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[Cancer Research 59, 4725-4731, September 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 4725-4731, September 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Contextual Effects of Transforming Growth Factor ß on the Tumorigenicity of Human Colon Carcinoma Cells1

Sheng-Chun Ye2, Jason M. Foster2, WenHui Li3, JiuRong Liang, Elizabeth Zborowska, Srinivas Venkateswarlu3, JianGen Gong, Michael G. Brattain3 and James K. V. Willson4

Department of Biochemistry and Molecular Biology, Medical College of Ohio at Toledo, Toledo, Ohio 43614-2595 [S-C. Y., W. L., J. L., S. V., J. G., M. G. B.], and Ireland Cancer Center and Department of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106 [J. M. F., E. Z., J. K. V. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor ßs (TGF-ßs) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-ß receptors have been shown to be indispensable for TGF-ß-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-ß1 and transfection of antisense TGF-ß1 to reduce TGF-ß1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-ß modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-ß system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-{Delta}RII and FET6-{Delta}RII), or with a vector containing the TGF-ß1 cDNA (CBS4-ß1S and FET-ß1S). Expression of the truncated RII reduced TGF-ß sensitivity, whereas overexpression of RII increased TGF-ß sensitivity. TGF-ß overexpression did not affect TGF-ß response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-{Delta}RII and CBS4-ß1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-{Delta}RII cells formed rapidly growing tumors, and FET-ß1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-ß signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-ß expression or TGF-ß signaling capability affects malignancy in a contextual manner.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF5 -ßs are a family of growth factors with multiple functions (1, 2, 3) . They have shown inhibitory effects on proliferation of most normal epithelial cells and function through binding to cell surface receptors. Three types of TGF-ß receptors, RI, RII, and RIII, have been identified in most mammalian cells (4) . Functional RI and RII have been determined to be indispensable for TGF-ß signal transduction (5, 6, 7, 8) . Elevated expression of various TGF-ß isoforms has been reported in tissues and/or cells from different cancers (9, 10, 11, 12, 13, 14, 15) . Arrick et al. (16) have shown TGF-ß1 overexpression in a tumorigenic murine cell stimulated tumor growth in vivo. The same group also observed that active but not latent TGF-ß1 transfection increased in vivo tumorigenicity of a sarcoma cell line, although the transfectant cells showed increased inhibitory effects on in vitro proliferation (17) . Steiner and Barrack (18) found that TGF-ß1 overexpression promoted prostate cancer growth, viability, and aggressiveness, despite increased autocrine negative TGF-ß activity in vitro. Thus, there is a strong positive correlation between high TGF-ß levels and tumor progression.

In contrast, other studies have demonstrated that transfection of antisense TGF-ß1 increased tumor progression, as assessed by stimulation of tumorigenicity in athymic mice by repressing autocrine TGF-ß activity in two human colon carcinoma cell lines (FET and CBS; Refs. 19 and 20 ). This suggested that TGF-ß signaling is an important deterrent to malignant progression because the TGF-ß antisense transfection resulted in decreased TGF-ß ligand and, therefore, subsequent loss of endogenous TGF-ß-mediated signaling. Importantly, antisense TGF-ß did not affect TGF-ß receptor expression or the ability to respond to exogenous TGF-ß, thus indicating that autocrine rather than exogenous mediated TGF-ß signaling was crucial to tumor suppression in these models.

Receptor studies on a variety of tumor types have demonstrated that RII deficiency, which results in loss of TGF-ß autocrine activity, is associated with malignant progression (21, 22, 23, 24, 25) . Other studies have shown that loss of TGF-ß sensitivity is also associated with malignant progression (26, 27, 28) . Sun et al. (29) showed that reintroduction of RII into the RII-deficient breast cancer cell line MCF7 reduced tumorigenicity in athymic mice in addition to the restoration of in vitro TGF-ß responsiveness. Wang et al. (30) demonstrated that reexpression of RII in the human colon carcinoma cell line HCT116 also reduced tumorigenicity. Interestingly, RII-transfected HCT116 cells showed restoration of autocrine TGF-ß inhibitory activity, but these cells lacked the ability to respond to exogenous TGF-ß with inhibition. As with the TGF-ß antisense studies, the result with RII-transfected HCT116 cells suggested that the autocrine response to endogenous TGF-ß is paramount to repression of malignant progression.

