the role of bile acids in carcinogenesis

Bile acids stimulate invasion and haptotaxis in human colorectal cancer

cells through activation of multiple oncogenic signaling pathways

PR Debruyne1, EA Bruyneel1, I-M Karaguni2, Xd Li1, G Flatau3, O Muller2, A Zimber4,C Gespach4 and MM Mareel*,1

1Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, B-9000Ghent, Belgium; 2Max-Planck-Institut fur molekulare Physiologie, 44227 Dortmund, Germany; 3INSERM U 452, BiologieCellulaire et Moleculaire des Microorganismes Pathogenes et de leurs Toxines, UFR de Medecine, 06107 Nice Cedex, France;4INSERM U482, Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, Hopital Saint-Antoine, 75571Paris Cedex 12, France

Bile acids are implicated in colorectal carcinogenesis asevidenced by epidemiological and experimental studies.We examined whether bile acids stimulate cellularinvasion of human colorectal and dog kidney epithelialcells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidneyMDCKT23 cells were seeded on top of collagen type Igels and invasive cells were counted after 24 hincubation. Activation of the Rac1 and RhoA smallGTPases was investigated by pull-down assays. Hapto-taxis was analysed with modified Boyden chambers.Lithocholic acid, chenodeoxycholic acid, cholic acid anddeoxycholic acid stimulated cellular invasion of SRC-and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originatingfrom a sporadic tumor, but were ineffective in premalig-nant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation ofhaptotaxis and was dependent on the RhoA/Rho-kinasepathway and signaling cascades using protein kinase C,mitogen-activated protein kinase, and cyclooxygenase-2.Accordingly, BA-induced invasion was associated withactivation of the Rac1 and RhoA GTPases andexpression of the farnesoid X receptor. We concludethat bile acids stimulate invasion and haptotaxis incolorectal cancer cells via several cancer invasionsignaling pathways.Oncogene (2002) 21, 6740 – 6750. doi:10.1038/sj.onc.1205729

Keywords: bile acids; invasion; colorectal cancer

Introduction

Bile acids (BA), commonly considered as trophicfactors for the enteric epithelium, and as detergents

for the emulsification and absorption of dietary lipidsand fat-soluble vitamins, participate also in coloncancer progression. High fat diet and cholesterol areimportant risk factors for the emergence of colorectalcancers, presumably because of their associated impacton the synthesis and secretion of BA (Hill et al., 1971;Reddy, 1981; Willett et al., 1990; Nagengast et al.,1995). Several epidemiologic studies have implicatedthe alterations of the production and composition ofBA in the intestinal lumen and blood with theincreased incidence of colorectal adenomas andadenocarcinomas (McMichael and Potter, 1985; Bayer-dorffer et al., 1993; Owen, 1997; Kamano et al., 1999).Compelling evidence for a causal link between BA andcolon cancer was supported by experimental carcino-genesis in rats. Cholic acid (CA), lithocholic acid(LCA), taurochenodeoxycholic acid (TCDCA) andchenodeoxycholic acid (CDCA) were considered astumor promoters because they did not induce tumorson their own, but increased the incidence of benignadenomas and malignant adenocarcinomas whenadministered after a carcinogen (Narisawa et al.,1974; Reddy et al., 1977).

Colorectal carcinogenesis is characterized by multiplemolecular alterations and cellular dysfunctions, includ-ing the loss of control of growth and differentiation, andby the acquisition of undesirable cellular activitiesduring the adenoma-adenocarcinoma transition, includ-ing cellular invasion, ectopic survival, neo-angiogenesis,and production of distant metastases. Invasion andmetastasis, the hallmarks of malignant tumors, are bothgreatly influenced by autocrine, paracrine and endocrinefactors in the tumor (Mareel et al., 1996). Accordingly,invasion of colorectal cancer cells into collagen type I isinduced by endogenous factors, such as hepatocytegrowth factor (HGF), leptin and trefoil peptides(Empereur et al., 1997; Kotelevets et al., 1998; Attoubet al., 2000; Emami et al., 2001). The possibleimplication of BA in cancer cell invasion is suggestedby the correlation between Dukes’ stage of human colontumors, and abnormal fecal deoxycholic acid (DCA)/cholic acid (CA) and LCA/DCA concentration ratios, aswell as the number of the BA binding sites in tumor cells(Summerton et al., 1983; Owen, 1997; Kamano et al.,

Received 10 March 2002; revised 16 May 2002; accepted 7 June2002

*Correspondence: M Mareel, Laboratory of Experimental Cancerol-ogy, Department of Radiotherapy and Nuclear Medicine, GhentUniversity Hospital (1P7), De Pintelaan 185, B-9000 Ghent, Belgium;E-mail: [email protected]

Oncogene (2002) 21, 6740 – 6750ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00

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1999). Recently, LCA and other BA were reported to beinvolved in the induction of cyclooxygenase-2 (COX-2),the secretion of matrix metalloproteinase 2 (MMP-2),and the migration of colorectal cancer cells (Halvorsenet al., 2000; Glinghammer and Rafter, 2001).

BA are ligands of the farnesoid X (FXR) andpregnane X (PXR) nuclear receptors (Makishima et al.,1999; Parks et al., 1999; Wang et al., 1999; Staudingeret al., 2001; Xie et al., 2001). In hepatocytes, expressionof the cholesterol 7a-hydroxylase (CYP7A1) gene isregulated through the coordinated activation of theFXR and the JNK/c-Jun signaling pathways (Gupta etal., 2001). In colorectal cancer cells, DCA and otherBA, induce the activation of the activator protein-1(AP-1) transcription factor, via a signaling cascadeusing protein kinase C (PKC) and the mitogen-activated p42/p44 and p38 protein kinases (MAPK)(Hirano et al., 1996; Qiao et al., 2000, 2001). The AP-1complex induced by DCA is composed of heterodimersincluding JunD, Fra-1 and c-Fos, which are actuallyconsidered as oncogenic transcription factors impli-cated in cellular invasion (Hennigan et al., 1994;Kustikova et al., 1998; Ozanne et al., 2000). One ofthe critical target genes of AP-1 is the cyclooxygenase-2(COX-2) gene, whose promoter activity and transcrip-tion are induced by BA in human colorectal andesophageal adenocarcinoma cells (Zhang et al., 1998;Glinghammer and Rafter, 2001). Induction andconstitutive overexpression of COX-2 is stronglyimplicated in the emergence of human colorectalpolyps and in cancer progression, through promotionof cell survival, invasion, and tumor angiogenesis(Fujita et al., 1998; Dannenberg and Zakim, 1999).The proinvasive potential of the COX-2 pathway isdirectly connected with activation of the MMP-2(Tsujii et al., 1997), and the Gaq cascade using thephospholipase C and PKC signaling elements (Rodri-gues et al., 2001).

