Activation of cellular invasion by trefoil peptides ... - The FASEB Journal

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways SYLVIE RODRIGUES,1 QUANG-DE´ NGUYEN,1 SANDRINE FAIVRE, ERIK BRUYNEEL,* LARS THIM,† BRUCE WESTLEY,‡ FELICITY MAY,‡ GILLES FLATAU,§ MARC MAREEL,* CHRISTIAN GESPACH,2 AND SHAHIN EMAMI INSERM U482, Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, Hoˆpital Saint-Antoine, 75571 Paris Cedex 12, France; *The Laboratory of Experimental Cancerology, Ghent University Hospital, B-9000 Ghent, Belgium; †Novo Nordisk, Bagsvaerd, DK-2880, Denmark; ‡ Department of Pathology, University of Newcastle, Newcastle upon Tyne NE1 4LP, UK; and § INSERM U452, Biologie Cellulaire et Mole´culaire des Microorganismes Pathoge`nes et de leurs Toxines, UFR de Me´decine, 06107, Nice Cedex, France We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX-1 and COX-2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U-73122), cyclooxygenases (SC-560, NS-398), and the thromboxane A2 receptor (TXA2-R) antagonist SQ-295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2-R mimetic U-46619, constitutively activated forms of the heterotrimeric G-proteins G␣q (AG␣q), G␣12, G␣13 (AG␣12/13), which are signaling elements downstream of TXA2-R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src-pS2 cells and human colorectal cancer cells HCT8/S11-pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2-R dependent. We detected a marked induction of COX-2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2R-dependent invasion pathway, which is monitored via a Rho- and G␣12/G␣13-independent mechanism using the G␣q/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.—Rodrigues, S., Nguyen, Q.-D., Faivre, S., Bruyneel, E., Thim, L., Westley, B., May, F., Flatau, G., Mareel, M., Gespach, C., Emami, S. Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways. FASEB J. 15, 1517–1528 (2001)

ABSTRACT

Key Words: inflammation 䡠 cancer progression 䡠 heterotrimeric G-proteins 䡠 Rho-like GTPases 䡠 phospholipase C

repair in the gastrointestinal tract (1–5). The three members of the trefoil factor family [pS2 (TFF1), spasmolytic polypeptide SP (TFF2), and intestinal trefoil factor ITF (TFF3)] are expressed in various tissues including hypothalamus, pituitary, breast, salivary gland, and prostate (5, 6). TFFs are abundantly cosecreted and tightly associated with mucins in a regionspecific fashion throughout the gastrointestinal tract and are heat, acid, and protease resistant (5, 7–9). For example, pS2 has shown to be expressed in mucussecreting cells of the human stomach (10). Similarly, SP is localized in gastric glands and duodenal Brunner’s glands, whereas ITF has been identified in goblet cells of the small intestine and colon (2, 11). In humans, TFFs are overexpressed or induced in inflammatory conditions such as peptic ulceration, colitis, Crohn’s disease, pancreatitis, and biliary disease (5, 12, 13). TFFs are thought to have beneficial effects on mucosal integrity via paracrine, local, and distant action in luminal digestive epithelial cells, after ulceration, and in wound healing during intestinal damage and inflammatory bowel disease (14 –16). Experimental and clinical data strongly suggest a close relationship between chronic inflammatory diseases and the incidence of solid tumors in the intestine, breast, and prostate (17, 18). Inflammatory breast cancer is one of the most aggressive types of human breast cancer, with a high incidence of local and systemic recurrence and distant metastases (19). Targeted disruption of the gene encoding the pertussis toxin (PTx) -sensitive heterotrimeric G-protein subunit G␣i2 induces inflammatory colitis that resembles the human disease in many aspects, including the development of adenocarcinoma in the colon (20). Several clinical studies have demonstrated that nonsteroidal 1

These authors contributed equally to this work. Correspondence: INSERM Unit U482, Hoˆpital SaintAntoine, 75571 Paris Cedex 12, France. E-mail: gespach@ st-antoine.inserm.fr 2

Trefoil peptides (TFFs) constitute a new class of regulatory peptides involved in mucosal protection and 0892-6638/01/0015-1517 © FASEB

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antiinflammatory drugs (NSAIDs: sulindac, indomethacin, piroxicam) reduce the number and size of intestinal polyps in patients with familial adenomatous polyposis (21–23) and inhibit chemically induced colon carcinogenesis in rodents (24). The common mechanism of action of NSAIDs is the inhibition of the cyclooxygenase pathway (COX). Inactivation of the COX-2 gene and selective COX-2 inhibitors provide protection against the development of intestinal polyps and tumors (25, 26). The cyclooxygenase pathway catalyzes the bis-oxygenation of arachidonic acid into prostaglandin PGH2, the immediate substrate for conversion into other PGs or thromboxane (TXA2) by specific synthases (27). These COX products are potent inflammatory agents. For example, TXA2 is suggested as a mediator of acute colitis in the rat colon. COX-1 is constitutively expressed in nearly all tissues, whereas COX-2 expression is induced in response to proinflammatory factors, tumor promoters, oncogenes, and cytokines (28 –31). COX-2 is abnormally elevated in colon adenomas, colorectal cancers, and azoxymethane-induced mouse tumors (32–34). Inflammatory situations and cancer progression are therefore associated with up-regulation of TFFs and COX in breast, prostate, and the gastrointestinal tract. We recently demonstrated intense expression of pS2 by Western blot and immunohistochemistry in human colorectal tumors and associated nonmalignant mucosa during the neoplastic progression, from adenoma through to liver metastases (35). Furthermore, we showed that pS2, SP and ITF induce cellular scattering and invasion of type I collagen in kidney and colonic epithelial cells, suggesting that TFFs can be involved in the progression of gastrointestinal tumors. The first steps in tumor invasion and metastasis involve destruction of the basement membrane, which contains type IV collagen, and subsequent degradation of type I collagen, the major structural component of the extracellular matrix that is degraded by cancer cells, stroma fibroblasts, and endothelial cells (36 –38). We have also shown that the small GTPase RhoA, the src oncogene, and phospholipase C/PKC pathways are essential signaling elements linking TFFs and cellular invasion (35). Thus, additional adverse effects for TFFs should be considered in view of their constitutive accumulation in inflamed digestive tract, human digestive tumors, and their potential actions in cell migration, scattering, tumor invasion, and metastasis. The present studies were prompted by our interest in defining the possible functional relations between the cancer-associated signaling pathways using TFFs, src, and COX on the invasive phenotype. Colonic and kidney epithelial cells were treated with exogenous TFFs and the cellular response was compared with stably transfected cells that express pS2 constitutively, reflecting the situation we observed for the pS2 protein in human colorectal cancers. Our hypothesis assumes that cyclooxygenases and their products are involved in the signaling pathways activated by TFFs and src during 1518

