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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-1110fje. |
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Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, Gent, Belgium; and
* INSERM U482, Hôpital Saint-Antoine, Saint-Antoine, Paris Cedex, France
2Correspondence: Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Gent, Belgium. E-mail: marc.mareel{at}UGent.be
SPECIFIC AIM
Dynamic and reciprocal communication between epithelial and stromal compartments is crucial for cancer progression. Myofibroblasts are stromal cells abundantly present at the invasion front of primary colon tumors. Their potential role in the transition from the noninvasive to invasive phenotype is suggested by their appearance before the invasive stage of the tumor. We have tried to better understand the cellular and molecular interactions between colon cancer cells and myofibroblasts.
PRINCIPAL FINDINGS
1. Myofibroblasts and rTGF-ß1-treated fibroblasts stimulate invasion of colon cancer cells into collagen and Matrigel in vitro
Myofibroblasts, in contrast to fibroblasts isolated from the adjacent normal colon of the same patient, stimulated invasion into collagen of HCT-8/E11, HCT-8/E11R1, and SW480 colon cancer cells in a 24 h assay. The presence of myofibroblasts in collagen changed the morphotype of colon cancer cells from round to elongated, with formation of cellular extensions. Invasion into Matrigel was significantly stimulated when both types of HCT-8 cells were mixed with myofibroblasts, a cellular response not observed in the presence of fibroblasts. TGF-ß transdifferentiates fibroblasts into myofibroblasts in vitro and in vivo. Indeed, fibroblasts treated for 7 days with rTGF-ß1 (0.1 and 1 ng/mL) stimulated invasion of HCT-8/E11 cells as myofibroblasts did. Acquisition of pro-invasive activity of fibroblasts was associated with the appearance of 
-SMA stress fibers, indicating fibroblast-to-myofibroblast transition.
2. Pro-invasive factors secreted by myofibroblasts are identified as TNC and SF/HGF, each of which is necessary but not sufficient for stimulation of human colon cancer cell invasion
In vitro, CMmyo stimulated invasion of various colon cancer cell types into collagen (Fig. 1
A) and changed the morphotype of HCT-8/E11 colon cancer cells from round to elongated, suggesting a role for pro-invasive factors secreted by myofibroblasts. That the ECM protein TNC may act as a pro-invasive molecule was strengthened by its presence in the CMmyo and on the surface of myofibroblasts (Fig. 1B
). The amount of TNC in the CMmyo used in collagen invasion experiments was 924 ng/2.5 x 106 cells. The pro-invasive activity of the CMmyo was annihilated by preincubation with the TNC-neutralizing antibody BC24 raised against its EGF-like repeats, but not with control IgG1 (Fig. 1C
). Blocking the EGF-R with the selective EGF-R tyrosine kinase inhibitor ZD1839 (Iressa) or by expression of its dominant negative form (HER-CD533) interfered with stimulation of invasion by CMmyo (Fig. 1D
).
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ELISA of SF/HGF performed in CMmyo, CMfib, and CMfib (rTGF-ß1 treated) showed equivalent amounts of SF/HGF (5–8 ng/2.5x106 cells). SF/HGF secreted by myofibroblasts or fibroblasts was bioactive since CMmyo, CMfib, or CMfib (rTGF-ß1 treated) stimulated c-Met phosphorylation in HCT-8/E11 cells (Fig. 2
A). Immunoprecipitation of CMmyo with an anti SF/HGF mAb (CMmyo, IP SF/HGF mAb), but not with control IgG1 (CMmyo, IP control IgG1), prevented phosphorylation of c-Met. Conversely, addition of rSF/HGF to CMmyo, IP SF/HGF mAb in an amount similar to that found in CMmyo restored c-Met activation. Moreover, preincubation of HCT-8/E11 with the selective SF/HGF antagonist NK4 prevented c-Met phosphorylation by CMmyo.
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CMmyo, IP SF/HGF mAb did not stimulate invasion of HCT-8/E11 and PC/AA/C1 in collagen type I, in contrast to CMmyo, IP control IgG1 or CMmyo, IP SF/HGF mAb supplemented with rSF/HGF (Fig. 2B
). In agreement, NK4 dose-dependently prevented invasion of HCT-8/E11 that were stimulated by CMmyo. These observationssuggest that myofibroblast-stimulated invasion requires SF/HGF; HCT-8/E11 and PC/AA/C1 cells are sensitive to the pro-invasive action of SF/HGF; and myofibroblasts may provide environmental stimuli, such as TNC, necessary for the pro-invasive activity of SF/HGF.
To mimic the effect of the pro-invasive factors secreted by myofibroblasts, we used purified TNC within a physiological range of concentrations and rSF/HGF at a concentration similar to that found in CMmyo. Neither TNC nor rSF/HGF alone stimulated invasion. Simultaneous addition of rSF/HGF with TNC stimulated invasion of HCT-8/E11 cells.