Here, we report that BA stimulate invasion ofcollagen type I gels by human colorectal cancer cellsthrough haptotaxis towards selected matrix compo-nents. Furthermore, we demonstrate that these cellularactivities are dependent upon several signaling path-ways directly implicated in colon cancer progression,namely, the RhoA and Rac1 small GTPases, PI3-K,PKC, MAPK and COX-2.

Results

Cooperation between BA and the oncogenes SRC andRHOA in cellular invasion by premalignant colorectal andkidney epithelial cells

We first investigated whether premalignant colon andkidney epithelial cells could be stimulated to invadecollagen type I gels in response to BA at thephysiologic concentration of 10 mM. As shown inFigure 1, the BA CDCA, LCA, and UDCA did notstimulate cellular invasion by premalignant humancolorectal adenoma PC/AA/C1 cells. Similar negativeresults were obtained with CDCA, LCA and UDCA in

the nontumorigenic kidney epithelial MDCKT23 cellline (Figure 1). Next, we examined the possiblecooperation between BA and SRC in the acquisitionof the invasive phenotype using PCmsrc cells whichwere obtained after transfection of PC/AA/C1 cells byan expression vector encoding the activated form of theSRC oncogene (Empereur et al., 1997). As shown inFigure 1, CDCA and LCA, but not UDCA, stimulatedthe invasiveness of PCmsrc cells in collagen type I gels.Furthermore, we found that MDCKT23-RhoAV14cells expressing a constitutively activated mutant formof the gene encoding the RhoA small GTPase (RHOA;Jou and Nelson, 1998) were also stimulated to invadecollagen type I gels in response to CDCA and LCA,but not to UDCA (Figure 1). As a control, we foundthat MDCKT23-RhoAN19 cells, which express a

Figure 1 Effects of BA on invasion of premalignant PC/AA/C1and MDCKT23 cells. Collaboration between BA and cellulartransformation induced by the SRC oncogene and the RhoAsmall GTPase. Invasion index in collagen type I gels was measuredafter 24 h at 378C, using PC/AA/C1 and MDCKT23 cells, andtheir counterparts PCmsrc and MDCKT23-RhoAV14, respec-tively transformed by constitutively activated SRC and RhoAV14oncogenes. Results were compared with MDCKT23-RhoAN19cells transformed by the dominant negative form of RhoA. Cellswere incubated in the presence of 0.1% DMSO (Control), orone of the following BA, at 10 mmol/l concentration: CDCA, che-nodeoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxy-cholic acid. Bars indicate mean values of invasion indices withupper bounds of 95% confidence intervals. *Statistical significancebetween controls and experimental data was indicated at P50.01

Bile acids and colorectal cancer progressionPR Debruyne et al

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dominant-negative form of RHOA, were not invasiveupon treatment with CDCA or LCA (Figure 1).

BA dose-dependently stimulate cellular invasion in humanmalignant PCmsrc and HCT-8/E11 cells

As a next step, we determined the effects of LCA,CDCA, and UDCA on human colorectal cancer HCT-8/E11 cells. As shown in Figure 2, cellular invasion wasstimulated, by LCA and CDCA in PCmsrc and HCT-8/E11 cells, following similar dose-response curves. Asdescribed above for PCmsrc cells, UDCA was alsoineffective in HCT-8/E11 cells. Maximal stimulation ofinvasion by BA occurred at 10 mmol/l, withoutapparent cytotoxicity (Figure 2). Higher concentrationsof LCA and CDCA (100 mmol/l) were toxic, asevidenced by trypan blue staining of about 20 – 30%

of the whole cell population. The potency of LCA andCDCA in stimulating cellular invasion was similar inboth cell types.

We compared the ability of the other non-conjugatedBA CA and DCA, as well as the glyco- and tauro-conjugated forms of CDCA and LCA to stimulateinvasion in PCmsrc and HCT-8/E11 cells (Figure 3).We found that both CA and DCA could stimulate thiscellular activity. In contrast, the glyco- and tauro-conjugated forms of CDCA (GCDCA and TCDCA),as well as the conjugated forms of LCA (GLCA andTLCA) were not competent in the two cellular modelspresented in Figure 3.

The hydrophilic bile acid UDCA was previouslyshown to protect hepatocytes against toxicity caused byless polar BA, such as CDCA, CA and DCA (Kurtz,1992). Combinations of BA with UDCA were, there-fore, tested in our assay. Stimulation of invasion byHCT-8/E11 and PCmsrc cells by CDCA or LCA wasnot altered by simultaneous addition of equimolar(10 mmol/l) concentrations of UDCA (data not shown).

Stimulation of invasion by BA and cell signaling

Because BA stimulate invasion in the transformedMDCKT23-RhoAV14 and in the tumoral PCmsrc andHCT-8/E11 cells, we tested the importance of the Rho-like GTPases for this effect. We first investigated thedirect effect of DCA on the activation status of RhoAand Rac1 in HCT-8/E11 colon cancer cells. Activation ofthese two GTPases by lysophosphatidic acid (LPA,2.5 mmol/l) was included as a control (Sander et al.,1999). As shown in Figure 4a, 10 – 500 mmol/l DCAincreased the GTP-bound state of RhoA up to about 18-fold, at concentrations that are efficient in stimulatingcell invasion, without alteration of cell viability (data notshown). In accordance with our data, Latta et al. (1993)also reported that 300 mmol/l DCA was not cytotoxic inseveral colorectal cancer cell lines. Thus, at concentra-tion as low as 10 mmol/l, DCA increased the GTP-boundstate of RhoA 11-fold, as observed with LPA, withoutany change in the total amount of this GTPase.Similarly, both LPA and 50 mmol/l DCA increased theGTP-bound state of the Rac1 GTPase up to about 30-fold, as illustrated in Figure 4b.

We next investigated the relationship between thepotential of BA to stimulate invasion and theexpression of the nuclear bile acid receptor FXR inthe human colon PC/AA/C1, PCmsrc and HCT-8/E11cells. As shown in Figure 5, the BA-sensitive PCmsrcand HCT-8/E11 cell lines, but not the BA-insensitivePC/AA/C1 cell line, expressed the FXR (Figure 5),suggesting a possible role for the FXR in thestimulation of invasion by BA.

