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J. Biol. Chem., Vol. 276, Issue 40, 37273-37279, October 5, 2001

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Transforming Growth Factor- Prevents Detachment-induced Inhibition of c-Src Kinase Activity, Bcl-X_L Down-regulation, and Apoptosis of Intestinal Epithelial Cells^*

Kirill Rosen, Mariano Loza Coll, Alwin Li, and Jorge Filmus

From the Sunnybrook and Women's College Health Sciences Centre, Division of Molecular and Cell Biology, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 and the Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada

Received for publication, July 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor- (TGF-), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF- exerts its anti-anoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X_L, another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Src-dependent manner. We conclude that TGF- has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X_L expression induced by detachment from the ECM.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Most epithelia rest upon and are tightly bound to a thin extracellular matrix (ECM)¹ called basement membrane. Detachment of epithelial cells from the basement membrane results in apoptosis, a phenomenon termed anoikis (1-3). Solid tumors grow in vivo as multicellular masses in which at least a proportion of cells is deprived of normal contacts with the basement membrane and is anoikis-resistant. Acquisition of such resistance is, therefore, an essential prerequisite for invasion and metastases in most cancers of epithelial origin.

Some of the molecular mechanisms by which epithelial cells become resistant to anoikis during tumor progression have been uncovered during the last few years (4-8). Our laboratory, in particular, has investigated the mechanisms of anoikis resistance in the context of colorectal cancer, and we have demonstrated that one such mechanism is based on the mutation and activation of the ras proto-oncogene (4, 6, 8).

A majority of colorectal cancers and cell lines established from these tumors express epidermal growth factor receptor (EGFR) and overproduce one or more of its ligands (9-12). In addition, blockade of such ligands inhibits growth of human colorectal carcinoma xenografts (13). In principle, these EGFR ligands could, at least in part, exert their oncogenic effect through the induction of resistance to anoikis, since their anti-apoptotic activity is well established (14-16). However, it has been shown in several cell systems that efficient EGFR signaling requires cell-ECM attachment. For example, in fibroblasts the capacity of EGF to induce EGFR phosphorylation, or trigger the activation of mitogen-activated protein kinases, strongly depends on integrin engagement (17-19). In addition, it has been demonstrated that EGF-induced activation of protein kinase B, another well established anti-apoptotic event, notably decreases upon detachment of kidney epithelial cells (5). Thus, it is unclear at the present time whether EGFR ligands have the capacity to inhibit anoikis in intestinal epithelial cells. We show here that TGF-, one of the members of the EGF family frequently secreted by colorectal tumors (10), is capable of suppressing anoikis of intestinal epithelial cells and that this growth factor exerts its anti-anoikis effect, at least in part, through the inhibition of detachment-induced down-regulation of c-Src kinase activity and Bcl-X_L expression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- The IEC-18 cells were obtained from Dr. A. Quaroni (Cornell University) and were cultured in -MEM containing 5% fetal bovine serum (FBS), 10 µg/ml insulin, and 0.4% glucose. RIE-1 cells were a gift of Dr. R. Coffey (Vanderbilt University). These cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. For suspension cultures 10⁶ cells were plated above a layer of 1% sea plaque-agarose polymerized in -MEM or Dulbecco's modified Eagle's medium. The IEC-18 clones transfected with a Bcl-X_L expression vector were described previously (8).

Vector Construction and Transient Transfection-- To generate the Bcl-X_L expression vector, the human Bcl-X_L cDNA was inserted into the EcoRI site of pcDNA3 (Invitrogen) in the sense orientation. For transient transfection 7.65 × 10⁵ IEC-18 cells were incubated for 17 h with 0.7 µg of the pEGFP-C1 expression vector (CLONTECH) and either 3.5 µg of pcDNA-3 or 3.5 µg of the Bcl-X_L expression vector in the presence of 6.25 µg/ml Lipofectin in OPTI-MEM medium. The transfection mixture was subsequently replaced by the normal -MEM growth medium, and the incubation was continued for another 24 h. GFP-positive cells were then isolated by FACS and plated in monolayer either immediately or after 48 h of suspension culture. The resulting cell colonies were visualized 10 days later by Crystal Violet staining and counted.

