoblotting using antibodies to phosphorylated MKK3/6. Blotting with total TAK1 was performed as a control. B, gene silencing was performed on cell populations using siRNA. Shown are cell populations either nontransfected (NT), transfected with siRNA targeting TAK1 (TAK1), or transfected with a scrambled control (Scram). Immunoblots were performed on 20 µg of total cell lysate to determine levels of target gene expression using the antibodies indicated. C, soft agar colony formation assays were performed using TAK1 knockdown R-Ras/EpH4 cells and colony formation was scored after 9 d. Knockdown of TAK1 expression significantly reduced colony formation in R-Ras/EpH4 cells. *, P < 0.01. D, 72-h proliferation assays were performed using TAK1 knockdown cells. Silencing TAK1 significantly reduced R-Ras/EpH4 cell proliferation. *, P < 0.01. Columns, mean values from transformation and proliferation assays from triplicate wells of a representative experiment; bars, SD.

 
From the above data, it is clear that pathways apart from JNK and p38 MAPK were required for the transformation of R-Ras/EpH4 cells. As we previously showed an important role for PI3K and mTOR signaling in the transformation of R-Ras2/TC21/EpH4 cells (9), we investigated the role of these pathways in the TGF-β–dependent transformation of R-Ras/EpH4 cells. R-Ras/EpH4 cells expressed elevated basal levels of Akt and mTOR activity relative to LZRS/EpH4 cells as determined by Western blot analysis (Fig. 6A ) and stimulation with TGF-β induced significantly more activation of these pathways in R-Ras/EpH4 cells. The roles of PI3K/Akt and mTOR in the transformation of R-Ras/EpH4 cells were determined by down-regulating these proteins using specific siRNAs (Fig. 6B) and performing colony formation and proliferation assays. As shown in Fig. 6C, silencing PI3K, Akt, or mTOR expression markedly reduced basal R-Ras/EpH4 colony formation and completely blocked the TGF-β–induced increase in R-Ras/EpH4 colony formation. Furthermore, the proliferation rate of R-Ras/EpH4 cells following knockdown of PI3K, Akt, or mTOR was reduced by 60% to 70% both basally and with TGF-β treatment (Fig. 6D). Blocking PI3K or Akt expression also reduced the proliferation rate in LZRS/EpH4 cells by 50%, whereas blocking mTOR expression had no effect. Similar effects on colony formation and cell proliferation were observed using specific inhibitors of PI3K (LY294002), Akt (Akt inhibitor II and III), or mTOR (rapamycin; data not shown). Thus, the PI3K/Akt and mTOR signaling pathways are also required for the transforming effects of TGF-β on R-Ras/EpH4 cells.


Figure 6
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  		Figure 6. PI3K/Akt and mTOR signaling in R-Ras–transformed cells. A, cell populations were analyzed for levels of activated and total Akt and mTOR by Western blot analysis. Serum-starved cells were stimulated with TGF-β (5 ng/mL) for the times indicated, and immunoblots were performed on 20 µg of total cell lysate using the antibodies indicated. A representative of three experiments performed is shown. B, gene silencing was performed on cell populations using siRNA. Shown are cell populations either nontransfected (NT), transfected with siRNA targeting either PI3Kβ, Akt, or mTOR (Target), or transfected with a scrambled control (Scram). Immunoblots were performed on 20 µg of total cell lysate to determine levels of target gene expression using the antibodies indicated. C, soft agar colony formation assays were performed using knockdown R-Ras/EpH4 cells and colony formation was scored after 9 d. Knockdown of PI3K, Akt, or mTOR expression significantly reduced colony formation in R-Ras/EpH4 cells. *, P < 0.001. D, 72-h cell proliferation assays were performed using knockdown cells. Silencing PI3K, Akt, or mTOR expression significantly reduced R-Ras/EpH4 cell proliferation. *, P < 0.01. Knockdown of PI3K or Akt significantly reduced proliferation in control cells. **, P < 0.05. Columns, mean values from transformation and proliferation assays from triplicate wells of a representative experiment; bars, SD.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Cooperation between TGF-β and oncogenic H-Ras plays a critical role in promoting transformation of EpH4 cells (23, 24); however, its role in transforming R-Ras2/TC21/EpH4 cells is far less significant (9). In this study, we show that like H-Ras and unlike R-Ras2/TC21, oncogenic R-Ras collaborates with TGF-β to highly transform EpH4 cells. R-Ras/EpH4 cells produce significantly increased levels of autocrine TGF-β and require TGF-β signaling to undergo transformation. The TGF-β–dependent effects on R-Ras–transformed EpH4 cells are mediated through activation of the TAK1/JNK/p38 MAPK, PI3K/Akt, and mTOR pathways. Thus, R-Ras and TGF-β cooperatively transform mammary epithelial cells through coordinate activation of Smad-independent signaling pathways.