The aE-catenin-negative derivative HCT-8/E11R1was not sensitive to stimulation by CDCA or LCA inthe collagen type I invasion assay (Figure 6). Thissuggests that a functional E-cadherin/catenin complexis needed for LCA-induced invasion. Accordingly, wecould inhibit LCA-stimulated invasion of HCT-8/E11cells, down to 31+9% of the control (LCA without

Figure 2 Effects of various concentrations of BA on invasion ofPCmsrc and HCT-8/E11 cells into collagen type I. Invasion indexin collagen gels as measured after 24 h at 378C, using PCmsrc andHCT-8/E11 cells incubated in the presence or absence of the indi-cated concentrations of the bile acids: CDCA (chenodeoxycholicacid), LCA (lithocholic acid), or UDCA (ursodeoxycholic acid).Ordinate: invasion index as mean values with upper bounds of95% confidence intervals


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antibody, arbitrary 100%) activity, and of PCmsrccells, down to 61+13% of the control activity, byaddition of an antibody that neutralizes the function ofE-cadherin (MB2).

In complementary experiments, we have examinedthe functional role of the b1 and aV integrins in LCA-stimulated invasion of human colon cancer cells. Theaddition of the neutralizing antibody P4C10 directedagainst the b1 integrin subunit inhibited LCA-stimulated invasion of HCT-8/E11 and PCmsrc cellsdown to 11+8% and 20+8% of the control activity,respectively. In comparison, the 69.6.5 antibody raisedagainst the aV integrin subunit was less efficient againstLCA-stimulated invasion in HCT-8 and PCmsrc cells(inhibition down to 95+22% and 57+15% of thecontrol activity, respectively).

Next, we investigated the possible contribution ofseveral oncogenic pathways directly implicated intumor cell motility and invasion, namely the Rho/Rho kinase (ROCK), PI3K, PKC, MAPK, and COX-2signaling cascades on LCA-induced invasion. As shownin Figure 7, pharmacological inhibitors of thesesignaling components reduced LCA-stimulated inva-sion down to 4 – 55% of the control (LCA withoutinhibitor) activity in PCmsrc and HCT-8/E11 cells.Finally, inhibition of matrix metalloproteinases by theGM6001 compound also reduced LCA-stimulatedinvasion down to 39+18 and 17+14% of the controlactivity in HCT-8/E11 and PCmsrc cells respectively.

LCA-induced changes in morphology and motilephenotype

As a first approach to identify the cellular mechanismsunderlying the potential of BA to stimulate invasion,

we have analysed the effects of the efficient invasionstimulator LCA on the morphology of PCmsrc andHCT-8/E11 cells (Figure 8). In HCT-8/E11 cells grownfor 20 h on glass coverslips in serum-free medium,treatment with LCA (10 mmol/l) induced cell roundingand appearance of smaller islands with more refringentborders. Furthermore, LCA inhibited filopodia forma-tion in HCT-8/E11 cells (Figure 8, lower panel). Theseeffects were less clear in PCmsrc cells. On coverslipscoated with collagen type I, HCT-8/E11 cells showedfew filopodia, but LCA still induced cell rounding(data not shown). Under all circumstances, islands ofcells were smaller and more numerous in the presenceof LCA.

As shown in Figure 9, migration of PCmsrc andHCT-8/E11 cells through Nucleopore filters wasdependent upon the concentration of collagen type Iused for coating the lower side of the filters.Directional cellular migration toward collagen type Iwas much lower in PC/AA/C1 and HCT-8/E11R1cells than in the invasion-competent cells PCmsrc andHCT-8/E11. Furthermore, this migration towards aninsoluble gradient of a substratum-bound attractant,i.e. haptotaxis, was further increased by LCA inPCmsrc and HCT-8/E11R1 cells, but not in PC/AA/C1 and HCT-8/E11 cells. In this assay, LCAincreased the number of transmigrated PCmsrc andHCT-8/E11 cells, not only to collagen type I, butalso to collagen type IV and laminin. Haptotaxis ofPCmsrc and HCT-8/E11 cells to fibronectin was verylow and insensitive to stimulation by LCA. Trans-filter migration was absent in the presence of poly-L-lysine coating used as a negative control (data notshown). In time-lapse videomicrographs, randommotility was not modified upon treatment with

Figure 3 Effects of the glyco- and tauro-conjugated forms of the BA CDCA and LCA on invasion of PCmsrc and HCT-8/E11 cellsinto collagen type I. Invasion index in collagen gels as measured after 24 h at 378C, using PCmsrc and HCT-8/E11 cells incubated inthe presence or absence of one of the following bile acids (10 mmol/l): CA (cholic acid), DCA (deoxycholic acid), CDCA (chenodeoxy-cholic acid), GCDCA and TCDCA (respectively, glyco- and tauro-chenodeoxycholic acid), LCA (lithocholic acid), GLCA and TLCA(glyco- and tauro-lithocholic acid), UDCA (ursodeoxycholic acid). Bars indicate mean values of invasion indices with upper bounds of95% confidence intervals; statistical significance between controls and experimental data was indicated at *P50.05 or **P50.01


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LCA, as evidenced by the unchanged track lengths ormembrane ruffling in HCT-8/E11 and PCmsrc cells(data not shown).

Discussion

The role of BA in normal and neoplastic growth ofdigestive epithelial cells is still controversial. Bothbeneficial and undesirable effects of BA were previouslydocumented (Nagengast et al., 1995; Zimber et al.,2000). Here, we show that premalignant cells exempli-fied by kidney epithelial MDCK cells and colonadenoma PC/AA/C1 cells are not sensitive to stimula-tion of invasion by BA. Our major finding is that theBA CDCA, LCA, CA, and DCA, but not UDCA,were acting as stimulators of invasion in PC/AA/C1and MDCK cells transformed by the SRC oncogene inPCmsrc cells or the constitutively activated, smallGTPase RHOA in MDCKT23-RhoAV14 cells. TheMDCK cells were used to express molecules of interest,the PC/AA/C1 cells were used as a model for colorectalcancer development. These observations support theidea that genetic alterations marking the transitionbetween the noninvasive and invasive stages do notnecessarily induce invasion but make the cellscompetent to subsequent stimulation by local media-tors, such as bile acids. Src kinase activation isconsidered as a frequent and early event in coloncancer progression. Furthermore, mutations at Tyr530,hampering the negative autoregulatory feedback of theSrc kinase activity, are found in advanced colorectalcancers (Irby et al., 1999).