Taken together, past work in this area raises the issue of whether "TGF-ß" in the general sense is helpful or detrimental in controlling malignancy. One possible explanation of this paradox is that it is a contextual phenomenon, i.e., TGF-ß autocrine inhibitory effects are an important deterrent to malignancy, whereas excessive TGF-ß ligand and disruption of the autocrine signal both promote malignant properties. The objective of this study was to determine whether both positive and negative modulation of malignant properties by manipulation of the TGF-ß signal transduction apparatus in TGF-ß-responsive cell lines can occur in the same isogenic model system.

The previously described CBS (19) and FET (20) human colon carcinoma model systems were used in this study. These cells show differentiated properties (including transport capability) and are weakly tumorigenic and nontumorigenic, respectively, in athymic nude mice (19 , 20 , 31) . CBS cells express the lowest amount of TGF-ß1 and TGF-ß2 mRNA and protein that we have encountered among human colon carcinoma cell lines, yet they demonstrated autocrine negative TGF-ß activity (19 , 24) . The weak tumorigenicity of CBS cells provides the potential for this model to be manipulated both positively (increasing malignancy) and negatively. FET cells are nontumorigenic and express average amounts of TGF-ß. As indicated above, both cell lines showed increased tumorigenicity after TGF-ß1 antisense transfection (19 , 20) .

The TGF-ß pathway was modulated three ways in this study through the generation of transfectant models. The first of these was designed to determine whether the overexpression of TGF-ß in TGF-ß-responsive CBS and FET cells would lead to increased tumorigenicity in this model system, as had been reported in other model systems (16, 17, 18) . The second model was designed to determine whether augmenting TGF-ß sensitivity by modulating the TGF-ß receptor system would lead to reduced tumorigenicity. This was accomplished through the stable transfection of RII into CBS cells. RII transfection of FET cells was not investigated, because these cells are nontumorigenic. The third model system was designed to determine whether blockade of TGF-ß signaling by dominant-negative RII expression in CBS and FET cells would result in enhanced tumorigenicity. This latter model differed from the TGF-ß antisense transfected models described previously (19 , 20) in that receptor removal would block only the autocrine TGF-ß effects. In addition, it has not yet been demonstrated that removal of receptor function in TGF-ß sensitive cells would lead to increased tumorigenicity.

These models were characterized for their effects on in vivo malignant properties as well as in vitro growth properties after modulation of their TGF-ß signaling capabilities. The results confirmed that increased amounts of TGF-ß ligand in TGF-ß-responsive cells lead to increased in vivo tumorigenicity. Additionally, increased tumorigenicity was observed in CBS cells resulting from autocrine disruption of the TGF-ß pathway with the {Delta}RII truncated receptor. Specifically, we showed for the first time that removal of TGF-ß receptor type II receptor function with the {Delta}RII construct leads to tumorigenicity in a nontumorigenic cell line model (FET). In addition, we demonstrated that autocrine augmentation of TGF-ß responsiveness through overexpression of RII function in weakly tumorigenic cells can ablate the slow tumor growth of the CBS isogenic model. Taken together, these results illustrate that the context of the TGF-ß signaling system as a whole (i.e., amount of TGF-ß ligand, level of functional RII receptors, and integrity of the autocrine downstream signaling) determines its positive or negative role in malignant progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The CBS and FET human colon carcinoma cell lines were established in vitro from primary tumors as described previously (32) . FET6 and CBS4 are typical limiting dilution clones derived from parental cells, thus allowing for transfection studies using an isogenic background to eliminate the effects of tumor cell heterogeneity present in uncloned cell lines.

Cells were adapted to continuous culture in a completely defined, serum-free medium consisting of McCoy’s 5A serum-free medium supplemented with pyruvate, vitamins, amino acids, antibiotics, 10 ng/ml epidermal growth factor, 20 µg/ml insulin, and 4 µg/ml transferrin, as described previously (32, 33, 34) . Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.