Caspase-8 Assay-- A caspase-8 colorimetric assay kit from R & D Systems was used according to the manufacturer's instructions. Cells growing as attached monolayers were treated with 4 µg/ml adriamycin for 12 h to generate a positive control.

Western Blot Analysis-- Cells were lysed for 30 min on ice in a buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 100 mM NaF, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, and 10 µg/ml leupeptin. After removing insoluble material, aliquots of supernatant containing 20-30 µg of protein were run under reducing conditions through a 7% polyacrylamide gel for EGFR analysis or a 12% polyacrylamide gel in all other cases. Proteins were transferred to a nylon membrane that was subsequently incubated for 1 h at room temperature in a TBST buffer (125 mM Tris-HCl (pH 8.0), 625 mM NaCl, 0.5% Tween 20) containing 4% skim milk. The membrane was then incubated with one of the following antibodies: anti-Bcl-X_L, anti-Fas (Transduction Laboratories), anti-Bak, anti-Src (Upstate Biotechnology, Inc.), anti-EGFR, anti-phosphotyrosine, anti-caspase-10, anti-caspase-3 (Santa Cruz Biotechnology). Incubation with antibodies was performed in a TBST buffer containing 5% bovine serum albumin in the case of anti-Bcl-X_L, anti-Bak, and anti-phosphotyrosine or 2.5% skim milk in all other cases for 1-2 h at room temperature or at 4 °C overnight. Binding of the antibodies was detected with the enhanced chemiluminescence system (PerkinElmer Life Sciences).

Apoptosis Assay-- 5 × 10⁴ cells were plated on a 24-well, 60- or a 100-mm dish in monolayer or in suspension culture. At the indicated times cells were removed from the plates, washed once with PBS, and assayed for the presence of nucleosomal fragments in the cytoplasm by a Cell Death Detection ELISA kit (Roche Molecular Biochemicals), according to the manufacturer's instructions.

In Vitro c-Src Kinase Assay-- c-Src activity was measured using an in vitro kinase assay as described previously (20). Briefly, 1 mg of cell lysate was immunoprecipitated with 1 µg of monoclonal anti-Src antibody and 30 µl of 50% agarose-bound protein A. Immunoprecipitates were washed three times with 100 µl of lysis buffer and three times with 100 µl of 10 mM HEPES (pH 8.0). Beads were then resuspended in 35 µl of reaction mixture (45 mM HEPES (pH 8.0); 150 mM NaCl; 50 mM MgCl₂; 10 µM Na₃VO₄; 2 µM ATP; and 10 µCi of [-³²P]ATP) containing 0.04 µg/µl acid-treated enolase (see below) and incubated at 30 °C for 15 min. Reactions were stopped by addition of 6 µl of 5× SDS-polyacrylamide gel electrophoresis loading buffer and boiling for 5 min. Samples were run through a 7.5% SDS-polyacrylamide gel, and the dried gels were analyzed by autoradiography. The protocol for acid treatment of enolase was adapted from Ref. 21. Briefly, 0.6 µl of enolase suspension (Sigma) was mixed with 0.6 µl of 60 mM HEPES (pH 8.0), 2.4 mM dithiothreitol, and 60% glycerol and added to 1.2 µl of 500 mM acetic acid. After incubation at 37 °C for 15 min, the reaction was stopped with 2.4 µl of 100 mM Tris-HCl (pH 8.0) and 20 mM MgCl₂.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TGF- Inhibits Anoikis of Normal Epithelial Cells-- To study the effect of TGF- on anoikis of intestinal epithelial cells, we used the non-malignant rat intestinal epithelial cell line IEC-18, which is highly sensitive to anoikis (4). First, we investigated whether TGF- has the ability to trigger EGFR phosphorylation in these cells when they are placed in suspension culture. We found that this growth factor induces high levels of EGFR phosphorylation in suspended IEC-18 cells (Fig. 1A). Furthermore, TGF- inhibited detachment-induced apoptosis of IEC-18 cells at all doses investigated (Fig. 1B). To ensure that the ability of TGF- to induce resistance to anoikis is not unique to IEC-18 cells, we tested the effect of this growth factor on detachment-triggered death in RIE-1 cells, another non-malignant cell line derived from normal rat intestinal epithelium (22). Similarly to what was found for IEC-18 cells, TGF- strongly inhibited anoikis of RIE-1 cells (Fig. 1C).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   TGF- triggers EGFR phosphorylation in suspension culture and inhibits anoikis of intestinal epithelial cells. A, IEC-18 cells were cultured in suspension for 10 h in the absence () or in the presence (+) of 65 ng/ml TGF-. EGFR phosphorylation was assessed by Western blot using an anti-phosphotyrosine antibody (top). Total levels of EGFR are shown in the bottom panel. B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h in the absence () or in the presence of the indicated amounts of TGF-. Apoptosis was then measured by the cell death ELISA. Results represent the average of two independent experiments plus the S.D. C, RIE-1 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h in the absence or in the presence of 65 ng/ml TGF-. Apoptosis was then measured by the cell death ELISA. Results represent the average of two independent experiments plus the S.D.