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cellular invasion. In this paper, we demonstrate that the COX and TXA2-R pathways exert a pivotal role in cellular invasion initiated by TFFs and src.

MATERIALS AND METHODS DNA constructs The cDNA encoding the constitutively activated form of the PTx-insensitive G␣-protein subunit G␣q(Q209L) was generously provided by Dr. E. Peralta (Harvard University, Cambridge, MA). This sequence was cloned into the EcoRI/XhoI site of the eukaryotic expression vector pcDNA3.1, which contains the neoresistance gene (InVitrogen, Groningen, The Netherlands). The G␣12(Q229L) and G␣13(Q226L) sequences encoding the activated G-proteins G␣12 (1.8 kb) and G␣13 (2.4 kb) were inserted into pcDNA3 at the EcoRI/ XbaI and BamHI/XbaI sites, respectively. These two pcDNA3/ G␣12Q229L and/G␣13Q226L vectors were generously provided by Dr. N. Dhanasekaran (Fels Institute for Cancer Research, Philadelphia, PA). These mutationally activated forms of GTPase-deficient G-proteins were designated AG␣q, AG␣12, and AG␣13. The human pS2 cDNA fragment (462 bp) was excised from the pGEM1 plasmid (39) by EcoRI/ BamHI digestion and cloned in-frame into pcDNA3. The new plasmid construct was called pcDNA3-hpS2. The structure and function of pcDNA3-hpS2 was checked by direct DNA sequencing and expression in HEK-293T cells. Cell lines and culture conditions The human embryonic kidney cell line HEK-293T was a generous gift from Dr. Silvio Gutkind (NIH, Bethesda, MD). Madin-Darby canine kidney epithelial cells MDCKts.src transformed by a temperature-sensitive mutant of v-src (MDCKts.src, clone 2) were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Cergy Pontoise, France) supplemented with 10% fetal calf serum (Boehringer Mannheim, Paris, France) plus L-gutamine and antibiotics (Gibco BRL), as described previously (40). MDCKts.src cells display an invasive phenotype at the permissive temperature of 35°C for src activity and are not invasive at the restrictive temperature 40°C. The parental MDCK cell line was a generous gift from Dr. Julian Downward (Imperial Cancer Research Fund, London, UK). The human colorectal cell line PCmsrc was grown routinely in 6 cm-diameter Petri dishes (41). After transfer of the activated c-src oncogene into the premalignant PC/AA/C1 cell line, PCmsrc cells became tumorigenic in athymic nude mice and invasive upon addition of HGF, leptin, and TFFs (35, 41, 42). HCT8/S11 human colonic cancer cells and MCF-7 human breast cancer cells were cultured as described (43, 44). Stable transfection of kidney epithelial cells About 3 ⫻ 106 MDCKts.src cells were stably transfected with activated forms of the PTx-insensitive G␣-protein subunits AG␣q, AG␣12, and AG␣13, using the corresponding pcDNA3 expression plasmids (3 ␮g), and 18 ␮l of LipofectAMINE Plus reagent (Gibco BRL). Control transfections were performed using the empty vector pcDNA3. MDCKts.src cells and human colonic HCT8/S11 cells (also designated HCT-8/E11 cells) were transfected under the same conditions using the pcDNA3-hpS2 vector (35). After 48 h, cultures were selected for 2 wk in 1 mg/ml neomycin (Gibco BRL); resistant colonies were ring-cloned as individual colonies or pooled for