DCA induced the GTP-bound active state of bothRhoA and Rac1, suggesting that these two smallGTPases play a key role in the pro-invasive signalingpathways controlled by BA. Cell rounding, loss offilopodia, and enhanced haptotaxis were observed in

Figure 4 Activation of the RhoA and Rac1 small GTPases bydeoxycholic acid (DCA) and lysophosphatidic acid (LPA) inHCT-8/E11 cells. Relative levels of RhoA and Rac1 in HCT-8/E11 cells that were treated for 10 min with increasing concentra-tions of deoxycholic acid (DCA) or lysophosphatidic acid (LPA).Western blot analysis showing (a) the rhotekin complexed activeRhoA (upper row) and the level of total RhoA (lower row),and (b) the PAK complexed active Rac1 (upper row) and the levelof total Rac1 (lower row). The amount of active RhoA and Rac1are increased upon treatment with 10, 50, 25 and 500 mmol/ldeoxycholic acid (DCA). LPA (2.5 mmol/l) is used as a positivecontrol. Quantitation of Western blots was by the Quantity OneSoftware. Values below the blots represent relative intensity ascompared to the control condition

Figure 5 Expression of the farnesoid X receptor in the invasion-competent PCmsrc and HCT-8/E11 cells. Western blot of the far-nesoid X receptor (FXR) was performed using total cell lysatesfrom PC/AA/C1, PCmsrc and HCT-8/E11 cells. Three bands,corresponding to the FXR (molecular weights around 66 kDa)were detected in rate brain homogenates (positive control) andin PCmsrc and HCT-8/E11 cells

Figure 6 Absence of bile acid stimulated cellular invasion in theaE-catenin-deficient HCT-8/E11R1 cells. HCT-8/E11 and the aE-catenin-deficient HCT-8/E11R1 cells were incubated in the pre-sence or absence (0.1% DMSO as control) of one of the followingBA (10 mmol/l): chenodeoxycholic acid (CDCA), lithocholic acid(LCA), and ursodeoxycholic acid (UDCA). Bars indicate meanvalues of invasion indices with upper bounds of 95% confidenceintervals. *Statistical significance between controls and experimen-tal data was indicated at P50.01


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colon cancer cells treated by BA, further indicatingthat these two small GTPases are an integral part ofthe mechanisms responsible for the acquisition of theinvasive phenotype. Members of the small Rho-likeGTPase family, namely RhoA, Rac1 and Cdc42, arewell recognized as key regulators of the cytoskeletalorganization that control epithelial integrity, cellmigration and invasion (Sander and Collard, 1999).RhoA is involved in the formation of focal contactsand contractile actin-myosin filaments through interac-tion with several downstream effectors such as Rhokinase (ROCK). Rac1 mediates a number of adhesion-and growth factor-dependent responses, including themembrane ruffling and lamellipodia formation thatfunction in the advancement of the leading edge ofmigrating cells (Hall, 1998). Accordingly, we foundthat MDCKT23-Rac1V12 cells expressing the consti-tutive activated form of Rac1 (Jou and Nelson, 1998)

are invasive without the addition of stimulators(Attoub et al., 2000). The involvement of the Rhosignaling pathway in cellular invasion induced by BAwas confirmed by our data using the pharmacologicalinhibitors of the Rho GTPases (C3T exoenzyme) andits downstream effector, ROCK (Y27632). It is,therefore, conceivable that BA stimulate tumor cellinvasion through RHOA- and SRC-dependent signal-ing pathways.

Haptotaxis or directional migration towards aninsoluble gradient of a substrate-bound attractant isimplicated in the invasive phenotype (Aznavoorian et al.,1990). Accordingly, we found a direct correlationbetween cell type sensitivity towards stimulation ofinvasion and haptotaxis in response to the different BA.Furthermore, invasion and haptotaxis were sensitive tothe same pharmacological inhibitors of the signalingelements (data not shown). In contrast, there were no

Figure 7 Lithocholic acid signaling and induction of cellular invasion in PCmsrc and HCT-8/E11 cells. Invasion index in collagentype I gels was measured at 378C, for PCmsrc cells (top) or HCT-8/E11 cells (bottom) incubated in the presence of lithocholic acid(LCA), either alone or combined with one of the pharmacological inhibitors of signal transduction pathways: Rho/ROCK (respec-tively C3 exoenzyme: 7 mg/ml and Y27632; 1 mmol/l), PI3K (Wortmannin: 10 nmol/l), PKC (GF109203X: 1 mmol/l or Go6976:40 nmol/l), MAPK p42/p44 (PD98059: 20 mmol/l) and p38 (SB203580: 1 mg/ml), and cyclooxygenase COX-2 (NS-398: 10 mmol/l). Data are mean+s.e.m. from three separate experiments. All results are statistically significant (P50.05) from the control stimu-lation measured in the presence of LCA


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changes in cell-cell and cell-matrix adhesion, andrandom motility upon treatment with LCA (data notshown). BA stimulate both invasion and haptotaxis inhuman colon cancer cells HCT-8/E11, possessing afunctional E-cadherin/catenin complex, but not in itsaE-catenin-deficient derivative HCT-8/E11R1 cell line(Vermeulen et al., 1999). These results indicate thatLCA-induced invasion and haptotaxis require functionalE-cadherin/catenin complexes, as shown previously forthe pro-invasive agents HGF, leptin and trefoil peptides(Emami et al., 2001). This is also supported by theneutralizing action of the E-cadherin-antibody MB2inhibiting the cooperation between E-cadherin andintegrins in wound-healing migration of intestinal cells(Andre et al., 1999). The putative integrin isotypesconcerned in our studies are likely a1b1 and a2b1, sinceLCA stimulates haptotaxis to collagen types I and IV aswell as to laminin, but not to fibronectin. In favor of thispossibility, we found that BA-stimulated cellular inva-sion is inhibited by the b1 integrin neutralizing antibody.

Stimulation of tumor cell invasion by BA requiresmultiple signaling pathways that are strongly impli-cated in cancer progression, including PI3K, PKC,MAPK, and COX-2 (Hirano et al., 1996; Kurz et al.,2000; Qiao et al., 2000, 2001). Activation of PKC byBA is well-documented in colorectal cancer cells(Huang et al., 1992; Pongracz et al., 1995; Hirano etal., 1996; Rao et al., 1997; Qiao et al., 2000).Implication of PKC-a in stimulation of haptotaxisand invasion has also been reported in colorectalcancer HT-29 M6 and RCM-1 cells (Batlle et al., 1998;Shimao et al., 1999). PKC activation by BA isachievable via signaling cascades involving PI3K andRhoA (Chang et al., 1998; Kurz et al., 2000; Rust etal., 2000). In the present study, we demonstrated thatLCA-stimulated invasion of PCmsrc and HCT-8/E11cells was prevented by wortmannin, further implicatingPI3-K as critical mediator of cellular invasionstimulated by oncogenes or epigenetic factors (Kotele-vets et al., 1998; Attoub et al., 2000; Emami et al.,