TGF- Prevents Detachment-induced Inhibition of c-Src Kinase Activity-- Recent work (17-19) has demonstrated that EGFR activity can be regulated by integrin engagement. In view of the ability of TGF- to inhibit anoikis in IEC-18 cells, we speculated that one of the molecular events that could trigger cell death upon detachment of these cells is a reduction in the levels of EGFR phosphorylation (Fig. 1A). However, we found that in 5% FBS, the conditions normally used to culture IEC-18 cells and to induce anoikis, detachment of these cells does not result in dephosphorylation of the receptor (Fig. 2A).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Detachment of intestinal epithelial cells results in the down-regulation of c-Src kinase activity. A, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h, and EGFR phosphorylation was assessed by Western blot using an anti-phosphotyrosine antibody (top). Total levels of EGFR are shown in the bottom panel. B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 4 h and analyzed for c-Src kinase activity using enolase as a substrate (top). The same cell lysates were probed for total c-Src expression by Western blot (bottom). C, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h in the presence of Me2SO or 10 µM PP1. Apoptosis was then measured by the cell death ELISA. Results represent the average of two independent experiments plus the S.D.

One well established mediator of EGFR signaling is the c-Src kinase (23, 24). In addition to being activated by the EGFR, this kinase is also regulated by integrins in fibroblasts (25). Since inhibition of c-Src activity can induce apoptosis in attached cells (26), we decided to investigate whether this activity is reduced upon detachment of IEC-18 cells. We found that this is indeed the case. Detachment of IEC-18 cells results in a strong inhibition of c-Src (Fig. 2B). Since the role of such inhibition in the induction of anoikis has not yet been investigated, we decided first to verify whether, like in other cell types, c-Src activity is required for the survival of IEC-18 cells growing in monolayer culture. To this end, we treated attached IEC-18 cells with PP1, a widely used specific inhibitor of the Src family kinases (27). Fig. 2C shows that PP1 induced significant levels of cell death, although not as much as those generated by cell detachment. These data suggest that c-Src is, at least in part, required for the survival of attached IEC-18 cells, and that inhibition of the activity of this kinase upon detachment represents one of the causes of anoikis. We further reasoned that if down-regulation of c-Src activity contributes to detachment-induced death, anoikis should be inhibitable by v-Src, a constitutively active mutant version of c-Src. In agreement with this, we found that expression of v-Src in IEC-18 cells strongly protects them from anoikis (data not shown).