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analysis of ectopic overexpression of the AG␣q, AG␣12, AG␣13, and pS2 transgenes by immunoblot analysis and further functional characterization (vide infra). Kidney MDCKts.src-pS2 cells (clone 2) and human colonic epithelial cells HCT-8/S11-pS2 cells (clone 2) stably transfected by human full-length hpS2 were cultured under standard conditions (35, 44). Conditioned media (CM) from parental MDCKts.src cells and their counterparts transformed by pS2, src, AG␣q, AG␣12, and AG␣13 were collected from subconfluent cell layers (70 –90% confluence in 3.5 cm Petri dishes) and used in the invasion and TXB2 assays described below. The CMs were designated as CM-pS2, CM-src, CM-AG␣q, CM-A G␣12, and CM-AG␣13, respectively. Western blot analyses For immunoblotting, cultured cells were homogenized at 4°C in RIPA buffer containing 0.1 mg/ml phenylmethysulfonyl fluoride (PMSF), 100 ␮M benzamidine, and 100 mM Na3VO4 as protease and phosphatase inhibitors. Insoluble material was removed by centrifugation for 15 min at 4°C and 12,000 g. Proteins were resolved in 12.5% SDS-PAGE gels and transferred to PVDF or Hybond-C Extra membranes (Amersham Pharmacia Biotech, Orsay, France). Membranes were blocked overnight in Tris-buffered saline (TBS: 20 mM TrisHCl, pH 8, 150 mM NaCl) containing 5% dried skimmed milk. The blots were then probed for 4 h at room temperature with one of the following primary rabbit polyclonal antibodies (pAbs) at the indicated dilutions: the pAb B860 –15 specific for G␣13 (1:1000) was from Dr. Paul Sternweis (University of Texas, Dallas, TX); the pAbs specific for G␣12 (S-20, 1:100), G␣q (E-17, 1:200), COX-1 (C-20, 1:500) and the goat pAb N-20 against COX-2 were from Santa Cruz Biotechnology (TEBU, Le Perray-en-Yvelines, France). The primary mouse mAbs against cyclooxygenases COX-2 (1:2000) and COX-1 (1:1000) were from Cayman Chemical (Spibio, Massy, France). The anti-pS2 mAb p2802 raised against the carboxyl-terminal domain of pS2 has been described (10). Membranes were washed in TBS containing 0.1% Tween 20 and probed for 90 min with secondary antibodies consisting of a goat anti-mouse IgGs pAb (1:2000, Santa Cruz Biotechnologies) or a donkey anti-rabbit IgGs pAb (1:2000, Amersham), then revealed by enhanced chemiluminescence Western detection (ECL, Amersham). Collagen invasion assays For invasion of collagen gels by renal and colorectal epithelial cells, Petri dishes were filled with 1.35 ml of neutralized type I collagen and incubated overnight at 37°C to allow gelling. Cells were harvested and isolated using Moscona buffer and trypsin/EDTA, then seeded on top of the collagen gels at the density of 0.3 ⫻ 106 cells per dish. Dispersed cells (0.3⫻106 cells) were cultured for 24 h at the indicated temperature in the presence or absence of the effectors and drugs. Invasive and superficial cells were counted in 12 fields of 0.157 mm2. The invasion index is the percentage of cells invading the gel over the total number of cells (45). None of the agents tested in the 24 h invasion assay (e.g., wortmannin, NS-398, SC-560, and SQ-295) interfered with cell growth and viability (trypan blue exclusion test). Other assays, peptides and reagents Conditioned culture media were prepared by plating 0.5–1 ⫻ 105 cells/well in 6-well culture dishes. Media from confluent layers was collected and cleared by centrifugation (5000 g, 10 min). Secretion of the cyclooxygenase-derived product TXA2

in the conditioned culture media was measured indirectly as its stable thromboxane B2 metabolite, using the EIA enzyme immunoassay kit from R&D Systems (Abingdon, UK). Intracellular TXB2 was quantified in the ice-cold cell lysates collected in 250 ␮l of the EIA buffer after cellular disruption through a 25 gauge hypodermic needle. Data are expressed as pg TXB2/␮g protein. Clostridium botulinum exoenzyme C3 transferase (abbreviated as C3T), which ADP-ribosylates and inactivates the small GTPases RhoA, B, and C but not the ras, Cdc42, or Rac GTPases (46), was isolated at 37°C from the BL21DE3 strain transformed by the pET28a plasmid recombined with the C3 gene. The toxin was purified according a modified version of the method published by Saito and Narumiya (47). Briefly, cells were lysed using a French press and the supernatant was subjected to anionic exchange chromatography (CM Sepharose fast flow, Amersham Pharmacia Biotech). Human ITF was produced in yeast and purified as described (48); recombinant hpS2 produced in Escherichia coli was purified by affinity chromatography (49). TFFs were used as monomers. Hepatocyte growth factor scatter factor (HGF) was a generous gift from Pr. Paolo Comoglio (University of Turin, Italy). Leptin was from R&D Systems Europe Ltd. (Oxon, UK). Wortmannin (WORT, a PI3⬘-kinase inhibitor), the PLC␤ inhibitor U-73122, the COX-1 and COX-2 inhibitors SC-560 and NS-398, Go¨6976 (a selective inhibitor of PKC␣ and PCK␤ I isoenzymes that has no effect on the atypical Ca2⫹-independent PKCs), and GF109203X (abbreviated as GF109, a PKC inhibitor with high selectivity for PKC␣, ␤I, ␤II, ␥, and ε isoenzymes) were from Calbiochem (Meudon, France). The prostaglandin H2/ thromboxane A2 receptor (TXA2-R) agonist U-46619 and the TXA2-R antagonist SQ29,548 (designated SQ-295 in the present study) were from Cayman Chemicals. PTx and PMSF were from Sigma (Saint-Quentin Fallavier, France). Collagen type I was from Upstate Biotechnology (Lake Placid, NY).

RESULTS Cellular invasion induced by external addition of TFFs or src oncogene is dependent on Rho, PLC, COX-1/COX-2, and TXA2-R signaling As shown in Fig. 1A, B, direct addition of intestinal trefoil factor (ITF, 0.1 ␮M) induced invasion of collagen gels by kidney epithelial cells MDCKts.src and human colorectal cancer cells PCmsrc (invasion index⫽ 11⫾1.7% and 10⫾0.9%, respectively). Control values were 1 ⫾ 0.5 and 1 ⫾ 0.6%, respectively. This marked stimulation of cellular invasion (⬃10-fold increase) was completely abolished by the PI3⬘-K inhibitor wortmannin, the RhoA inhibitor C3T, the PLC inhibitor U-73122, and the COX-2 inhibitor NS-398 (25 ␮M). Cellular invasion index measured in MDCKts.src cells was also increased to 7 ⫾ 1.6% (n⫽4 experiments) after exogenous addition of pS2 (Fig. 2A) and to 11 ⫾ 1.4% after constitutive activation of src (Fig. 2B). The COX-1 inhibitor SC-560 blocked the invasive phenotype induced by pS2 in a dose-dependent manner. It become apparent that the selective COX-1 and COX-2 inhibitors NS-398 (Fig. 1A, B) and SC-560 (Fig. 2A) abolish cellular invasion induced by ITF and pS2 in kidney and colonic epithelial cells. Our findings raise the possibility that the cyclooxygenase-derived products TXA2 and PGH2 are down-