Mutant R-Ras and H-Ras behave in a similar manner with respect to their cooperation with TGF-β in cell transformation. EpH4 cells expressing oncogenic H-Ras undergo an epithelial-fibroblastoid conversion following TGF-β stimulation, which is maintained through autocrine TGF-β production (24). Furthermore, these cells require TGF-β signaling to induce tumor formation in vivo (25). The transformation of EpH4 cells with activated mutants of H-Ras (9) or R-Ras does not affect TGF-β signaling through the Smad pathway, and R-Ras/EpH4 cells do not require this pathway for transformation to occur. This contrasts with reports that oncogenic transformation by H-Ras alters the TGF-β–dependent phosphorylation of Smad2/3 in RGM1 cells derived from the rat gastric epithelium (31), and in mammary and lung epithelial cells (32). Thus, interactions between Ras proteins and the Smad signaling network are highly dependent on the cell type.

We show that TAK1 is activated by both R-Ras and TGF-β in EpH4 cells and that TAK1 activation is required by TGF-β to mediate the transformation of R-Ras/EpH4 cells. Like TGF-β, TAK1 can act as either a tumor suppressor or tumor promoter under varying conditions. For example, TAK1 enhances the in vitro migration and lung metastasis of colon CT26 cancer cells (33) and is required for the TGF-β–dependent invasion and metastasis of MDA-MB-231 breast cancer cells (34). However, TAK1 also mediates TGF-β–induced apoptosis via p38 MAPK in PC-3U prostate cancer cells (35) and targets the SnoN oncoprotein for degradation, thereby relieving its inhibition of TGF-β–dependent Smad signaling (36). Although it is currently unknown how R-Ras alters TAK1 signaling, it is possible that R-Ras directly interacts with TAK1 as TAK1 can form a tertiary complex with H-Ras and PI3K at the plasma membrane (37). Thus, TAK1 may provide a link through which signals from TGF-β and R-Ras converge to promote increased cell proliferation and transformation.

In this study, we established novel roles for JNK and p38 MAPK as mediators of R-Ras–induced growth and transformation, which is not surprising as these kinases are key regulators of both tumor promotion and suppression (38, 39). Unlike studies where JNK and p38 MAPK were shown to mediate TGF-β–dependent phosphorylation of Smad2 and Smad3 (31, 40), we did not observe any changes in Smad signaling following transformation with R-Ras. The observation that R-Ras/EpH4 cells are reliant on JNK and p38 MAPK signaling for cell transformation may be due to the ability of these kinases to regulate TGF-β expression levels (41, 42).

We report that Akt and mTOR signaling are required for R-Ras–induced transformation. These observations are different from R-Ras2/TC21, which induces EpH4 cell transformation by a PI3K-dependent and Akt-independent pathway (9), but are consistent with studies showing that constitutive Akt activity is required for R-Ras to induce estrogen-independent proliferation in MCF-7 breast cancer cells (43) and that PI3K/Akt and mTOR activity is critical for the increased proliferation and migration of cervical epithelial cells transfected with mutant R-Ras (15). Furthermore, TGF-β regulation of cell size, migration, invasion (44), and protein synthesis (19) requires activation of both Akt and mTOR. Although it has been reported that Akt and mTOR can inhibit TGF-β–dependent Smad signaling by interacting with Smad3 and preventing its phosphorylation (45), this behavior was not observed in R-Ras/EpH4 cells.

Our observations that R-Ras is significantly less transforming than R-Ras2/TC21 yet more tumorigenic than H-Ras in EpH4 cells in vivo and in vitro (9) agree with earlier studies performed in NIH 3T3 and Rat-1 cells (1, 5). The reason for the differences in oncogenic potential between these closely related Ras proteins is not known. One possibility is the divergence in amino acid sequence within the hypervariable COOH terminus of these GTPases, a region that determines their subcellular localization (46, 47). Another possibility is that the unique NH2-terminal 26–amino acid extension of R-Ras confers specificity of R-Ras function, as these amino acids have been shown to play a role in Rac activation, cell spreading, and cell migration (48). Finally, the hypervariable COOH terminus may cooperate with the effector loop to create signaling specificity (49).

In conclusion, we have shown that noncanonical TGF-β signaling plays a significant role in promoting the transformation of a mammary epithelial cell line by oncogenic R-Ras. Furthermore, we show a critical role for TAK1 in this process, suggesting a novel role for this kinase in TGF-β–mediated oncogenic transformation.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: CA 085492 and CA 102162 (H.L. Moses), RO1-DK 69921 (R. Zent), R01-CA94849 (A. Pozzi), RO1-DK074359 (A. Pozzi), and a Merit Award from the Department of Veterans Affairs (R. Zent).

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.

Received 2/10/08. Revised 5/13/08. Accepted 5/23/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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