We then investigated whether TGF- can revert detachment-induced reduction of c-Src kinase activity. As shown in Fig. 3A, treatment with this growth factor notably suppressed such reduction. We reasoned that if the effect of TGF- on c-Src activity is required for the anti-anoikis activity of this growth factor, c-Src inhibitors, such as PP1, should block TGF--induced protection from anoikis. Indeed, we observed that PP1 completely abolished the anti-anoikis effect of this growth factor (Fig. 3B), suggesting that TGF- protects IEC-18 cells from anoikis in a c-Src-dependent manner.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   TGF- prevents detachment-induced inhibition of c-Src kinase activity. A, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 4 h in the absence () or in the presence (+) of 65 ng/ml TGF-, and analyzed for c-Src kinase activity using enolase as a substrate (top). The same cell lysates were probed for total c-Src expression by Western blot (bottom). B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h in the presence of Me2SO (), 65 ng/ml TGF- (TGF-), or 65 ng/ml TGF- and 10 µM PP1 (TGF- +PP1). Apoptosis was then measured by the cell death ELISA. Results represent the average of duplicates plus the S.D. This experiment was performed three times with similar results.

TGF- Inhibits Detachment-induced Activation of Caspase-3-- One of the molecular events most frequently associated with apoptosis is the activation of caspases (28). Consistent with this, anoikis of IEC-18 cells was strongly inhibited by the broad spectrum caspase inhibitor Z-VAD-FMK (Fig. 4A). Caspase-3 is an executioner caspase that triggers a number of key apoptosis-specific events such as chromosomal DNA fragmentation (28, 29). As shown in Fig. 4B, this caspase is activated upon detachment of IEC-18 cells, and this activation can be reverted by TGF- treatment (Fig. 4C). These data suggest that TGF- suppresses anoikis by blocking pro-apoptotic molecular events that result in the activation of caspase-3.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   TGF- inhibits detachment-induced activation of caspase-3. A, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for 17 h in the presence of Me2SO (mon, susp) or 250 µM Z-VAD-FMK (susp+zVAD). Apoptosis was then measured by the cell death ELISA. Results represent the average of duplicates plus the S.D. This experiment was performed three times with similar results. B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times and assayed for caspase-3 cleavage by Western blot. C, IEC-18 cells were cultured in suspension for 10 h in the absence () or in the presence (+) of 65 ng/ml TGF- and assayed for caspase-3 cleavage by Western blot.

TGF- Has No Impact on the Molecular Events Associated with Detachment-induced Fas Activation-- One of the molecular events potentially capable of triggering caspase-3 is the detachment-induced activation of the death receptor Fas. Such activation has been shown to contribute to anoikis of human umbilical vein endothelial cells (30). In addition, anoikis of Madin-Darby canine kidney cells, MCF-10A mammary cells, and HaCaT skin cells is regulated by components of the pathway triggered by Fas activation (7, 31, 32). Since the induction of this pathway seems to be a common event during anoikis, we investigated whether Fas-dependent signaling is also involved in this form of apoptotic cell death in the case of IEC-18 cells.

Treatment of the leukemic HUT78, and Burkitt's lymphoma BL-60 cells with an agonistic anti-Fas antibody results in a shift in the mobility of this receptor in a polyacrylamide gel from a monomeric form toward a slower migrating species of an apparent molecular mass of ~110 kDa (33). A similar change in Fas electrophoretic mobility is observed when human umbilical vein endothelial cells are detached from the ECM (30). Such alteration in mobility has been interpreted as an indication of oligomerization and activation of Fas (30, 33). In agreement with this, we found that detachment of IEC-18 cells results in a similar retardation of the mobility of Fas in a polyacrylamide gel, suggesting that Fas is activated in detached cells (Fig. 5A).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Detachment of IEC-18 cells triggers pro-apoptotic signaling events associated with the activation of Fas. A, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times, and the activation-associated aggregation of Fas was assessed by Western blot using an anti-Fas antibody. B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times, and the cleavage of IETD-pNA tetrapeptide, a known substrate of caspase-8, was measured in the respective cell lysates by a colorimetric assay. Adherent IEC-18 cells treated with adriamycin (mon+Adr) were used as a positive control. Results represent the average of two independent experiments plus the S.D. C, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times and assayed for caspase-10 cleavage by Western blot.