TGF-ß1 Expression Vector Construction and Transfection.
A full-length human TGF-ß1 cDNA was inserted downstream of the Rous sarcoma virus long terminal repeat promoter of the RLDN-plasmid, which also carries the neomycin resistance gene under the control of the ß-globin promoter (20) . The control vector is the plasmid without the TGF-ß1 cDNA insert. The TGF-ß1 expression plasmid or the control plasmid was linearized and transfected into CBS4 cells. Electroporation was carried out at 250 V, 960 microfarads with a Gene Pulser apparatus (Bio-Rad). The transfected cells were allowed to grow for 48 h before being subjected to selection with 600 µg/ml geneticin (G418 sulfate; Life Technologies, Inc.). Stable cell clones resistant to G418 sulfate were ring cloned after 2–3 weeks and expanded for screening of TGF-ß1 expression. The control clones were pooled, expanded and designated as CBS4-Neo or FET6-Neo.

RII and {Delta}RII Expression Vector Construction and Transfection.
The RII expression vector was described previously (29) . A full-length RII cDNA was subcloned into a tetracycline-controllable expression system kindly provided by Dr. H. Bujard at the University of Heidelberg (35) . The plasmid containing the RII cDNA was cotransfected with the vector containing the tetracycline-controllable transactivator and the neomycin resistance gene using the electroporation method described above. The control cotransfections, therefore, contain all transfecting elements except the RII cDNA. FET6 cells were not transfected with RII cDNA because they are nontumorigenic in athymic nude mice, and thus, RII would not be a modulator of FET6 tumorigenicity.

The {Delta}RII expression vector construction was described previously (36) . Nucleotides 298-1188 from the human RII cDNA were amplified by PCR, except that the nucleotide 1187 was changed from thymidine (T) to adenosine (A). The amplified sequence was subcloned into the tetracycline-controllable expression system as well as the control system described above. The truncated RII ({Delta}RII) encoded amino acid residues 1–283 of the human RII; thus, most of the serine/threonine kinase domain and COOH-terminal tail of the normal human RII is absent from the {Delta}RII protein. Overexpression of the {Delta}RII repressed TGF-ß signaling pathways in a dominant-negative fashion. The transfectants were subjected to the same selection conditions as described above. After 2 weeks, antibiotic-resistant clones were ring cloned and expanded for screening of RII or {Delta}RII expression, respectively.

RNA Analysis.
Total RNA from transfectant cells in culture and xenografts from tumor-bearing nude mice was extracted by guanidine thiocyanate homogenization and ultracentrifuged through a cesium chloride gradient as described previously (37) . The RII and TGF-ß1 riboprobe plasmids for RNase protection assay were as described previously (20 , 29) . The probe for RII is 350 bases; the protected fragments for endogenous RII and transfected RII are 265 and 282 bases, respectively. Because the transfected fragment of {Delta}RII only starts at nucleotide 298 of the RII cDNA, the protected fragment for {Delta}RII transfection was 253 bases, i.e., from bases 298 to 550.

RNase protection assays were performed as described previously (20 , 29) . Radioactive riboprobes were allowed to hybridize with RII, TGF-ß1, and {Delta}RII mRNA in 40 µg of total RNA. The hybridization mixture was then treated with RNase A and RNase T1, followed by proteinase K treatment. The protected fragments of the probes were analyzed by urea-PAGE and visualized by autoradiography. Actin mRNA protected by an actin riboprobe was used for normalization.

Receptor Cross-Linking.
Purified TGF-ß1 was iodinated by the chloramine-T method and used to visualize TGF-ß receptors after cross-linking with DSS, as described previously (38) . The expression of cell surface TGF-ß receptors was determined by incubation of 125I-labeled TGF-ß1 (200 pM) with cell monolayers in 35-mm tissue culture wells, followed by cross-linking to receptors with DSS as described by Segarini et al. (39) . Labeled cell monolayers were solubilized in 200 µl of 1% Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride, and equal amounts of cell lysate protein were electrophoresed in 4–10% gradient SDS-polyacrylamide gel under reducing conditions and exposed for autoradiography.

Measurement of TGF-ß1 in Conditioned Medium.
To measure the amount of total TGF-ß1 in the conditioned media, cells were cultured in six-well culture plates using serum-free McCoy’s 5A medium until 48 h prior to the exponential, confluent, or quiescent growth stage. The conditioned medium was then collected in siliconized Eppendorf tubes, and the cell number was determined by hemocytometer counts. The total TGF-ß1 in the conditioned medium was quantitated with a TGF-ß1 ELISA kit from Promega (Madison, WI), according to the manufacturer’s specifications.

