Marc Mareel

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-β or wounding

Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel™ when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-β (TGF-β) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-β. Functional N-cadherin activity is implicated in TGF-β stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-β1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-β stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling

We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube‐like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor‐a. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX‐2 (NS‐398) and epidermal growth factor receptor (EGF‐R) tyrosine kinase (ZD1839), but is independent of KDR/Flk‐1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX‐2 and EGF‐R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.—Rodrigues, S., Van Aken, E., Van Bocxlaer, S., Attoub, S., Nguyen, Q.‐D., Bruyneel, E., Westley, B. R., May, F. E. B., Thim, L., Mareel, M., Gespach, C., Emami, S. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase −2 and EGF receptor signaling. FASEB J. 17, 7–16 (2003)

Low Doses of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Upregulation of MMPs by soluble E-cadherin in human lung tumor cells.

Loss of E‐cadherin/catenin mediated cell–cell adhesion and overexpression of matrix metalloproteinases (MMPs) are largely involved in tumor invasion. It has been recently shown that high levels of a soluble 80 kDa fragment of E‐cadherin, resulting from a cleavage by MMPs, are found in serum and in urine from cancer patients. Additionally, this soluble E‐cadherin (sE‐CAD) promotes cell invasion into chick heart and into collagen type I gels. The aim of our study was to examine the mechanism of sE‐CAD‐induced cell invasion. Since MMPs play a crucial role in invasion, we looked for induction of MMPs by sE‐CAD in noninvasive human lung tumor cells 16HBE. An induction of MMP‐2, MMP‐9 and MT1‐MMP expression was observed both at the mRNA and at the protein level in the presence of sE‐CAD (in conditioned medium form or in E‐cadherin HAV peptide form). No induction of MMP‐1, ‐3 and ‐7 or variation of the levels of their inhibitors, TIMP‐1 and TIMP‐2, were detected. The biologic relevance of the sE‐CAD‐induced MMP upregulation was tested by demonstrating that sE‐CAD promotes in vitro cell invasion in a modified Boyden chamber assay. These data provide new insight into mechanisms of tumor invasion by ectodomain shedding of the cell–cell adhesion molecule E‐cadherin.

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell–cell aggregation, and suppressed the invasive phenotype in an E-cadherin–dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin–neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

CagA Associates with c-Met, E-Cadherin, and p120-Catenin in a Multiproteic Complex That Suppresses Helicobacter pylori–Induced Cell-Invasive Phenotype

BACKGROUNDnHelicobacter pylori induces an invasive phenotype in gastric epithelial cells through a mechanism that requires the type IV secretion system and the phosphorylation of c-Met. The E-cadherin-catenin complex is a major component of the adherens junctions and functions as an invasion suppressor. We investigated whether E-cadherin has a role in H. pylori-induced, c-Met phosphorylation-dependent cell-invasive phenotype.nnnMETHODSnAGS cells that lack E-cadherin and that are invasive to H. pylori stimulation were transduced with E-cadherin and infected with H. pylori. NCI-N87 cells, which endogenously express E-cadherin, were also used for infection experiments.nnnRESULTSnE-cadherin was sufficient to suppress not only H. pylori-mediated cell-invasive phenotype but also c-Met and p120-catenin tyrosine phosphorylation. H. pylori infection led to increased interactions between E-cadherin and p120-catenin, c-Met and E-cadherin, and c-Met and p120-catenin. Using in vitro infection assays, we showed that H. pylori CagA interacts with E-cadherin, p120-catenin, and c-Met. Finally, using small interfering RNA, we showed that interactions between CagA and E-cadherin and between CagA and p120-catenin were established through c-Met.nnnCONCLUSIONSnWe suggest that H. pylori alters the E-cadherin-catenin complex, leading to formation of a multiproteic complex composed of CagA, c-Met, E-cadherin, and p120-catenin. This complex abrogates c-Met and p120-catenin tyrosine phosphorylation and suppresses the cell-invasive phenotype induced by H. pylori.