Fas-mediated apoptosis is known to occur via the activation of caspase-8 and caspase-10 (34-39). Since Fas appeared to be activated by detachment of IEC-18 cells, the status of these caspases in suspension culture was assessed. As shown in Fig. 5B, compared with what was observed in monolayer culture, caspase-8 was found to be relatively weakly activated by cell detachment, suggesting that this is not the only Fas-dependent initiator caspase triggered in these cells. Indeed, we found that detachment results in a strong activation of caspase-10 (Fig. 5C). It should be noted that in addition to being the initiators of the Fas-induced proteolytic cascade, both caspase-8 and caspase-10 can be activated downstream of caspase-3 in response to cytochrome c release from the mitochondria (40). However, in the case of anoikis of IEC-18 cells both caspase-8 and caspase-10 were activated prior to caspase-3 induction (compare Fig. 4B to Fig. 5, B and C). Collectively, these data suggest that detachment of IEC-18 cells results in the induction of pro-apoptotic Fas-mediated signaling events.

Next we investigated whether TGF--induced suppression of anoikis was due to the ability of this growth factor to inhibit the initial events associated with Fas activation, such as Fas aggregation and cleavage of caspase-10. We found that TGF- was unable to inhibit any of these events (Fig. 6), suggesting that TGF--induced suppression of anoikis occurs independently of the initial steps of the Fas-induced signaling pathway.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   TGF- treatment does not prevent detachment-induced pro-apoptotic events associated with Fas activation. A, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times in the absence () or in the presence (+) of 65 ng/ml TGF-, and the activation-associated aggregation of Fas was assessed by Western blot using an anti-Fas antibody. B, IEC-18 cells were cultured in monolayer (mon) or in suspension (susp) for the indicated times in the absence () or in the presence (+) of 65 ng/ml TGF-, and assayed for caspase-10 cleavage by Western blot.

TGF- Inhibits Detachment-induced Down-regulation of Bcl-X_L-- Caspase-3 activity can be suppressed by anti-apoptotic members of the Bcl-2 family such as Bcl-X_L (38). Recently, we found (8) that detachment of IEC-18 cells results in a strong down-regulation of Bcl-X_L expression, and that this down-regulation partially contributes to the induction of anoikis of these cells. A similar down-regulation of Bcl-X_L has been observed during anoikis of human intestinal epithelial cells, keratinocytes, as well as normal ovarian epithelial cells (8, 41, 42). In addition, we have demonstrated that activated ras partially contributes to anoikis resistance of intestinal epithelial cells by preventing detachment-induced inhibition of Bcl-X_L expression (8). Previously we found (8) that individual clones of IEC-18 cells expressing exogenous Bcl-X_L are significantly more resistant to anoikis than controls transfected with vector alone. To further corroborate the evidence of the critical role of Bcl-X_L in anoikis, we decided to test its involvement in this process by an independent method. To this end, IEC-18 cells were co-transfected with a green fluorescent protein (GFP) expression vector and either pcDNA-3 or pcDNA-3 carrying the Bcl-X_L cDNA. The transfected GFP-positive cells were then isolated by FACS and plated in monolayer culture either immediately or after incubation in suspension culture for 48 h. After 10 days the resulting cell colonies were counted, and the ratio between the number of colonies formed by cells that were cultured in suspension and the number derived from cells plated immediately after sorting was determined for each vector. As shown in Fig. 7A, the number of clonogenic cells that survived through the suspension culture was significantly higher in case of the Bcl-X_L-transfected cells compared with vector control. This result provides additional evidence demonstrating that detachment-induced down-regulation of Bcl-X_L does indeed play a causal role in anoikis of IEC-18 cells.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   TGF- prevents detachment-induced down-regulation of Bcl-XL. A, IEC-18 cells were co-transfected with a GFP expression vector and either pcDNA-3 or pcDNA-3 carrying the Bcl-XL cDNA. The transfected GFP-positive cells were then isolated by FACS and plated in monolayer culture either immediately or after incubation in suspension culture for 49 h. The resulting cell colonies were counted 10 days later, and the ratio between the number of colonies formed by cells that were cultured in suspension and the number derived from cells plated immediately after sorting was determined for each vector. This ratio was arbitrarily defined as 1.0 for the pcDNA-3-transfected cells. Results represent the average of two independent experiments plus the S.D. B, IEC-18 were cultured in monolayer (mon) or in suspension (susp) for 1 h in the absence () or in the presence (+) of 65 ng/ml TGF- and assayed for Bcl-XL expression by Western blot. The membrane was re-probed with an anti-Bak antibody as a loading control. C, IEC-18 were cultured in suspension (susp) for 1 h in the absence () or in the presence (+) of 65 ng/ml TGF- and 10 µM PP1 and assayed for Bcl-XL expression by Western blot. The membrane was re-probed with an anti-Bak antibody as a loading control.