TGF-ß Sensitivity.
The p3TP-Lux promoter reporter construct containing a TGF-ß response element was used to determine TGF-ß sensitivity in transient transfection assays as described previously (8) . Briefly, the p3TP-Lux plasmid (20 µg) and a ß-galactosidase plasmid (4 µg) were transiently cotransfected in control cells or transfectants by electroporation as described above. The cells were then divided equally into replicate cell culture dishes, half of which were treated with 10 ng/ml TGF-ß1 for 48 h. The treated and untreated cells were lysed in receptor lysis buffer (Promega). The luciferase activity was assayed using a luminometer (Berthold Lumat LB 9501) for 10 s. ß-Galactosidase activity in the cell lysates was assayed according to published methods (40) . Luciferase activity was normalized to ß-galactosidase activity and expressed as relative activity in arbitrary units.

TGF-ß sensitivity was also determined by anchorage-independent growth assays in semi-solid medium. Assays were carried out in 9-cm2 plates using 0.8% Sea Plaque agarose in medium as an underlayer. Cells (6 x 103) were plated with or without 10 ng/ml TGF-ß in 0.4% agarose layered on top of the 0.8% agarose underlayer. Two weeks after plating, resultant colonies were stained with p-iodonitrotetrazolium and visually counted. Autocrine TGF-ß activity was determined by treating cells with a combination of TGF-ß1 and -ß2 neutralizing antibodies (30 µg/ml each) because CBS cells express these two family members but do not express TGF-ß3. The protocol for determining autocrine activity has been described previously (19) . Cells were plated in six-well plates at 104 cells/well. At 24 h after plating, antibodies or control IgG antibody was added to the plate, and 24 h later, DNA synthesis was determined by incorporation of [3H]thymidine for 1 h into trichloroacetic acid-precipitable DNA.

In Vivo Characterization.
Control CBS4 cells and CBS4 transfected cells from exponentially growing cultures were injected s.c. behind the anterior forelimb of 5–6-week old athymic mice (Ireland Cancer Center Colony, Cleveland, OH) at an inoculum of 5 x 106 or 10 x 106 cells. FET6 controls and transfectants were injected at an inoculum of 107 cells. Mice were maintained in microisolators on laminar flow racks. Tumors were measured externally using calipers, and the volume was determined by the equation V = (L x W2) x 0.5, where L is the largest dimension and W is the largest dimension perpendicular to L. Growth curves were plotted from the mean volume of 9–10 xenograft tumors for each group and measured every 2–3 days. The number of xenografts in each group that formed tumors and then either continued to grow or underwent regression was determined. Tumor formation was defined as a xenograft that attained a minimal volume of 100 mm3 at any point in the experiment. Xenograft regression was defined as any tumor that attained a volume of 100-mm2 and then regressed below 100 mm3.

Transgene expression was modulated in xenografts by treating mice with tetracycline in the drinking water ad libitum at a concentration of 1 mg/ml. Mice were treated with tetracycline for 48 h prior to inoculation and cells that were injected were maintained in medium with tetracycline at a concentration of 0.1 µg/ml for 3 days before injection.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of TGF-ß, RII, and {Delta}RII.
To determine whether modulation of TGF-ß signaling affects in vivo tumorigenicity both positively and negatively in the same isogenic model system, we stably transfected TGF-ß1, RII, and {Delta}RII into human colon carcinoma cell lines. Positive clones with high levels of transfected TGF-ß1 (not shown), RII, or {Delta}RII mRNA expression were obtained (Fig. 1)Citation . The addition of tetracycline in the medium almost completely suppressed the ectopic RII or {Delta}RII mRNA expression (Fig. 1Citation , Lanes 4–7, from left), indicating the effectiveness of the tetracycline repressible system. TGF-ß1 overexpression in transfectants was confirmed by ELISA using conditioned medium from transfected cells compared with control cells (Fig. 2A)Citation . Cell surface TGF-ß receptor expression was determined by 125I-labeled TGF-ß cross-linking assay using CCL64 cells as a positive control. RII mobility in human cells is slightly slower than CCL64 RII mobility because of differences in glycosylation (Fig. 2B)Citation . Receptor cross-linking assays confirmed that the RII-transfected clone expressed higher levels of RII protein relative to controls. Tetracycline suppressed the transfected RII expression to control cell levels (Fig. 2B)Citation . The {Delta}RII cDNA encodes the first 283 amino acid residues of the human RII protein because the change from thymidine to adenosine in position 1187 introduced a stop codon at residue 284 (from TAT to TAA). {Delta}RII transfectants expressed a tetracycline-regulatable protein corresponding to the size of truncated RII protein in the cross-linking gel, which disappeared after treatment with 0.1 µg/ml tetracycline (Fig. 2B)Citation . TGF-ß1 transfection did not affect TGF-ß receptor levels.