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signaling (Fig. 3A, B), whereas the TXA2-R mimetic U-46619 dose-dependently induced invasiveness (Fig. 3C). An apparent maximal effect (invasion index⫽10⫾0.8%) was induced at 10 ␮M U-46619. However, it is noteworthy that invasiveness induced by this concentration of U-46619 was not inhibited by C3T, indicating that an additional invasion pathway, independent of Rho, is operative. This is addressed in more detail in the experiments described below. Cellular invasion induced by ectopic pS2 overexpression is dependent on PLC, COX-1/COX-2, but is Rho independent

Figure 1. Induction of cellular invasion by external addition of intestinal trefoil factor. Invasion index in collagen gels was measured using kidney MDCKts.src cells (A) or human colonic PCmsrc cells incubated for 24 h (B) in the presence of ITF (100 nM) either alone or combined with one of the following inhibitors of signal transduction pathways: PI3⬘-K (wortmannin, WORT: 10 nM), Rho (C3 exotransferase, C3T: 3 ␮g/ml), PLC (U-73122: 1 ␮M), and COX-2 (NS-398: 25 ␮M). The percentage of invasive cells in collagen type I gels was determined in the presence and absence (control) of the indicated effectors, as indicated in Materials and Methods. Data are means ⫾ se from 5 experiments.

stream effectors of cellular invasion induced by TFFs and src. To explore this hypothesis, we first observed in Fig. 2A that the TXA2-R agonist U-46619 induced activation of cellular invasion in MDCKts.src cells (9⫾1%) that was comparable to activation by TFFs and src and was abolished by the COX-1 inhibitor SC-560. Second, we found that cellular invasion induced by src in MDCKts.src cells was reversed by NS-398 and SC-560 (Fig. 2B). Third, the PI3⬘-K, Rho, and PLC inhibitors wortmannin, C3T, and U-73122 abolished src-induced cellular invasion in MDCKts.src cells (Fig. 2B). Most important, the TXA2-R antagonist SQ-295 also blocked cellular invasion induced by the src oncogene. Therefore, we sought to extend these results to trefoil factors pS2 and ITF. As shown in Fig. 3, the COX-2 inhibitor NS-398 prevented the induction of invasion by pS2 and ITF (Fig. 3A, B), but was ineffective against HGF-induced invasion in MDCKts.src cells (Fig. 3C) or leptin (Fig. 5B, vide infra). Similarly, the TXA2-R antagonist SQ-295 (10 and 30 ␮M) failed to control invasion induced by HGF and leptin (data not shown), indicating that COX and the TXA2-R selectively play an essential role in TFFand src-induced invasion. This implies that these two COX/TXA2-R pathways are not involved in this cellular activity induced by the leptin receptor Ob-Rb or the HGF receptor Met (42). In contrast, the TXA2-R antagonist SQ-295 dose-dependently reversed pS2 and ITF 1520

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As shown in Fig. 4A, B), MDCKts.src-pS2 cells (clone 2) and human colonic epithelial cells HCT8/S11-pS2 (clone 2) stably transfected by the human pS2 cDNA exhibited constitutive invasiveness (10⫾0.5 and 11⫾0.3%) that was abrogated by wortmannin, U-73122, and the COX-2 inhibitor NS-398. Abrogation of invasiveness by HCT8/S11-pS2 cells was also found in the presence of the COX-1 inhibitor SC-560 (not shown). In contrast, the Rho inhibitor C3T failed to reverse invasion induced by the ectopic expression of pS2. Overexpression of pS2 was confirmed by immunoblotting in the clonal derivatives isolated from MDCKts.srcpS2 cells (Fig. 4C: pool 1 and clones 2, 5) and HCT8/

Figure 2. Induction of cellular invasion by external addition of pS2 and activation of the src oncogene. A) Invasion index was measured in MDCKts.src cells incubated at 40°C in the presence of pS2 (100 nM) or 10 ␮M of the TXA2-R agonist U-466, either alone or combined with the indicated concentrations of the COX-1 inhibitor SC-560. B) MDCKts.src cells were incubated at the permissive temperature 35°C for src activation in the presence or absence of PI3⬘-K inhibitor WORT (10 nM), Rho inhibitor C3T (3 ␮g/ml), PLC inhibitor U-73122 (1 ␮M), COX-2/COX-1 inhibitors NS-398 (25 ␮M) and SC-560 (100 nM), or the TXA2-R antagonist SQ-295. Data are means ⫾ se from 4 – 6 experiments.

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pared from MDCKts.src-pS2 cells in our invasion assay, using MDCKts.src cells and parental MDCK cells as targets (Fig. 5A, B). As shown in Fig. 5A, CM-pS2 induced a remarkable activation of invasiveness in MDCKts.src cells (invasion index⫽10.3⫾1.3%) that was blocked by wortmannin and the PLC inhibitor U-73122. Invasion induced by CM-pS2 was resistant to C3T and PTx, suggesting that the Rho-dependent pathway and PTx-sensitive heterotrimeric proteins (G␣o/G␣i1–3) are not involved. The CM from MDCKts.src-pcDNA3 cells used as a negative control was ineffective. Furthermore, the effect of CM-pS2 was not inhibited by C3T, strongly suggesting that the invasiveness of MDCKts.src challenged with CM-pS2 is not related to the biological activity of pS2 in the CM-pS2. To investigate this, the effect of the CM-pS2 on invasiveness of the parental MDCK cells that are not

Figure 3. Role of the TXA2-R antagonist SQ-295 and Rho inhibitor C3T on cellular invasion induced by pS2, ITF, and the TXA2 mimetic U-466. Invasion index was measured using MDCKts.src cells incubated at 40°C in the presence of pS2 (A) or ITF (B) either alone or combined with the COX-2 inhibitor (NS-398: 25 ␮M) or various concentrations of the TXA2-R antagonist SQ-295 (1–30 ␮M). C) MDCKts.src cells were incubated in the presence of HGF (10 U/ml) alone or combined with NS-398 or in the presence of various concentrations of the TXA2-R agonist U-466 (0.1–50 ␮M). The Rho inhibitor C3T was also tested in combination with 10 ␮M U-466. Data are means ⫾ se from 4 – 6 experiments.