3. TNC constitutes the crucial difference between fibroblasts and myofibroblasts
Since SF/HGF was found in CMfib and CMmyo in roughly equal concentrations, it seemed logical to assume that TNC was responsible for the change in pro-invasive activity at the transition between fibroblasts and myofibroblasts. Production of TNC in the CM, used for collagen invasion, is enhanced by treatment of fibroblasts with rTGF-ß1 (51–1163 ng/2.5x106 cells). CMfib is made pro-invasive by exogenous addition of 1 µg purified TNC. The pro-invasive effect of CMfib (rTGF-ß1 treated) is annihilated by preincubation with the TNC-neutralizing antibody BC24 but not with control IgG1. Blocking EGF-R signaling by the selective tyrosine kinase inhibitor ZD1839 (Iressa) and expression of the EGF-R dominant negative mutant (HER-CD533) reversed the pro-invasive action CMfib (rTGF-ß1 treated).
4. SF/HGF acts through activation of Rac, TNC through inactivation of RhoA
In our present experiments invasion of HCT-8/E11 cells induced by CMmyo was associated with an elongated morphotype, suggesting Rac-dependent and RhoA/ROCK-independent signaling pathways. To test this, we measured GTP-bound forms of Rac and RhoA in HCT-8/E11 cells under the same experimental conditions as the collagen invasion assay. When TNC was admixed to collagen type I, almost no active RhoA was found and addition of rSF/HGF had no effect. CMmyo inhibited RhoA activity in HCT-8/E11 cells seeded on top of collagen type I gels. This inhibition was TNC dependent and SF/HGF independent as evidenced by the TNC-neutralizing mAb BC24 and by CMmyo, IP SF/HGF mAb. Inversely, Rac activity was remarkably enhanced by rSF/HGF and inhibited by TNC in HCT-8/E11 cells on top of collagen type I gels. In comparison, CMmyo stimulated Rac activity through SF/HGF-dependent and TNC-independent pathways as evidenced by CMmyo, IP SF/HGF mAb and by the TNC-neutralizing mAb, respectively.
The differential control of Rho and Rac exerted by the CMmyo and its corresponding pro-invasive agents TNC and SF/HGF was validated by invasion assays using pharmacological modulators and stable expression of dominant negative and constitutively active forms of RhoA/ROCK and Rac in HCT-8/E11 cells. Treatment of HCT-8/E11 cells with C3T, inactivating RhoA, or Y27632, inhibiting ROCK, permits rSF/HGF or CMfib to exert its pro-invasive activity in the absence of TNC. This suggests that TNC-induced RhoA inhibition and subsequent permissive pro-invasive signals can be mimicked by pharmacological inhibitors of the RhoA-ROCK cascade. Both C3T and Y27632 failed to inhibit CMmyo stimulated invasion. Conversely, the RhoA activator CNF-1 alleviates the pro-invasive effect of CMmyo. HCT-8/E11 cells that express dominant negative RhoA (RhoAT19N) or constitutive active RhoD (RhoDG26V), an inhibitor of RhoA, were sensitive to the pro-invasive effect of rSF/HGF. Similarly, blockade of the downstream RhoA effector ROCK by the dominant negative ROCK (ROCK
RB/PH) sensitized HCT-8/E11 cells to the pro-invasive effect of rSF/HGF. Invasion stimulated by CMmyo or by rSF/HGF+TNC was not affected by the expression of RhoAT19N, RhoDG26V, or ROCKRB/PH. In contrast, expression of constitutively active RhoA (RhoAG14V) or constitutively active ROCK (ROCKCAT) was incompatible with stimulation of invasion by rSF/HGF, rSF/HGF+TNC, or CMmyo. Expression of dominant negative Rac (RacT17N) renders HCT-8/E11 cells resistant to stimulation of invasion by myofibroblast-derived factors.
CONCLUSIONS
Our present in vitro experiments confirm that myofibroblasts derived from human colon cancer or rTGF-ß-treated colon fibroblasts stimulate invasion of colon cancer cells. We investigated the presence of pro-invasive factors in CMmyo. CMmyo stimulates invasion and causes c-Met phosphorylation and Rac activation in a SF/HGF-dependent manner, as suggested by using the SF/HGF antagonist NK4 and immunodepletion of SF/HGF from CMmyo. rSF/HGF or CMfib also cause c-Met phosphorylation and Rac activation but are not pro-invasive, suggesting that SF/HGF is required for pro-invasive activity of the CMmyo but not sufficient. We explored the possible contribution of the matricellular protein TNC as a candidate pro-invasive helper molecule. Indeed, TNC is up-regulated in fibroblasts by treatment with rTGF-ß1. Like rSF/HGF, TNC alone is not sufficient to stimulate invasion, but the combination of both molecules is clearly pro-invasive. CMmyo stimulates invasion and causes RhoA inactivation in HCT-8/E11 in a TNC-dependent manner, suggesting that TNC-mediated RhoA inactivation confers a permissive signal to SF/HGF. We confirmed the validity of this mechanism by the RhoA inhibitor C3T exoenzyme, the dominant negative mutant RhoAT19N, and the constitutively activated RhoA inhibitor RhoDG26V. Taken together, our study identifies a new mechanism involved in cellular invasion in which the cross-signaling between cancer cells and stromal fibroblast-like cells may contribute to the progression of human solid tumors.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1110fje; 
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