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  		Fig. 1. RNase protection assays of TGF-ß1, RII, and {Delta}RII transfected clones. Total RNA (40 µg) from control cells or transfectants (Lanes 2, 4, 6, and 7) was hybridized with 32P-labeled TGF-ß1, RII, or actin probes, followed by RNase treatment, resolved on urea-PAGE, and exposed to autoradiography. RII probe, 350 bases; endogenous RII protected fragment, 264 bases; transfected RII protected fragment, 282 bases; {Delta}RII protected fragment, 253 bases; TGF-ß1 probe, 275 bases; TGF-ß protected fragment, 242 bases; actin probe and protected fragment, 155 and 135 bases, respectively. RNA samples (Lanes 3, 5, and 8) were also isolated from transfected cells plated and maintained in serum-free medium plus 0.1 µg/ml tetracycline for 24 h.

 


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  		Fig. 2. TGF-ß and receptor levels in CBS4 transfectants. A, ELISA results for conditioned medium from CBS4-Neo and CBS4-ß1S transfectants. TGF-ß1 levels are expressed as pg protein per 1 x 106 cells; bars, SD. B, cell surface expression of TGF-ß receptors in CBS4-Neo and transfectants. Receptor cross-linking assays were performed to verify cell surface expression of RI, RII, and {Delta}RII. Confluent monolayer cultures of CBS4-Neo and transfectants were incubated with 200 pM 125I-labeled TGF-ß1 and cross-linked with DSS as described in "Materials and Methods." Cell lysates containing 100 µg of protein were electrophoresed on a 4–10% gradient SDS-polyacrylamide gel under reducing conditions and autoradiographed. Receptor expression was also examined in cells treated with tetracycline, as described for Fig. 1Citation .

 
Responsiveness to Exogenous TGF-ß1.
To compare the effects of the various transfections on TGF-ß1 response, 3TP-Lux promoter-reporter transient transfection assays were performed (Fig. 3A)Citation . The 3TP-Lux promoter-reporter construct contains a TGF-ß response element from the plasminogen activator inhibitor gene in tandem with the luciferase cDNA. Thus, increasing sensitivity to TGF-ß is reflected by increased luciferase activity. Ectopic expression of RII resulted in an increased sensitivity to TGF-ß because CBS4-RII cells showed a 6-fold induction of luciferase activity over CBS4-RII cells without TGF-ß treatment. CBS4-Neo cells showed only a 2.8-fold induction of 3TP-Lux activity after TGF-ß1 treatment, thus indicating that increasing TGF-ß sensitivity resulted from RII transfection. Ectopic expression of the {Delta}RII construct completely eliminated any measurable 3TP-Lux response to exogenous TGF-ß. This result indicated that the dominant-negative activity from this construct was effective in preventing TGF-ß response. TGF-ß1 sense transfectants showed little difference in sensitivity from that of CBS4-Neo cells because TGF-ß1 treatment generated a 3.1-fold increase in luciferase activity.



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  		Fig. 3. TGF-ß sensitivity. A, the 3TP-Lux vector was transiently cotransfected with the ß-galactosidase vector into RII, {Delta}RII, and TGF-ß1 transfected and control cells. Luciferase activity was normalized to ß-galactosidase activity and expressed as means for three replicate wells; bars, SD. Induction of luciferase activity after TGF-ß1 treatment was expressed as the ratio between the relative luciferase activity in the presence and absence of TGF-ß1. B, anchorage-independent growth assays were performed in six-well plates, as described previously (30) . Cells (6 x 103) were plated in 0.4% agarose over an underlayer of 0.8% agarose with and without 10 ng/ml TGF-ß. Colonies were determined 14 days after plating and counted visually after staining with p-iodonitrotetrazolium; bars, SD. C, an example of a six-well plate (no TGF-ß, wells A, B, C, and D; with TGF-ß, wells E, F, G, and H) as described above is shown here for CBS4-Neo (A and E), CBS4-RII (B and F), CBS4-ß1 (C and G), and CBS4-{Delta}RII (D and H).