S11-pS2 cells (Fig. 4D: pool 1 and clones 2, 6). The same signal was identified in two independent positive controls, using a human gastric tumor sample and the MCF-7 human breast cancer cell line. We next tested whether we could detect the pS2 protein in the CM from kidney and colonic epithelial cells stably transfected by the pS2 cDNA. The presence of the main 9.6 kDa band and a diffuse band with higher molecular weight was evident in the CM-pS2 isolated from HCT8/ S11-pS2 and MDCKts.src-pS2 cells, but was absent in their nontransfected counterparts (Fig. 4E). Specificity of the detected pS2-immunoreactive band (9.6 kDa) was established using transient transfections in the human embryonic kidney cell line HEK-293T (Fig. 4F). Induction of cellular invasion by the conditioned media from MDCKts.src-pS2 cells stably transfected by the human pS2 cDNA We next assayed the biological activity of secreted products in the conditioned medium (CM-pS2) pre-

Figure 4. Induction of cellular invasion by ectopic overexpression of the pS2 cDNA. Invasion index in collagen type I gels was measured using pS2-transfected cells MDCKts.src-pS2 kidney cells (A) or HCT8/S11-pS2 human colonic cells (B) incubated for 24 h in the presence or absence (control) of WORT (10 nM); C3T (3 ␮g/ml); U-73122 (1 ␮M), and NS-398 (25 ␮M). Data are means ⫾ se from 3– 6 experiments. C–F) Immunoblot analysis of the human pS2 protein after transfection of kidney MDCKts.src cells (C) and human colonic HCT8/S11 cells (D) stably transfected by the pcDNA3-hpS2 vector. No immunoreactive band was obtained in mock-transfected cells, using the pcDNA3 empty vector. E) detection of the pS2 protein in conditioned media collected from the pS2-transfected cell lines HCT8/S11-pS2 (clone 2) and MDCKts.src-pS2 (clone 2). No immunoreactive band was detected in the culture media collected from their mock- and nontransfected counterparts (F): the pS2 protein was identified by Western blot as a single 9.6 kDa band in transiently transfected HEK-293T.pS2 cells, and similar signals were observed in a human gastric tumor sample and human breast cancer cells MCF-7 as positive controls.


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Figure 5. Induction of cellular invasion by the conditioned media isolated from MDCKts.src-pS2 cells overexpressing the pS2 protein. The conditioned media prepared from MDCKts.src-pS2 cells incubated for 24 h at 40°C (CM-pS2) was collected and tested for the ability of fresh cultures of MDCKts.src cells (A) or parental MDCK cells (B) to invade collagen type I gels. The CM-pS2 was tested either alone or combined with either WORT (10 nM), C3T (3 ␮g/ml), U-73122 (1 ␮M), the G␣o/G␣i inhibitor PTx (200 ng/ml), or the TXA2-R antagonist SQ-295 (30 ␮M). As controls, MDCKts.src and parental MDCK cells were exposed to the thromboxane mimetic U-466 (10 ␮M) and leptin (100 ng/ ml) either alone or combined with C3T or the COX-2 inhibitor NS-398. Data are means ⫾ se from 4 experiments.

sensitive to ITF (35) or pS2 (Fig. 5B) was examined. CM-pS2 markedly enhanced invasiveness in nontransformed MDCK cells (invasion index⫽10⫾0.5%). This induction by CM-pS2 was completely blocked by the TXA2-R antagonist SQ-295. As controls, we verified that stimulation of MDCK cells by the TXA2-R agonist U-46619 led to efficient stimulation, whereas the COX-2 inhibitor NS-398 was ineffective against leptin receptor activation. Activation of cellular invasion by the GTPasedeficient forms of the PTx-insensitive G-proteins G␣q, G␣12, and G␣13 Having established the causal role of the COX and TXA2-R pathways in cellular invasion stimulated by TFFs and src, we next investigated how TXA2-R might stimulate the ability of parental MDCK cells and MDCKts.src cells to invade collagen gels. The cyclooxygenase-derived products TXA2 and PGH2 are potent activators of the TXA2-R. Various authors have proposed that the G-protein-coupled Tp␣ and Tp␤ recep1522