We decided then to investigate whether TGF- is able to rescue IEC-18 cells from anoikis by suppressing the detachment-induced down-regulation of Bcl-X_L expression. As shown in Fig. 7B, TGF- treatment of IEC-18 cells noticeably inhibits the reduction of Bcl-X_L expression caused by the loss of cell-ECM interaction. Since the anti-anoikis effect of TGF- can be prevented by the c-Src inhibitor PP1 (Fig. 3B), we investigated whether the effect of TGF- on Bcl-X_L expression requires c-Src kinase activity. As depicted in Fig. 7C, TGF--induced increase in Bcl-X_L expression was strongly inhibited by PP1.

If the inhibitory effect of TGF- on anoikis is mediated by the effect of this growth factor on Bcl-X_L expression in detached cells, ectopic expression of this anti-apoptotic molecule in IEC-18 cells should have a similar impact on activation of caspase-3 and caspase-10 to that of TGF- treatment. Fig. 8 shows that this is indeed the case. Two anoikis-resistant IEC-18-derived clones expressing ectopic Bcl-X_L (8) displayed a significantly lower degree of detachment-induced caspase-3 activation than the controls (Fig. 8A). Likewise, in agreement with what was seen in TGF--treated cells, exogenous Bcl-X_L had no effect on detachment-triggered caspase-10 cleavage (Fig. 8B). These data indicate that both TGF- and Bcl-X_L protect cells from anoikis by a mechanism that prevents caspase-3 activation and that this protection occurs independently of the activation of Fas.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Ectopic Bcl-XL mimics the effect of TGF- on detachment-induced activation of caspases-3 and -10. IEC-18 cells, IEC-18 cells transfected with vector alone (vector 22), and two independently derived clones of IEC-18 cells expressing ectopic Bcl-XL (Bcl-x 3 and Bcl-x 27) were cultured in suspension for 10 h and assayed for caspase-3 (A) or caspase-10 (B) cleavage by Western blot.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have shown in this study that TGF- has the ability to inhibit anoikis of normal intestinal epithelial cells. It has been reported that the capacity of the EGFR, which is the receptor for this growth factor, to induce various signaling events is strongly reduced in the absence of cell-ECM attachment (5, 17-19). Thus, it is possible that certain components of the TGF--driven signaling system are not activated at optimal levels in intestinal epithelial cells when they are not attached to the ECM. However, our data clearly demonstrate that this growth factor is capable of triggering at least one anti-apoptotic pathway that is sufficient for significant inhibition of anoikis of these cells.

We have demonstrated here that detachment of IEC-18 cells does not result in a reduction of the levels of EGFR phosphorylation, indicating that changes in the activity of the receptor per se are not involved in the induction of anoikis. Thus, these data suggest that TGF--induced activation of the EGFR leads to the inhibition of this form of cell death by triggering signals that override detachment-induced pro-apoptotic events initiated in an EGFR-independent manner. Our results indicate that one of these events is the inhibition of c-Src kinase activity. This kinase is known to be independently regulated by both EGFR and integrin-ECM interactions (23-25). The activation of c-Src by integrins can be mediated by focal adhesion kinase, a cytoplasmic enzyme that is known to be regulated by integrin engagement (25).

We have also shown here that TGF- suppresses anoikis, at least in part, by preventing detachment-induced inhibition of c-Src kinase. These data are consistent with the fact that both EGFR and c-Src have a well established ability to inhibit apoptosis in adherent cells (26). These results are also consistent with the finding demonstrating that v-Src, a constitutively active form of Src, is capable of inhibiting anoikis of Madin-Darby canine kidney cells (2, 5).