2001). Our results obtained with Go 6976, an inhibitorblocking the ATP binding site within the catalyticdomain of these ser/thr kinases, suggest that PKC-aand -bI are the isoenzymes likely involved in BA-stimulated collagen invasion (Hofmann, 1997). PKCinhibitors selectively interfere with positive invasionpathways (Attoub et al., 2000; Emami et al., 2001),presumably through interruption of transcriptionsignals controlled by the AP-1 sites (also designatedas TRE, for TPA-response elements) in the regulatoryregion of several genetic elements implicated in cancerprogression (Ozanne et al., 2000). For example, bothCDCA and DCA were shown to increase expression ofthe cyclooxygenase COX-2 in colorectal cancer cells, ina PKC- and MAPK-dependent manner, presumablythrough the action of AP-1 on a cyclic AMPresponsive element (CRE) located at – 56 in theCOX-2 promoter (Zhang et al., 1998; Glinghammer

Figure 8 Effects of lithocholic acid on the morphology ofPCmsrc and HCT-8/E11 cells. Phase-contrast micrographs ofHCT-8/E11 and PCmsrc cells grown on glass coverslips in thepresence or absence of lithocholic acid (10 mmol/l; LCA) for20 h. Arrowheads indicate filopodia. Scale bar=100 mm

Figure 9 Effects of various concentrations of BA on haptotaxisto collagen type I in PCmsrc and HCT-8/E11 cells. Cells were in-cubated with 0.1% DMSO (control) or lithocholic acid (10 mmol/l; LCA). Abscissa: concentration of collagen type I used for sub-strate coating. Ordinate: mean number of cells per microscopicfield as an index for cell migration with error bars for 95% con-fidence intervals


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and Rafter, 2001). This CRE is critical for both Ras-and Rac1-induced transactivation of the COX-2promoter. Alternatively, the COX-2 promoter mightbe activated through cis-acting elements locatedbetween nucleotides – 80 and – 40 that are requiredfor transactivation by the RhoA and Rac1 smallGTPases (Slice et al., 2000). Downstream effectors ofthe invasion pathways controlled by BA are COX-2and matrix metalloproteases (MMPs), as suggested byour data using the non-steroidal anti-inflammatorydrug NS-398, and GM6001, acting on MMP1, -2, -3, -8, and -9. A recent report that forced overexpression ofthe COX-2 cDNA activates MMP2 is consistent withthis possibility (Tsujii et al., 1997). It has been alsoreported that the BA LCA activates the secretion ofMMP2 in human colon cancer Caco-2 cells (Halvorsenet al., 2000). Induction of COX-2 and accumulation ofits products, such as thromboxane-A2 and prostaglan-dins have been implicated in the acquisition of theinvasive phenotype, during the adenoma/adenocarci-noma transition and subsequent progression towarddistant metastasis (Tsujii et al., 1997; Rodrigues et al.,2001).

Most interestingly, the expression of the nuclearFXR receptor (farnesoid X receptor) for BA (Makishi-ma et al., 1999; Parks et al., 1999; Wang et al., 1999) ininvasion-competent PCmsrc and HCT-8/E11 cells ledus to postulate that other transcriptional mechanismsmight be implicated in the stimulation of invasion byBA. Recent data identified a BA signaling pathwayleading to the activation of the FXR receptor, therebyregulating the expression of genes encoding the BAbinding protein I-BABP, and CYP7a, which is involvedin the major biosynthetic pathway of BA in the liver(Makishima et al., 1999; Parks et al., 1999; Wang et al.,1999). We have also demonstrated recently that BAcooperate with retinoic acid and 1,25-dihydroxy-vitamin D3 in the monocytic differentiation of humanleukemic cells, through a PKC-dependent pathway,and down-regulation of the myeloblastin gene whichcontains a BA-responsible element (BARE-II) in itspromoter (Zimber et al., 2000). Thus, the difference inactivity of the various BA in cellular invasion andhaptotaxis in the present study (Figure 3) might berelated to the expression of distinct bile acidtransporters, receptors or receptor co-activators incolorectal cancer cells (Makishima et al., 1999; Parkset al., 1999; Wang et al., 1999; Staudinger et al., 2001;Xie et al., 2001). This is in agreement with the selectiveimplication of the LCA-specific PXR receptor in thetranscriptional activation of the hepatic CYP3A4 gene,which is responsible for detoxification by hydroxylationof cholestatic BA such as LCA and other xenobiotics(Staudinger et al., 2001; Xie et al., 2001).

In the present study, we presented some clues forelucidating the possible functional role of BA acting asinvasion stimulators in SRC- and RHOA-transformedhuman colon and kidney epithelial cells and in coloncancer cells. In contrast, BA do not exhibit suchactivity in premalignant epithelial cells. Regarding toclinical relevance of our data, one can postulate that

some BA, (CA, DCA, CDCA and LCA), but notUDCA, may stimulate invasion in human sporadictumors, in addition to their known roles as mitogens,inducers or inhibitors of apoptosis in colon cancer celllines, and in normal or adenomatous colorectal mucosachallenged with carcinogens (Kozoni et al., 2000;McMillan et al., 2000; Schlottmann et al., 2000). Infavor of this hypothesis, secondary BA are convertedfrom primary BA by the anaerobic bacterial flora, atthe site of emergence of colorectal adenomas andcarcinomas. The stimulation of invasion and hapto-taxis by BA, reported here, occurred at concentrations(10 – 20 mmol/l) that are observed in the portalcirculation (Moseley, 1999). It is also tempting tospeculate that UDCA might be useful in the treatmentof colorectal cancer. Indeed, upon chronic administra-tion, UDCA becomes the principal BA in bile, serumand stool (Kurtz, 1992; Batta et al., 1998), presumablythrough a negative feedback on BA synthesis. Consis-tent with this observation, feeding with UDCA in a ratmodel of colon carcinogenesis resulted in a decreasedproportion of DCA in the stool, and absence ofinvasive tumors (Earnest et al., 1994).

In conclusion, our study contributes to the under-standing of a role for BA in the progression of humancolon tumors, through the activation of the SRConcogene and Rho-like small GTPases, directlyimplicated in cellular invasion.