 
Sensitivity to exogenous TGF-ß treatment by the various transfectants was confirmed by anchorage-independent growth in soft agarose. The relative TGF-ß sensitivities, manifested by the inhibition of colony formation, of CBS4-Neo control cells and the various transfectant models paralleled the TGF-ß response observed in the 3TP-Lux reporter assay (Fig. 3B)Citation . Dominant-negative RII transfectants were not sensitive to inhibition of colony formation by TGF-ß1 (Fig. 3BCitation and Fig. 3CCitation , wells D and H), whereas CBS4-RII transfectants showed a reduction of 80% in colony formation when treated with 10 ng/ml TGF-ß (Fig. 3BCitation and Fig. 3CCitation , wells B and F). Colony formation was inhibited by 56 and 62% in CBS4-Neo (Fig. 3BCitation and Fig. 3CCitation , wells A and E) and CBS4 TGF-ß1 (Fig. 3BCitation and Fig. 3CCitation , wells C and G) sense-transfected cells, respectively. Basal colony formation was not affected in TGF-ß sense-transfected cells relative to CBS4-Neo because autocrine negative TGF-ß1 is principally associated with quiescent cells in this model.

Autocrine negative TGF-ß activity of the CBS4-Neo cells and transfectants was determined by treating with TGF-ß neutralizing antibodies to TGF-ß1 and TGF-ß2 as described previously (19) . Neutralization with TGF-ß1 antibody should result in the blockade of autocrine negative activity, thus stimulating DNA synthesis. Antibodies to these ligands were used because previous work showed that TGF-ß1 and TGF-ß2, but not TGF-ß3, are expressed by CBS cells (24) . Ectopic expression of both RII and TGF-ß1 increased autocrine negative TGF-ß activity in CBS4 cells (Fig. 4)Citation by 56 ± 3% and 70 ± 3%, respectively, relative to RII and TGF-ß1-transfected cells treated with control IgG protein. This compared with an increase of 30 ± 2% [3H]thymidine incorporation in CBS4 cells treated with TGF-ß1 and -ß2 neutralizing antibodies. In contrast, CBS4-{Delta}RII cells showed decreased autocrine negative activity because neutralizing antibody treatment led to an increase of only 18 ± 1% [3H]thymidine incorporation relative to IgG control cells. Taken together, these experiments as well as those determining response to exogenous TGF-ß demonstrate the expected modulations of TGF-ß sensitivities resulting from the transfections used for this study.



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  		Fig. 4. Autocrine negative TGF-ß activity. TGF-ß autocrine negative activity was determined by treatment of CBS4-Neo control cells and CBS4 transfectants with TGF-ß1 + TGF-ß2 neutralizing antibody (30 µg/ml each). Cells were plated at low density (104 cells) in six-well plates in the presence and absence of antibodies. [3H]Thymidine incorporation was determined 24 h after antibody treatment.

 
Effects of RII, {Delta}RII, and TGF-ß1 Expression on Tumorigenicity in Weakly Tumorigenic CBS and Nontumorigenic FET Colon Cancer Cells.
CBS4-Neo and FET6-Neo cells displayed the in vivo growth characteristics observed previously (Refs. 19 and 20 ; Fig. 5Citation ). Although both cells are growth factor-dependent malignant cells derived from invasive human colon cancers, they differ in their individual growth patterns in athymic mice. CBS4-Neo is weakly tumorigenic in athymic mice, forming small xenografts that progress slowly over time, as reported by Wu et al. (19) and illustrated in Fig. 5ACitation . In contrast, FET6-Neo, when injected at 107 cells (a 2-fold higher inoculum than required for CBS4-Neo tumorigenicity), forms a nodule at the site of inoculation that does not grow and in the majority of cases regresses over 2–3 weeks (Fig. 5B)Citation .