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tor subtypes for thromboxane A2 and PGH2 (designated here as TXA2-R) might couple to several G-proteins, including the PTx-insensitive G-proteins G␣q, G␣12/ G␣13, G␣16, and to the PTx-sensitive G␣i2 (50 –53). The data presented in Fig. 5A support the notion that the PTx-sensitive G-protein G␣i2 is not involved. The major signaling pathway used by both Tp␣/␤ subtypes of the TXA2-R is the G␣q-dependent stimulation of the ␤-isoforms of phospholipase C (PLC␤) by TXA2/PGH2 agonists, resulting in protein kinase C activation (PKC) and mobilization of intracellular calcium. It is therefore plausible that TFFs and src promote invasiveness through activation of the TXA2-R signaling pathway using the PTx-insensitive G-proteins G␣q, G␣12, and G␣13. We therefore extended our studies to investigate the effects of the GTPase-deficient mutants of G␣q, G␣12, and G␣13 encoding activated forms (AG␣q, AG␣12, AG␣13) of these three G-protein subunits. Immunoblot analysis identified several positive MDCKts.src clones expressing AG␣q (clones 2, 3, and 9), AG␣12 (clones 1 and 4), and AG␣13 (clones 2 and 3). Densitometric analysis revealed that these ectopic Gprotein mutants were overexpressed two- to threefold over endogenous levels (data not shown). Stably transfected MDCKts.src-AG␣q cells (clone 9) exhibited constitutive invasiveness (invasion index⫽9⫾1.3%) that was resistant to the Rho inhibitor C3T (Fig. 6A). All the elements related to the TFF and src signaling pathways, including PI3⬘-K, PLC, COX-1, COX-2, and the TXA2-R, appear to be essential components for cellular invasion induced by the G-protein AG␣q (Fig. 6A). As expected, the PKC inhibitors Go¨6976, GF109, and down-regulation of PKC by phorbol ester abolished invasion caused by AG␣q in MDCKts.src cells (not shown). The activated forms of AG␣12 and AG␣13 (Fig. 6B, C) also induced activation of invasiveness in MDCKts.src cells (invasion index⫽10⫾1.7 and 10⫾1%), but showed a different pattern of inhibition. Whereas U-73122, NS-398, and SQ-295 had no effect on AG␣12/ AG␣13-induced invasion, the Rho and PI3⬘K inhibitors completely reversed this induction. This last result is consistent with the identification of RhoA as a downstream target through which activated G␣12 and G␣13 induce cytoskeletal reorganization. Finally, it is now well accepted that the ubiquitously expressed G␣12/ G␣13 families of heterotrimeric G-proteins have been implicated in cell transformation (54, 55). Taken together, our data support the notion that G␣q and its downstream targets, including PLC-␤/PKC, play an essential role in the induction and maintenance of the invasive phenotype in kidney and colonic epithelial cells stimulated by TFFs, src, and the TXA2-R. To confirm this hypothesis, we compared the ability of conditioned medium from AG␣q, AG␣12, and AG␣13transfected MDCKts.src cell cultures to initiate invasiveness in the parental MDCK cell line. As shown in Fig. 7A, CM-G␣q mimicked the invasion induced by ectopic pS2 in MDCKts.src cells and HCT8/S11 cells (Fig. 4A,

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Expression of cyclooxygenases and TXB2 in kidney and colonic epithelial cells transformed by pS2, src, and AG␣q As shown in Fig. 8A, immunoreactive COX-2 protein expression in MDCKts.src cells was markedly induced by activated src and AG␣q. Elevation of COX-2 levels by src was not blocked by C3T, U73122, or the cyclooxygenase inhibitors NS398 and SC-560, but was reversed by wortmannin, suggesting that PI3⬘-K is implicated in src-induced COX-2 expression. To ascertain the functionality of immunoreactive COX-2 induction, accumulation of the stable PGH2/TXA2 metabolite TXB2 was measured in CM-src. Activation of src resulted in a remarkable increase of TXB2 release in CM of MDCKts.src cells (33-fold control), from a basal level of 0.44 ⫾ 0.22 to 14.8 ⫾ 2.9 pg/␮g protein (n⫽7 experiments). There was no elevation of TXB2 levels in response to HGF, leptin, and AG␣q. Accumulation of TXB2 induced by src in CM-src was blocked by wort-

Figure 6. Induction of cellular invasion by constitutively activated forms of the G-proteins G␣q, G␣13, and G␣12. Cellular invasion patterns were compared using MDCKts.src cells stably transfected by the constitutively activated, GTPasedeficient forms of G␣q-Q209L (AG␣q: A), G␣13-Q226L (AG␣13: B), or G␣12-Q229L (AG␣12: C). The invasion index was then measured in the corresponding cell lines incubated at 40°C in the presence or absence (control) of the following inhibitors of signal transduction pathways: PI3⬘-K (wortmannin, WORT), RhoA (C3 exotransferase, C3T); PLC (U-73122), COX-1 (SC-560), COX-2 (NS-398), and the TXA2-R antagonist SQ-295. Data are means ⫾ se from 5–9 experiments.

B), because the same Rho-independent signaling cascades using PI3⬘-K, PLC and COX are involved. In contrast, CM from MDCKts.src cells transformed by AG␣13 or AG␣12 (CM-AG␣12 or -AG␣13) did not induce invasiveness (Fig. 7B). Similar to the response observed with CM-G␣q, conditioned medium isolated from the src-transformed MDCKts.src cells cultured at the permissive temperature 35°C (CM-src) led to marked invasiveness in parental MDCK cells (invasion index⫽11%), as compared to leptin as positive control (Fig. 7C). Consistent with these findings, invasion induced by CM-src was also blocked by pharmacological inhibitors of the PLC and COX pathways, but not by the Rho inhibitor C3T.

Figure 7. Cellular invasion patterns in response to the conditioned media isolated from MDCKts.src cells expressing constitutively activated forms of the G-proteins G␣q, G␣12/G␣13, and src. Invasion index was measured in parental MDCK cells exposed for 24 h at 37°C to the conditioned media prepared from MDCKts.src cells stably transfected by AG␣q (CM-AG␣q: A), AG␣12 or AG␣13 (CM-AG␣12 or -AG␣13: B), or expressing the src oncogene (CM-src: C). The CM-AG␣q and CM-src was tested alone or combined with WORT, C3T, U-73122, NS-398, SC-560, or the TXA2-R antagonist SQ-295. As a positive control, MDCK cells were exposed to leptin (100 ng/ml: C). Data are means ⫾ se from 4 experiments.