Materials and methods

Cell cultures

The human colon cancer HCT-8/E11 cell line and its aE-catenin-deficient round subclone HCT-8/E11R1 have beendescribed (Vermeulen et al., 1995, 1999). Both HCT-8/E11and HCT-8/E11R1 cell lines were maintained in RPMI-1640medium (Life Technologies, Ghent, Belgium), supplementedwith 10% heat-inactivated fetal bovine serum (FBS), 1 mM

sodium pyruvate, 100 mg/ml streptomycin, 250 IU/ml peni-cillin and 2.5 mg/ml fungizone. The PC/AA/C1 cell line isderived from an adenoma in a patient with familialadenomatous polyposis coli (FAP) (Williams et al., 1990).The PCmsrc cell line was established after stable transfectionof PC/AA/C1 cells by an expression vector encoding theactivated (Tyr527?Phe527) form of the SRC oncogene(Empereur et al., 1997). The PC/AA/C1 and PCmsrc celllines were cultured in Dulbecco’s Modified Eagle Medium(DMEM, Life Technologies) supplemented with 20% FBS,8 mM L-glutamine, 100 mg/ml streptomycin, 250 IU/ml peni-cillin, 2.5 mg/ml fungizone, 1.75 mmol/l insulin and 1 mg/mlhydrocortisone. The dog epithelial kidney MDCKT23 cellline and its derivatives transfected with mutant cDNAsencoding the dominant-constitutive and -negative forms ofthe gene encoding for the RhoA GTPase (RHOA) under thecontrol of a tetracycline-repressible transactivator(MDCKT23-RhoAV14 and MDCKT23-RhoAN19 cells)were obtained from Dr WJ Nelson (Department ofMolecular and Cellular Physiology, Stanford, CA, USA).These MDCK cell lines were cultured in DMEM mediumsupplemented with: 10% FBS, 100 IU/ml streptomycin,250 IU/ml penicillin, 2.5 mg/ml fungizone, 200 mg/ml hygro-mycin (Roche Diagnostics, GmbH, Mannheim, Germany),


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and 20 ng/ml doxycycline (Sigma, St Louis, MO, USA) (Jouand Nelson, 1998). Before the invasion assay, MDCKT23 celllines were cultured in the continuous presence of doxycycline(for continuous repression of the ectopic expression of theRho mutants by the tetracycline repressible transactivator),or in its absence for 48 h (for induction of the RHOAmutants). All cell lines used were mycoplasma-free, as testedby DAPI-staining (Sigma).

Reagents and antibodies

BA, lysophosphatidic acid, poly-L-lysine, human fibronectin,and collagen type IV were from Sigma. Clostridium botulinumexoenzyme C3 transferase (C3 exoenzyme) that ADP-ribosylates and inactivates the small GTPase Rho proteinswas previously described (Rodrigues et al., 2001). The Rhokinase inhibitor Y27632 was kindly provided by YoshitomiPharmaceutical Industries Ltd (Osaka, Japan). The pharma-cological inhibitors of the PKC isoforms -a and -bI (Go6976), and -a, -bI, -bII, -g, and -e isoforms (GF109203X), PI-3 kinase (wortmannin), COX-2 (NS-398) were from Calbio-chem (La Jolla, CA, USA). The mitogen-activated proteinkinase (MAPK) inhibitors 2’-amino-3’methoxyflavone(PD98059, specific for p42/p44) and SB203580 (specific forp38 MAPK) were from Alexis Corporation (San Diego, CA,USA) and New England Biolabs, Inc. (Beverly, MA, USA)respectively. Laminin was from Roche Diagnostics GmBH.PD98059, SB203580 and BA were first dissolved in DMSOand further diluted in culture medium. Final concentrationsof DMSO did not exceed 0.1%. BA were used at 10 mmol/l,,unless otherwise indicated. The matrix metalloproteaseinhibitor MMP GM6001 (Galardyn) was from ChemiconInternational (Temecula, CA, USA). Mouse IgG1 mono-clonal antibody (mAb P4C10) against the b1 integrin subunitwas from Life Technologies. Rat IgG2a mAb 69.6.5 againstaV integrin subunit was a gift from J-C Lissitzky (Lehmannet al., 1994). Mouse IgG2b mAb MB2, a functionally-neutralizing E-cadherin antibody was used, as describedpreviously (Bracke et al., 1993).

Collagen invasion assay

Collagen type I gels (from rat tail, Upstate Biotechnology,Lake Placid, NY, USA) were prepared in DMEM (0.22%w/v),using 6-well plates, as described (Vakaet et al., 1991). Cellswere seeded on top of the collagen gels, with or withoutdrugs or antibodies. After 24 h incubation at 378C, theinvasion index was calculated from the mean number ofinvasive cells, over the total number of cells, counted in atleast 12 microscopic fields. Cell viability was evaluated bythe trypan blue exclusion test (Sigma).

Morphotype on solid substrate

For seeding on solid substrate, cells were detached withtrypsin/EDTA (Life Technologies) from a nearly confluentmonolayer, washed in PBS, suspended in FBS-free mediumand seeded on glass coverslips that were coated at 378C for2 h with 10 mg/ml collagen type I in PBS. Cultures wereincubated for 20 h.

Assays for motility

For time-lapse videomicroscopy, we used freshly seeded orconfluent cell cultures in 6-well plates. Time-lapse imageswere recorded with an inverted microscope (phase-contrast;objective 206 to evaluate random motility and 406 to

evaluate membrane ruffling). The inverted microscope wasconnected with an MTI CCD72 camera (Dage MTI,Michigan City, IN, USA) and a U-matic VO-5850Pvideorecorder (Sony, Tokyo, Japan) at 30 s intervals. Tracklengths were measured as described (Bracke et al., 1997).

Haptotaxis assays (Aznavoorian et al., 1990) wereperformed in 48-well microchemotaxis chambers (Neuro-Probe Inc., Bethesda, MD, USA) with 8-mm pore sizeNucleopore filters (Costar, Cambridge, MA, USA). Thelower surface of each filter was precoated for 1 – 2 h at 378Cwith purified matrix proteins, at the indicated concentrations.The lower compartment was filled with 33 ml mediumcontaining BA, drugs or antibodies. Cells (12 500 for HCT-8/E11 and HCT-8/E11R1 cells; 50 000 for PC/AA/C1 andPCmsrc cells) were dissolved in 50 ml medium containing BA,drugs or antibodies, added to the upper compartment, andincubated for 4 – 6 h. Membranes were then fixed in methanoland stained with 10% Giemsa. Non-migratory cells on theupper surface of the filter were wiped with a cotton swab andcell number on the lower surface of the filter was quantifiedby counting 10 fields per well (objective: 640, ocular: 610).All experiments were performed at least in triplicate.

Western blotting

Cells were lysed in the Laemmli buffer. Proteins (50 mg) wereseparated by 7.5% or 12% SDS–PAGE under reducingconditions and transferred onto Hybond nitrocellulose orPVDF membranes (Amersham Pharmacia Biotech, Buck-inghamshire, UK). After blockage with 5% non-fat dry milkin PBS with 0.5% Tween-20, membranes were incubated withmAbs directed against Rac1 (Upstate Biotechnology, LakePlacid, NY, USA) or RhoA (Santa Cruz Biotechnology), orwith antibodies against the mouse, rat and human FXRreceptor (1 : 200; sc-1204; Santa Cruz Biotechnology, SantaCruz, CA, USA), followed by washing, and incubation withanti-mouse (Santa Cruz Biotechnology) or anti-goat anti-bodies (Santa Cruz Biotechnology) conjugated withhorseradish peroxidase. The ECL staining reagent was fromAmersham Pharmacia Biotech.