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  		Fig. 5. Effect of ectopic RII expression on tumorigenicity. The CBS4-Neo or CBS4-RII cells were inoculated at 5 x 106 cells (A). CBS4-RII cells formed xenografts in 9 of 20 inocula sites, whereas 18 of 19 CBS4 inocula resulted in tumor formation. The CBS4-RII xenografts that formed regressed over time. Treatment of athymic mice bearing the CBS4-RII xenografts with tetracycline (CBS4 RII-TET) led to a reversal of the RII effect and restoration of the parental xenograft growth pattern (A). FET6-Neo cells formed small nodules that failed to grow and eventually regressed (B). Values are means; bars, SE.

 
The overexpression of RII in CBS led to a reduction in tumorigenicity (nine xenografts from 20 inocula with CBS4-RII versus 18 of 19 formed with CBS4-Neo). All CBS4-RII xenografts that did form nodules were observed to regress over a short interval (Fig. 5A)Citation . The growth-inhibitory effects of RII could be reversed and the tumorigenicity and growth pattern of CBS4-Neo restored when CBS4-RII cells were inoculated into athymic animals receiving tetracycline (Fig. 5A)Citation . Because FET6-Neo is nontumorigenic, it was not possible to evaluate the inhibitory effect of RII on in vivo growth in this cell line.

Disruption of TGF-ß signaling by introduction of the dominant-negative RII receptor led to a dramatic increase in both tumorigenicity and xenograft growth of CBS4-{Delta}RII and FET6-{Delta}RII cell lines. The CBS4-{Delta}RII cells form large xenografts that grow rapidly (Fig. 6A)Citation . Similarly, FET6-{Delta}RII cells were highly tumorigenic, forming tumors at all sites of inoculation, and all grew to large xenografts (Fig. 6B)Citation . The tumorigenicity and accelerated growth of CBS4-{Delta}RII was repressed in athymic mice treated with tetracycline (Fig. 6A)Citation .



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  		Fig. 6. Effect of blocking autocrine TGF-ß signaling on tumorigenicity. Clones expressing the dominant-negative RII receptor CBS4-{Delta}RII (A) and FET6-{Delta}RII (B) were inoculated s.c. in athymic mice. Formation of xenografts and in vivo growth is compared with parental cell lines CBS4-Neo (A) and FET6-Neo (B). Tetracycline treatment of mice bearing the CBS4-{Delta}RII xenografts (CBS4 {Delta}RII-TET) reversed the effect of the dominant-negative RII receptor and restored the growth characteristics of the parental cell line. Values are means for 9–10 xenografts in each group; bars, SE.

 
Verification of transgene expression and tetracycline modulation was performed by human-specific RNase protection assay of RNA harvested from xenografts (Fig. 7)Citation . In untreated cells, there is an endogenous RII band as well as a separate band that represented the {Delta}RII transgene as shown in Fig. 7Citation , whereas in the tetracycline-treated group, the transgene band is not present.



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  		Fig. 7. RNase protection analysis of CBS4-{Delta}RII xenografts untreated and treated with tetracycline. Tumor xenografts were pulverized, and RNA was extracted with guanidine. All xenograft tumors show the endogenous RII RNA doublet, as indicated. A second doublet with faster mobility reflects the truncated RII ({Delta}RII), which is absent in the tetracycline-treated group.

 
Overexpression of TGF-ß in CBS4 or in FET increased both tumorigenicity and the xenograft growth rate as compared with controls (Fig. 8)Citation . Most impressive was the increased tumorigenicity and aggressive xenograft growth observed in two FET clones with ectopic expression of TGF-ß1 when compared with the nontumorigenic FET parental cells.



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  		Fig. 8. Effect on of ectopic TGF-ß1 expression tumorigenicity. CBS4-ß1 (A) or FET-ß1 (B) cells were inoculated s.c. in athymic mice treated with and without tetracycline. Values are means for 10 xenografts in each group; bars, SE.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGF-ß has been shown to negatively regulate the proliferation of most epithelial cells (2 , 3) but has also been shown to be stimulatory in some cell types and/or physiological contexts (41, 42, 43) . TGF-ß has also been shown to have immunosuppressive effects and, therefore, could inhibit immunological surveillance (44, 45, 46, 47, 48) . Given the wide range of TGF-ß effects, it is not surprising that both tumor-suppressive and tumor-promoting activities have been described. The question arises as to whether the role of the growth factor in malignancy is determined by the physiological context or is specified by the malignant cell type. To address this question, we have used isogenic models in which malignancy could be potentially increased or decreased, depending upon modulation of specific components of the TGF-ß system. Our results are consistent with the view that effects of TGF-ß on tumor progression are contextual, i.e., TGF-ß autocrine inhibitory effects are an important deterrent to malignancy, but too much TGF-ß promotes malignant properties.