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Figure 8. Expression of the cyclooxygenases COX-1/COX-2, secretion of the COX-produced eicosanoid TXB2, and cellular invasion in MDCKts.src and HCT8/S11 cells transformed by src, pS2, and AG␣q. A) Western blot analysis for the levels of COX-2 and COX-1 expression in total protein extracts (50 –200 ␮g protein) prepared from MDCKts.src and HCT8/ S11 cells before and after transformation by the src oncogene, ectopic overexpression of pS2, and AG␣q. Kidney and colonic epithelial cells were cultured for 24 h in the presence or absence (control) of the following inhibitors of signal transduction pathways: RhoA (C3 exotransferase, C3T); PLC (U73122); COX-2 (NS-398); COX-1 (SC-560); PI3⬘-K (wortmannin, WORT). Each whole-cell lysate was immunoblotted with primary antibodies directed against COX-2 (72–74 kDa) or COX-1 (72 kDa) and revealed by the ECL detection method. B) Invasiveness (o) and extracellular TXB2 levels (⌬) were induced by src in MDCKts.src cells incubated for 24 h in the presence or absence of various concentrations of SC-560 or NS-398. Data are from 3– 6 experiments and are expressed as percent of control stimulation induced by the src oncogene.

mannin, attenuated by the PLC inhibitor U73122 (P⬍0.02), and was C3T-insensitive (data not shown). Src-induced TXB2 levels were abolished by SC-560 and NS-398, suggesting that the functional activities of COX-1 and COX-2 are interdependent (Fig. 8B). Halfmaximal inhibition by SC-560 was observed at 3 nM for invasion and TXB2 accumulation. Regarding the COX-2 inhibitor, half-maximal inhibition of src-induced invasion and TXB2 accumulation was observed at 2–5 nM NS-398 in the endogenous COX-2/COX-1 system presented here. Similarly, intracellular TXB2 levels induced by src in MDCKts.src cells were dose-dependently inhibited by the COX inhibi1524

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tors, with inhibitory potencies of 5 nM SC-560 and 1 nM NS-398 (not shown). Whereas overexpression of pS2 led to a much lower extent of COX-2 protein induction in MDCKts.src-pS2 cells (Fig. 8A) and only a twofold increase of TXB2 levels in MDCKts.src cells and HCT8/ S11 cells (basal level⫽0.039⫾0.02 pg/␮g protein, n⫽7 experiments), cellular invasion induced by pS2 in MDCKts.src-pS2 cells (ectopic overexpression) and HCT8/ S11 cells (external addition) was abrogated by 100 nM SC-560 or NS-398 (IC50⫽3–5 nM SC-560 and 1– 4 nM NS-398). Induction of COX-2 protein in HCT8/S11pS2 cells and MDCKts.src-AG␣q cells (Fig. 8A) was insensitive to C3T, U-73122, COX-1/COX-2 inhibitors, and wortmannin. It appears that depletion of TXB2 and blockade of COX-1/COX-2 enzymes by NS-398 and SC-560 may prove to be causal in the abolition of invasiveness induced by src and TFFs in kidney and colonic cancer cells. The cyclooxygenase COX-1 is constitutively expressed in most cell types, whereas the immediate-early response gene COX-2 is induced by v-src, ras, lipopolysaccharide (LPS), cytokines, and tumor promoters, such as phorbol esters or bile acids (28 –31, 56 –58). Accordingly, when MDCKts.src or HCT8/S11 cells are transformed by src, pS2, or G␣q, COX-1 protein levels are unchanged (Fig. 8A) even in the presence of pharmacological inhibitors of the src- and TFF-dependent invasion pathways. Ectopic overexpression of the COX-1 or COX-2 cDNAs also resulted in the activation of membrane metalloproteinase MMP-2, tumorigenesis, and metastatic potential (59, 60).

DISCUSSION Acquisition of the invasive phenotype is the hallmark of adenoma-adenocarcinoma conversion during the progression of colorectal tumors toward local invasion and metastasis in distant vital organs. The starting point of this study was the possible implication of cyclooxygenases on cellular invasion induced by src and TFFs. The current data indicate that persistent activation of the src oncogene and ectopic overexpression of pS2 lead to constitutive invasiveness via the induction of a Rhoindependent mechanism using cyclooxygenases and the G-protein coupled receptor TXA2-R (Fig. 9). Because the TXA2-R and its associated G-proteins G␣q, G␣12, and G␣13 are candidate effectors linking COXs and cancer progression, we have demonstrated that COX-2 and TXB2 levels are remarkably elevated in MDCKts.src cells during src-induced invasiveness. Thus, the src oncogene can generate, first, a COX/TXA2dependent intracrine/autocrine activation loop via the TXA2-R/G␣q pathway, and second, a paracrine activation of cellular invasion via increased levels of TXA2 in the CM-src. Downstream activation of the TXA2-R associated G␣q subunit is directly connected with the PLC pathway and its downstream elements, including PKCs and calcium-dependent signaling. There are several lines of evidence that these routes are indeed

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Figure 9. Model depicting the cyclooxygenase- and TXA2-Rdependent invasion pathways induced by constitutive activation of src and TFFs. In normal digestive mucosa, trefoil peptides exert a beneficial role on wound healing and restitution of injured digestive epithelia. During the early steps of the neoplastic progression (A), transient elevation of TFFs can induce cellular invasion in premalignant digestive epithelial cells via a RhoA- and TXA2-R/PLC-dependent mechanism and contribute to the neoplastic progression, depending on the transformed status of the colonic epithelial cells. Chronic inflammation and cancer progression are associated with constitutive overexpression of pS2 (B), leading to increased metastatic potential via a Rho-independent and PLC-dependent intracrine/autocrine loop using the TXA2-R and its associated G␣q subunit. This autonomous invasive phenotype is exacerbated in src-transformed cells and is monitored in panel C by COX-2 induction, increased intracellular and external TXA2 levels, and autocrine/paracrine activation of the serpentine TXA2-R and G␣q via a Rhoindependent and PLC-dependent mechanism.