Determination of Rac1 and RhoA activation by pull-down assays

The levels of the active small GTPases Rac1 and RhoA underGTP-bound forms were measured as described (Sander et al.,1998; Zondag et al., 2000). GTP-bound Rac1 or GTP-boundRhoA GTPases were respectively precipitated by PAK (p21-activated kinase-1) or the C21 fragment of rhotekin, eachfused to glutathione-S transferase precoupled to glutathione-Sepharose beads. The total amount of these two GTPases incell lysates, and their corresponding GTP-bound fractionswere detected by Western blot analysis. Immunoblots werequantitated with the Quantity One Software (Bio-RadLaboratories, Hercules, CA, USA).

Statistical analysis

Data on collagen type I invasion and haptotaxis wereanalysed by the Student t-test and the non-parametricMann –Whitney U test, with P50.05 considered as statisti-cally significant.

AcknowledgmentsWe thank L Baeke, R Colman, G Desmet, G De Bruyneand A Verspeelt for technical assistance, Dr F Andre and


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Dr T Boterberg (Laboratory of Experimental Cancerology,Ghent University Hospital, Ghent, Belgium) and Dr VRigot (Unite de Biologie Cellulaire, Universite Catholiquede Louvain, Brussels, Belgium) for helpful suggestions, andDr J-C Lissitzky (Laboratoire de Biochimie Cellulaire,CNRS, Marseille, France) for providing the antibodyagainst aV integrin.This work was supported by the‘FWO-Vlaanderen’ (Brussels, Belgium), the ‘Sportvereni-

ging tegen Kanker’ (Brussels, Belgium), the ‘ASLK/VIVA-verzekeringen’ (Brussels, Belgium), the ‘G.O.A. from theVlaamse Gemeenschap’ (Brussels, Belgium), INSERM andARC (France) and the ‘Deutsche Krebshilfe e.V.’ (Bonn,Germany). Ph. Debruyne is a Research Assistant with the‘Bijzonder Onderzoeksfonds Universiteit Gent’ (Ghent,Belgium).

References

Andre F, Rigot V, Thimonier J, Montixi C, Parat F,Pommier G, Marvaldi J and Luis J. (1999). Int. J. Cancer,83, 497 – 505.

Attoub S, Noe V, Pirola L, Bruyneel E, Chastre E, Mareel M,Wymann MP and Gespach C. (2000). FASEB J., 14,

2329 – 2338.Aznavoorian S, Liotta LA and Kupchik HZ. (1990). J. Natl.Cancer Inst., 82, 1485 – 1492.

Batlle E, Verdu J, Dominguez D, del Mont Llosas M, Diaz V,Loukili N, Paciucci R, Alameda F and Garcia de HerrerosA. (1998). J. Biol. Chem., 273, 15091 – 15098.

Batta AK, Salen G, Holubec H, Brasitus TA, Alberts D andEarnest DL. (1998). Cancer Res., 58, 1684 – 1687.

Bayerdorffer E, Mannes GA, Richter WO, Ochsenkuhn T,Wiebecke B, Kopcke W and Paumgartner G. (1993).Gastroenterology, 104, 145 – 151.

Bracke ME, Depypere H, Labit C, Van Marck V, VennekensK, Vermeulen SJ, Maelfait I, Philippe J, Serreyn R andMareel MM. (1997). Eur. J. Cell. Biol., 74, 342 – 349.

Bracke ME, Vyncke BM, Bruyneel EA, Vermeulen SJ, DeBruyne GK, Van Larebeke NA, Vleminckx K, Van RoyFM and Mareel MM. (1993). Br. J. Cancer, 68, 282 – 289.

Chang J-H, Pratt JC, Sawasdikosol S, Kapeller R andBurakoff SJ. (1998). Mol. Cell. Biol., 18, 4986 – 4993.

Dannenberg AJ and Zakim D. (1999). Sem. Oncol., 26, 499 –504.

Earnest DL, Holubec H, Wali RK, Jolley CS, Bissonette M,Bhattacharyya AK, Roy H, Khare S and Brasitus TA.(1994). Cancer Res., 54, 5071 – 5074.

Emami S, Le Floch N, Bruyneel E, Thim L, May F, WestleyB, Rio M-C, Mareel M and Gespach C. (2001). FASEB J.,15, 351 – 361.

Empereur S, Djelloul S, Di Gioia Y, Bruyneel E, Mareel M,Van Hengel J, Van Roy F, Comoglio P, Courtneidge S,Paraskeva C, Chastre E and Gespach C. (1997). Br. J.Cancer, 75, 241 – 250.

Fujita T, Matsui M, Takaku K, Uetake H, Ichikawa W,Taketo MM and Sugihara K. (1998). Cancer Res., 58,

4823 – 4826.Glinghammar B and Rafter J. (2001). Gastroenterology, 120,401 – 410.

Gupta S, Stravitz RT, Dent P and Hylemon PB. (2001). J.Biol. Chem., 276, 15816 – 15822.

Hall A. (1998). Science, 279, 509 – 514.Halvorsen B, Staff AC, Ligaarden S, Prydz K and Kolset SO.(2000). Biochem. J., 349, 189 – 193.

Hennigan RF, Hawker KL and Ozanne BW. (1994).Oncogene, 9, 3591 – 3600.

Hill MJ, Drasar BS, Aries V, Crowther JS, Hawksworth Gand Williams REO. (1971). Lancet, 1, 95 – 100.

Hirano F, Tanaka H, Makino Y, Okamoto K, Hiramoto M,Handa H and Makino I. (1996). Carcinogenesis, 17, 427 –433.

Hofmann J. (1997). FASEB J., 11, 649 – 669.

Huang XP, Fan XT, Desjeux JF and Castagna M. (1992).Int. J. Cancer, 52, 444 – 450.

Irby RB, Mao W, Coppola D, Kang J, Loubeau JM,Trudeau W, Karl R, Fujita DJ, Jove R and Yeatman TJ.(1999). Nat. Genet., 21, 187 – 190.

Jou T-S and Nelson WJ. (1998). J. Cell. Biol., 142, 85 – 100.Kamano T, Mikami Y, Kurasawa T, Tsurumaru M,Matsumoto M, Kano M and Motegi K. (1999). Dis. ColonRectum, 42, 668 – 672.

