Marc M. Mareel

The Two-Handed E Box Binding Zinc Finger Protein SIP1 Downregulates E-Cadherin and Induces Invasion

Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Here, we report that expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and showed similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors.

Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)‐β stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF‐β‐upregulated extracellular matrix glycoprotein tenascin‐C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast‐stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)‐like repeats of TNC through EGF‐receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c‐Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA‐Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness

We recently established the critical role of the lipid phosphatase activity of the PTEN tumor suppressor in stabilizing cell‐cell contacts and suppressing invasiveness. To delineate the effector systems involved, we investigated the interaction of PTEN with E‐cadherin junctional complexes in kidney and colonic epithelial cell lines. PTEN and the p85 regulatory subunit of phosphatidylinositol 3‐OH kinase (PI3K) co‐immunoprecipitated with E‐cadherin and catenins. By using a yeast two‐hybrid assay, we demonstrated that PTEN interacted indirectly with β‐ catenin by binding the scaffolding protein MAGI‐1b. This model was corroborated in various ways in mammalian cells. Ectopic expression of MAGI‐1b potentiated the interaction of PTEN with junctional complexes, promoted E‐cadherin‐dependent cell‐cell aggregation, and reverted the Src‐induced invasiveness of kidney MDCKts‐src cells. In this model, MAGI‐1b slightly decreased the activity of AKT, a downstream effector of PI3K. By using dominant‐negative and constitutively active AKT expression vectors, we demonstrated that this kinase was included in the pathways involved in Src‐induced destabilization of junctional complexes and was necessary and sufficient to trigger invasiveness. We propose that the recruitment of PTEN at adherens junctions by MAGI‐1b and the local down‐regulation of phosphatidylinositol‐3,4,5‐trisphosphate pools and downstream effector systems at the site of cell‐cell contacts are focal points for restraining both disruption of junctional complexes and induction of tumor cell invasion.

The invasive phenotypes.

The expression of the invasive (I+ or I-) phenotypes determines cancer metastasis (M+ or M- phenotype). The invasive (I+ or I-) phenotypes can be divided according to time and site of expression into subphenotypes, which can be assessed separately. At various sites along the metastatic pathway the expression of the I phenotypes can be accompanined by the presence of uncontrolled growth (G+ phenotype) or its absence (G- phenotype). Various combinations of the I and G phenotypes determine the behaviour of metazoan or parasitic cells under normal, pathological non-neoplastic and neoplastic conditions. Although the G+I+M+ combination correlates with full malignancy, the sequence of events leading to the acquisition of these phenotypes during tumor development is not clear. Conditional invasion in experimental systems indicates that a tumor may be invasive and metastatic when part of its population temporarily expresses the I+ phenotype. These experiments further stress the importance of the tumor-host ecosystem for the regulation of the I phenotypes. As distinct from some parasites, the invasive morphotype of vertebrate cells cannot be simply identified. Nevertheless, within the tumor-host ecosystem morphological correlates of the activities of invasive cells may be recognized. They reflect one or more of the I+ functions, namely: motility; loss of homotypic cell-cell adhesion; establishment of alternative cell-substrate and heterotypic cell-cell adhesion; breakdown of extracellular matrices. These functions are not exclusive for I+ tumor cells, and neither are the molecular markers investigated so far. Oncogene activation leads mainly to G+ expression, and in this way serves as a signal amplifier for the I and M phenotypes. Attractive candidate molecular markers of I phenotypes are: regulators of hydrolase activities; cell-cell adhesion molecules; cell surface receptors. From data presently available, we hypothesize that invasion depends upon the balance between an I+ and an I- pathway, with both pathways being sensitive to stimulation and inhibition.

The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cell-cell adhesion and to suppress invasion.

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF–LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF–LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a β-catenin-dependent activation of the WNT pathway.

Invasion promoter versus invasion suppressor molecules: the paradigm of E-cadherin

ConclusionMetastasis determines cancer malignancy. Neoplastic cells metastasize through a multistep process of invasion. At each step, these cells create a dynamic micro-ecosystem in which the elements of the host are considered to paricipate actively invasion. Within such micro-ecosystems, invasion is believed to be governed by a balance between the activation of promoter (i-minus) and suppressor (i-plus) genes. Products of such genes regulate the expression of the invasive (I-plus) and the nonivasive (I-minus) phenotypes. Experimentsin vitro andin vivo, as well as observations on human cancers have put forward the cell-cell adhesion molecule E-cadherin (L-CAM; uvomorulin) as a regulator of epithelial organization and an invasion suppressor.