involved in cellular invasion and metastasis. Accordingly, invasion induced by G␣12/G␣13 is Rho dependent and unaffected by PLC and COX inhibitors. In addition, the CM from G␣12/G␣13-transfected MDCKts.src cells does not induce invasion. Most important, the Rho inhibitor C3T failed to abolish invasion induced by ectopic overexpression of pS2 and AG␣q, suggesting that these two signaling pathways may overcome and circumvent this blockade. Although src activation initiated a remarkable increase in TXB2 and COX-2 protein levels in MDCKts.src cells, this cyclooxygenase product is not increased consistently in the conditioned medium from pS2- and AG␣q-transformed kidney and colonic epithelial cells. We therefore performed additional experiments showing that the dose-response of NS-398 and SC-560 for inhibition of invasion and TXB2 accumulation induced by src in MDCKts.src cells are superimposed, according to IC50 values that are consistent with their respective potencies on the COX-1 and COX-2 enzymes. Regarding the COX-1 inhibitor, half-maximal inhibition by SC-560 was observed at 3 nM for invasion and TXB2 accumulation. The IC50 of SC-560 on recombinant COX-1 protein is 10 nM, and SC-560 inhibits rCOX-2 at 600-fold higher concentration (61). Regard-

ing the COX-2 inhibitor, half-maximal inhibition of src-induced invasion and TXB2 accumulation was observed in the interdependent COX-2/COX-1 system presented here at 3–5 nM NS-398. The potency of NS-398 on baculovirus-purified COX-2 is 100 nM (62). Furthermore, we found that cellular invasion induced by pS2 in MDCKts.src cells and HCT8/S11 cells was abolished by the COX-1/COX-2 inhibitors within the same range of concentrations. Therefore, our data provide a direct functional link between cyclooxygenases and cellular invasion induced by src and TFFs, and underscore that invasiveness is abrogated when TXB2 levels and cyclooxygenase activities are fully downregulated. This relationship strongly suggests that transient, local, and marginal elevation of TXA2 levels that are not associated with a detectable increase of cellular and external TXB2 in the CM can exert a crucial role in the activation of cellular invasion and the TXA2-R/G␣q signaling system via local diffusion within the plasma membrane. In vitro, the biological half-life of TXA2 is ⬃30 s. Because PGH2 is a freely diffusible molecule in the membrane lipid bilayer, one can speculate that this TXA2-R ligand can be rapidly withdrawn from the external milieu after interaction with this membrane receptor. Diffusion of endogenous PGH/TXA2 products from discrete intracellular/intramembrane pools, leading to intracrine- and autocrine-dependent activation of cellular invasion is also consistent with our data. COX-2 protein is rapidly degraded via the proteasome pathway in human colon carcinoma cells, which constitutively express COX-2 (63). A recent report by Coffey and co-workers (64) demonstrated that EGF-R activation leads to the basolateral release of prostaglandins in intestinal epithelial cells. Such a polarized secretory pathway for COX-produced eicosanoids can be related to the short lifetime of TXA2 and COX-2, linked to a compensatory local delivery, at the immediate vicinity of this transmembrane receptor. In favor with this proposition, recent reports identified COX-2 expression in both the epithelial and stromal compartments of sporadic human colorectal cancers and prostate adenocarcinomas (65– 67). Administration of the proinflammatory agent LPS induced COX-2 expression in subepithelial fibroblasts, which are immediately adjacent to intestinal epithelial cells (68). Consistent localization of COX-2 was also demonstrated in superficial interstitial macrophages in human sporadic colorectal adenomas (65). Therefore, exogenous TXA2 and PGH2 produced by the tumor stroma, including mesenchymal, endothelial, immune cells, and platelets, may participate in paracrine activation of the TXA2-R signaling pathways in cancer cells. Our findings also imply that the TXA2-R and G␣qdependent activation mechanism presented in Fig. 9 is controlled by the COX pathways by preferential coupling of enzyme activity to discrete prostanoid/thromboxane/prostaglandin synthases, according to the transformation phenotype induced by TFFs and the src oncogene. Indeed, we have been unable to detect any


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elevation of TXB2 levels in MDCKts.src-AG␣q cells despite the marked induction of COX-2 protein by this constitutively activated G-protein. In fact, other products of the COX pathways that are natural ligands of the TXA2-R (such as PGH2, other prostanoids and isoprostanes) are potential effectors for this activation loop. The COX-1/COX-2-derived prostane isomers 8-epiPGE2 and 8-epi-PGF2␣ (69, 70) whose effects can be prevented by TXA2-R antagonists are also induced by chronic cigarette smoking, oxidative stress, and reactive oxygen species. Alternatively, our data also argue that the functional competence of the TXA2-R exerts a permissive synergism on invasion promoters and signaling pathways. As previously observed in MDCKts.srctransformed cells and human breast cancer MCF-7 cells, the CM-src in our studies also contains other invasion promoters, such as E-cadherin fragments (71). Similarly, pro-EGF has shown to be activated by proteases via a G-protein receptor activation loop similar to the TXA2-R/G␣q cascade in the present study (72). Several studies have addressed the role of cyclooxygenases in neoplasia, but the molecular mechanisms involved are still poorly understood. The data reported here indicate that the COX and TXA2-R signaling pathways may participate to the persistent activation of G␣q linked to the acquisition of the invasive phenotype controlled by TFFs and src in transformed kidney and colonic epithelial cells. We propose that this cyclooxygenase- and TXA2-R-dependent mechanism is exacerbated by the src oncogene and is operative in TFF-transformed cells (Fig. 9). This intracrine and paracrine activation loop constitutes a potential link for our understanding of the molecular and cellular mechanisms involved in the chemoprevention of colorectal cancers and inflammation by NSAIDs. There is a growing interest in developing NSAID-like drugs for prevention of many types of human cancers. Because of the gastrointestinal toxicity and frequency of complications associated with NSAIDs, one can envisage that the TXA2-R signaling cascade might represent a new therapeutic target with which to control the multistep progression induced by the cyclooxygenase pathways in solid tumors. This work was supported by INSERM, Research Grants from l’Association de la Recherche sur le Cancer (to C.G. and S.E.), and the Fortis Bank, Verzekeringen (Brussels, Belgium).

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The FASEB Journal

Received for publication November 27, 2000. Revised for publication March 7, 2001.

RODRIGUES ET AL.