Kotelevets L, Noe V, Bruyneel E, Myssiakine E, Chastre E,Mareel M and Gespach C. (1998). J. Biol. Chem., 273,

14138 – 14145.Kozoni V, Tsioulias G, Shiff S and Rigas B. (2000).Carcinogenesis, 21, 999 – 1005.

Kurtz W. (1992). Verdauungskrankheiten, 10, 153 – 162.Kurz AC, Block C, Graf D, vom Dahl S, Schliess F andHaussinger D. (2000). Biochem. J., 350, 207 – 213.

Kustikova O, Kramerov D, Grigorian M, Berezin V, Bock E,Lukanidin E and Tulchinsky E. (1998).Mol. Cell. Biol., 18,7095 – 7105.

Latta RK, Fiander H, Ross NW, Simpson C and SchneiderH. (1993). Cancer Lett., 70, 167 – 173.

Lehmann M, Rabenandrasana C, Tamura R, Lissitzky J-C,Quaranta V, Pichon J and Marvaldi J. (1994). Cancer Res.,54, 2102 – 2107.

Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM,Luk A, Hull MV, Lustig KD, Mangelsdorf DJ and Shan B.(1999). Science, 284, 1362 – 1365.

Mareel M, Berx G, Van Roy F and Bracke M. (1996). J. CellBiochem., 61, 524 – 530.

McMichael AJ and Potter JD. (1985). J. Natl. Cancer Inst.,75, 185 – 191.

McMillan L, Butcher S, Wallis Y, Neoptolemos JP and LordJM. (2000). Biochem. Biophys. Res. Commun., 273, 45 – 49.

Moseley RH. (1999). Textbook of Gastroenterology. 3rd edn.Chapter 17. Yamada T (eds), Alpers DH, Owyang C,Laine L and Powell DW (ass. eds.). Philadelphia, NewYork, Baltimore: Lippincott Williams & Wilkins, pp.380 – 403.

Nagengast FM, GrubbenMJAL and vanMunster IP. (1995).Eur. J. Cancer, 31A, 1067 – 1070.

Narisawa T, Magadia NE, Weisburger JH and Wynder EL.(1974). J. Natl. Cancer Inst., 53, 1093 – 1097.

Owen RW. (1997). Scand. J. Gastroenterol. Suppl., 222, 76 –82.

Ozanne BW, McGarry L, Spence HJ, Johnston I, Winnie J,Meagher L and Stapleton G. (2000). Eur. J. Cancer, 36,1640 – 1648.

Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, ConslerTG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM,Moore DD and Lehmann JM. (1999). Science, 284, 1365 –1368.

Pongracz J, Clark P, Neoptolemos JP and Lord JM. (1995).Int. J. Cancer, 61, 35 – 39.


6749

Oncogene

Qiao D, Chen W, Stratagoules ED and Martinez JD. (2000).J. Biol. Chem., 275, 15090 – 15098.

Qiao D, Stratagouleas ED and Martinez JD. (2001).Carcinogenesis, 22, 35 – 41.

Rao Y-P, Stravitz RT, Vlahcevic ZR, Gurley EC, Sando JJand Hylemon PB. (1997). J. Lipid. Res., 38, 2446 – 2454.

Reddy BS. (1981). Cancer Res., 41, 3766 – 3768.Reddy BS, Watanabe K, Weisburger JH and Wynder EL.(1977). Cancer Res., 37, 3228 – 3242.

Rodrigues S, Nguyen Q-D, Faivre S, Bruyneel E, Thim L,Westley B, May F, Flatau G, Mareel M, Gespach C andEmami S. (2001). FASEB J., 15, 1517 – 1528.

Rust C, Karnitz LM, Paya CV, Moscat J, Simari RD andGores GJ. (2000). J. Biol. Chem., 275, 20210 – 20216.

Sander EE and Collard JG. (1999). Eur. J. Cancer, 35, 1302 –1308.

Sander EE, ten Klooster JP, van Delft S, van der KammenRA and Collard JG. (1999). J. Cell. Biol., 147, 1009 – 1021.

Sander EE, van Delft S, ten Klooster JP, Reid T, van derKammen RA, Michiels F and Collard JG. (1998). J. Cell.Biol., 143, 1385 – 1398.

Schlottmann K, Wachs F-P, Krieg RC, Kullmann F,Scholmerich J and Rogler G. (2000). Cancer Res., 60,

4270 – 4276.Shimao Y, Nabeshima K, Inoue T and Koono M. (1999).Clin. Exp. Metastasis, 17, 351 – 360.

Slice LW, Bui L, Mak C and Walsh JH. (2000). Biochem.Biophys. Res. Commun., 276, 406 – 410.

Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D,MacKenzie KI, LaTour A, Liu Y, Klaassen CD, BrownKK, Reinhard J, Willson TM, Koller BH and Kliewer SA.(2001). Proc. Natl. Acad. Sci. USA, 98, 3369 – 3374.

Summerton J, Flynn M, Cooke T and Taylor I. (1983). Br. J.Surg., 70, 549 – 551.

Tsujii M, Kawano S and DuBois RN. (1997). Proc. Natl.Acad. Sci. USA, 94, 3336 – 3340.

Vakaet Jr L, Vleminckx K, Van Roy F andMareel M. (1991).Invasion Metastasis, 11, 249 – 260.

Vermeulen SJ, Bruyneel EA, Bracke ME, De Bruyne GK,Vennekens KM, Vleminckx KL, Berx GJ, Van Roy FMand Mareel MM. (1995). Cancer Res., 55, 4722 – 4728.

Vermeulen SJ, Nollet F, Teugels E, Vennekens KM, MalfaitF, Philippe J, Speleman F, Bracke ME, Van Roy FM andMareel MM. (1999). Oncogene, 18, 905 – 915.

Wang H, Chen J, Hollister K, Sowers LC and Forman BM.(1999). Mol. Cell, 3, 543 – 553.

Willett WC, Stamper MJ, Colditz GA, Rosner BA andSpeizer FE. (1990). N. Engl. J. Med., 323, 1664 – 1672.

Williams AC, Harper SJ and Paraskeva C. (1990). CancerRes., 50, 4724 – 4730.

Xie W, Radominska-Pandya A, Shi Y, Simon CM, NelsonMC, Ong ES, Waxman DJ and Evans RM. (2001). Proc.Natl. Acad. Sci. USA, 98, 3375 – 3380.

Zhang F, Subbaramaiah K, Altorki N and Dannenberg AJ.(1998). J. Biol. Chem., 273, 2424 – 2428.

Zimber A, Chedeville A, Abita J-P, Barbu V and Gespach C.(2000). Cancer Res., 60, 672 – 678.

Zondag GCM, Evers EE, ten Klooster JP, Janssen L, van derKammen RA and Collard JG. (2000). J. Cell. Biol., 149,775 – 781.


6750

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