Plasmin Produces an E-Cadherin Fragment That Stimulates Cancer Cell Invasion

Abstract Matrix metalloproteases from the cell surface cleave an 80 kDa Ecadherin fragment (sECAD) that induces invasion of cancer cells into collagen type I and inhibits cellular aggregation. Conditioned media from MDCKts.srcCl2 cells at 40 C and 35 C, PCm.src5 and COLO-16 cells at 37 C contained spontaneously released sECAD; these 48 h old conditioned media were capable of inhibiting Ecadherin functions in a paracrine way. Here we show direct cleavage of the extracellular domain of Ecadherin by the serine protease plasmin. sECAD released by plasmin inhibits Ecadherin functions as evidenced by induction of invasion into collagen type I and inhibition of cellular aggregation. This functional inhibition by sECAD was reversed by aprotinin or by immunoadsorption on protein Sepharose 4 fast flow beads with antibodies against the extracellular part of Ecadherin. Our results demonstrate that plasmin produces extracellular Ecadherin fragments which regulate Ecadherin function in cells containing an intact Ecadherin/ catenin complex.

Inhibition by Platelet-activating Factor of Src- and Hepatocyte Growth Factor-dependent Invasiveness of Intestinal and Kidney Epithelial Cells PHOSPHATIDYLINOSITOL 3′-KINASE IS A CRITICAL MEDIATOR OF TUMOR INVASION

This study was designed to characterize platelet-activating factor receptor (PAF-R) expression and function in normal and cancerous human colonic epithelial cells. PAF-R gene transcripts were analyzed by reverse transcription-polymerase chain reaction and Southern blot, using three sets of primers corresponding either to the coding region of the human PAF-R sequence (polymerase chain reaction product: 682 base pairs (bp)) or to the leukocyte- and tissue-type transcripts of 166 and 252 bp, respectively. An elongated splice variant was identified in the 5′-untranslated region of the tissue-type PAF-R transcript (334 bp) in colonic epithelial crypts and tumors. In human colonic PCmsrc cells transformed by c-src oncogene, the hepatocyte growth factor (HGF)-dependent invasiveness of collagen gels was abolished by 0.1 μm PAF and restored by the PAF-R antagonists WEB2086 and SR27417. PAF blocked HGF-induced tyrosine phosphorylation of p125 focal adhesion kinase. The phosphatidylinositol 3′-kinase (PI3′-K) inhibitors wortmannin and LY294002 totally blocked the HGF-induced invasion. Similar effects were observed in ts-srcMDCK kidney epithelial cells transformed by a v-Src temperature-sensitive mutant: (i) PAF and wortmannin exerted additive inhibitory effects on Src-induced invasion and (ii) activated and dominant negative forms of p110α PI3′-K, respectively, amplified and abrogated the Src- and HGF-dependent invasiveness of parental and ts-srcMDCK cells. We also provided the first evidence for the contribution of rapamycin-insensitive, pertussis toxin-dependent G-protein pathways to the integration of the signals emerging from activated Met and PAF receptors. These results indicate that PI3′-K is a critical transducer of invasiveness and strongly suggest that PAF exerts a negative control on invasion by inhibiting this signaling pathway. A possible beneficial role of PAF analogs on tumor invasion is therefore proposed.

Bile acids stimulate invasion and haptotaxis in human colorectal cancer cells through activation of multiple oncogenic signaling pathways.

Bile acids are implicated in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. We examined whether bile acids stimulate cellular invasion of human colorectal and dog kidney epithelial cells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidney MDCKT23 cells were seeded on top of collagen type I gels and invasive cells were counted after 24 h incubation. Activation of the Rac1 and RhoA small GTPases was investigated by pull-down assays. Haptotaxis was analysed with modified Boyden chambers. Lithocholic acid, chenodeoxycholic acid, cholic acid and deoxycholic acid stimulated cellular invasion of SRC- and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originating from a sporadic tumor, but were ineffective in premalignant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation of haptotaxis and was dependent on the RhoA/Rho-kinase pathway and signaling cascades using protein kinase C, mitogen-activated protein kinase, and cyclooxygenase-2. Accordingly, BA-induced invasion was associated with activation of the Rac1 and RhoA GTPases and expression of the farnesoid X receptor. We conclude that bile acids stimulate invasion and haptotaxis in colorectal cancer cells via several cancer invasion signaling pathways.

The αE-catenin gene ( CTNNA1 ) acts as an invasion-suppressor gene in human colon cancer cells

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the αE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second αE-catenin allele. The αE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.