We have shown that TGF- and c-Src prevent anoikis of intestinal epithelial cells by inhibiting detachment-induced down-regulation of Bcl-X_L. This is in agreement with reports indicating that Bcl-X_L expression is dependent on the activities of the EGFR and c-Src in adherent keratinocytes and fibroblasts (26, 41, 43, 44). In addition, the expression of this anti-apoptotic molecule has been shown to be induced by v-Src (45). c-Src itself is known to induce the activities of several signaling molecules including mitogen-activated protein kinase and STAT-3 (23, 46), and both have the ability to stimulate Bcl-X_L expression (43, 45). The potential involvement of these two mechanisms in the anti-anoikis effect of TGF- is the subject of our ongoing research.

Our data indicate that detachment of intestinal epithelial cells results in the activation of at least two pro-apoptotic molecular pathways. One of these pathways is triggered by the activation of Fas, and the other by the down-regulation of Bcl-X_L. Caspases-8 and -10, which are the initiator caspases in the Fas-induced cell death pathway, can each directly activate caspase-3, one of the key effector caspases (40, 47). Since caspase-3 is known to be inhibited in the presence of high Bcl-X_L levels (through both cytochrome c-dependent and -independent events) (38, 48, 49), it seems reasonable to speculate that both the activation of Fas and the down-regulation of Bcl-X_L ultimately converge on the activation of caspase-3. Certainly, the involvement of other effector caspases in this process is also likely. The cooperative effect of these signaling events on caspase-3 could then elevate its activity beyond the threshold level required for the induction of apoptosis. If, however, one of the caspase-3-inducing molecular events is inhibited, as it occurs in response to the TGF--triggered increase in Bcl-X_L expression, caspase-3 activity can drop below anoikis-permissive levels. Naturally, the possibility that TGF- has the ability to inhibit anoikis through alternative, Bcl-X_L-independent, mechanisms cannot be excluded. For example, anoikis of mammary epithelial cells has been shown to occur as a result of the insertion of Bax into the mitochondria (50). Whether this molecular event can be regulated by TGF- remains to be investigated.

TGF- is frequently secreted by human colorectal tumor cells (9, 10, 12), and it is thought to play an important role in the progression of this type of malignancy (14). EGFR has recently been validated (51) as a relevant therapeutic target in colorectal cancer treatment. In addition, development and progression of this type of cancer are often associated with an increased c-Src activity and enhanced Bcl-X_L expression (52-56). Based on our data, it is tempting to speculate that TGF- contributes to colorectal cancer progression, at least in part, by inhibiting anoikis through a c-Src-dependent induction of Bcl-X_L expression. It is also important to note that the role of EGF family members in tumor progression is not limited to colorectal cancer (57-59). In addition, TGF- transgenic mice are known to be prone to neoplastic transformation in several tissues (60, 61).

While this manuscript was in preparation, two studies (62, 63) were published demonstrating that, similar to what was observed here for intestinal epithelium, EGF family members can inhibit anoikis of normal breast and skin cells. Collectively, these data suggest that the ability to contribute to tumor progression through the inhibition of anoikis may represent a general property of the members of this family of growth factors.

	ACKNOWLEDGEMENT

We thank Penny Papadakos for assistance in the preparation of this manuscript.

	FOOTNOTES

^* This work was supported by the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Sunnybrook and Women's College Health Sciences Center, Division of Molecular and Cell Biology, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada. Tel.: 416-480-6100, ext. 3350; Fax: 416-480-5703; E-mail: filmus@sten.sunnybrook.utoronto.ca.

Published, JBC Papers in Press, August 3, 2001, DOI 10.1074/jbc.M106424200

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; EGFR, epidermal growth factor receptor; TGF-, transforming growth factor ; FBS, fetal bovine serum; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; -MEM, -minimum Eagle's medium; ELISA, enzyme-linked immunosorbent assay; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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