RhoA- and RhoD-dependent regulatory switch of Gα subunit signaling by PAR-1 receptors in cellular invasion

Thrombin and proteinase‐activated receptors (PAR) specifically regulate several functions that markedly enhance the transformation phenotype such as inflammation, cell proliferation, tumor growth, and metastasis. We recently reported that thrombin inhibits cellular invasion induced by src, hepatocyte growth factor (HGF), and leptin in kidney and colonic epithelial cells via predominant activation of the pertussis toxin (PTx) ‐sensitive G‐proteins Gαo/Gαi. We provide pharmacological and biochemical evidence that in the presence of PTx, PAR‐1 induced cellular invasion through Gα12/Gα13‐ and RhoA/Rho kinase (ROCK) ‐dependent signaling. However, inhibition of the endogenous small GTPase RhoA by the C3 exoenzyme, dominant‐negative N19‐RhoA, activated G26VRhoD, and activators of the nitric oxide/cGMP pathways conferred invasive activity to PAR‐1 via a signaling cascade using Gαq, phospholipase C (PLC), Ca2+/ calmodulin myosin light chain kinase (CaM‐MLCK), and phosphorylation of MLC. We found that cellular invasion induced by the src oncogene is abrogated by inhibitors of the RhoA/ROCK pathway and is independent of PLC/CaM‐MLCK signaling. Our data demonstrate that the RhoA and RhoD small GTPases are acting as a molecular switch of cellular invasion and reveal a novel critical mechanism by which PAR‐1 bypass Gαo/i and RhoA inhibition via differential coupling to heterotrimeric G‐proteins linked to divergent or convergent biological responses. Our data also indicate that Rho GTPases and ROCK mediate a src‐dependent invasion signal in kidney and colonic cancer cells. We conclude that dynamic regulation of Rho GTPases activation and inactivation by oncogenes, growth factors, cGMP‐inducing agents, and adhesion molecules can initiate convergent invasion signals controlled by the thrombin PAR‐1 in cancer cells.—Nguyen, Q.‐D., Faivre, S., Bruyneel, E., Rivat, C., Seto, M., Endo, T., Mareel, M., Emami, S., Gespach, C. RhoA‐ and RhoD‐dependent regulatory switch of Gα subunit signaling by PAR‐1 receptors in cellular invasion. FASEB J. 16, 565–576 (2002)

Cancer invasion and metastasis: interacting ecosystems

Malignant tumors invade and metastasize. They consist of cancer cells, evolving through genetic and epigenetic modulation, mixed with tumor-associated host cells, emerging from resident or bone marrow-derived precursors. These cells establish ecosystems to activate cellular programs for local invasion and distant metastasis. Characteristic of such malignancy-related activities is communication inside ecosystems between cells, ligands, receptor protein complexes, and signaling pathways as well as between ecosystems comprising the primary tumor, lymph node and distant metastasis, bone marrow and blood and lymph circulation. Complexity is another characteristic, resulting from: heterogeneity of the cell populations; the numbers of promoter and suppressor genes, their levels of regulation, and the pleiotropic activities of their products; biological redundancy of the molecular mechanisms underpinning invasion-related activities. Clinical attention is paid to putative new targets, namely host cells, individual molecules and their signaling pathways, as well as the effects of current treatment on invasion and metastasis.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1‐O‐octadecyl‐2‐O‐methyl‐glycerophosphocholine (ET‐18‐OMe) is an analogue of the naturally occurring 2‐lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET‐18‐OMe modulates cell‐cell adhesion of human breast cancer MCF‐7 cells. In the present study, we tested the effect of ET‐18‐OMe on adhesion, invasion and localisation of episialin and E‐cadherin in MCF‐7/AZ cells expressing a functional E‐cadherin/catenin complex. The MCF‐7/6 human breast cancer cells were used as negative control since their E‐cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E‐cadherin, a calcium‐dependent transmembrane cell‐cell adhesion and signal‐transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell‐associated proteoglycans or mucin‐like molecules such as episialin. Episialin, also called MUC1, is an anti‐adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET‐18‐OMe inhibited the E‐cadherin functions of MCF‐7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E‐cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET‐18‐OMe had no influence on tyrosine phosphorylation of β‐catenin and the E‐cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET‐18‐OMe induced finger‐like extensions with clustering of episialin together with E‐cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET‐18‐OMe caused a shift in the flow‐cytometric profile of episialin toward a lower intensity for MCF‐7/AZ cells. In contrast with MCF‐7/AZ cells, the adhesion‐deficient and noninvasive MCF‐7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET‐18‐OMe. Co‐localisation of episialin with E‐cadherin was rarely observed. We conclude that in the human breast cancer cells MCF‐7/AZ, E‐cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell‐cell adhesion and invasion.