There are several potential mechanisms that could lead to the disruption of TGF-ß balance in tumors. For example, TGF-ß overexpression in tumors as modeled by the CBS and FET TGF-ß1 transfectants in this study is one obvious mechanism. The evidence for overexpression of TGF-ß in tumors and/or tumor-bearing patients is extensive. It should be noted that the TGF-ß1 cDNA used in this study encodes for a native TGF-ß, which would then have to undergo the appropriate processing to become activated. As a consequence, another mechanism generating a TGF-ß imbalance could be enhanced processing and activation. However, there is little in vitro or in vivo evidence for such mechanisms playing a major role in tumorigenesis.

Loss of RII has been noted in several systems as discussed above. Loss of RII would clearly create an imbalance in TGF-ß response, as noted in {Delta}RII transfectants in this study. However, there is also the possibility that loss of receptor could also have the effect of generating higher levels of free TGF-ß in the tumor because there would be reduced receptor binding. One reason we used the CBS system in this study was to minimize the potential for this type of phenomenon because the CBS cell line produces very small amounts of TGF-ß (24) . Moreover, the {Delta}RII encodes for a product that has been shown to bind TGF-ß. Nonetheless, this type of mechanism could be operable in tumors that have lost RII expression. Along this line, there is also the possibility that RIII loss could contribute to free TGF-ß levels. Chen et al. (49) have shown that introduction of RIII into mammary carcinoma cells that lack RIII expression regenerates autocrine TGF-ß and leads to tumor regression. These investigators hypothesize that some of the effects of RIII overexpression on in vivo growth might be derived from sequestration of free TGF-ß.

In the isogenic models tested in this study, TGF-ß1 overexpression led to increased tumorigenicity, regardless of the fact that TGF-ß transfection increased autocrine TGF-ß activity and reduced cell growth in vitro, as in other TGF-ß overexpression cell models (16, 17, 18) . This suggested that paracrine effects of TGF-ß are responsible for increased tumorigenicity. This result could involve enhanced angiogenesis or immunosuppression, as discussed above.

Several novel observations were made in this study in addition to the demonstration of the contextual nature of TGF-ß effects on malignancy. One of these was the demonstration that removal of TGF-ß sensitivity through dominant-negative RII transfection resulted in the formation of very large tumors from the nontumorigenic FET cell line model. This is the first demonstration that removal of RII alone can lead to tumorigenicity in a nontumorigenic cell. In addition, dominant-negative RII transfection also dramatically enhanced the tumorigenicity of the weakly tumorigenic CBS cells. Importantly, these effects were reversed in both cell line models by tetracycline treatment of the mice. TGF-ß1 overexpression has been shown to increase tumorigenicity in a variety of models; however, this is the first study to demonstrate tumor formation by nontumorigenic cells. These results raise the possibility that malignant cells (e.g., occult metastasis and others) may become activated by the disruption of the TGF-ß physiological context.


   FOOTNOTES
 
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.

1 This work was supported by NIH Grants CA68316, CA43703, CA38173, and CA50457. S-C. Y. performed this work as part of the requirement for the Ph.D. degree at Medical College of Ohio. J. M. F. is a Dudley Allen Scholar from the Department of Surgery, Case Western Reserve University. Back

2 These authors contributed equally to this work. Back

3 Present address: Department of Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Carl Drive, San Antonio, TX 78284. Back

4 To whom requests for reprints should be addressed, at Ireland Cancer Center, University Hospitals of Cleveland and Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44116. Phone: (216) 844-8562; Fax: (216) 844-4975. Back

5 The abbreviations used are: TGF, transforming growth factor; RI, RII, and RIII, receptor types I, II, and III, respectively; DSS, disuccinimidyl suberate. Back

Received 3/31/99. Accepted 7/22/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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