The mediating role of joint attention in the relationship between motor skills and receptive and expressive language in siblings at risk for autism spectrum disorder.

Language problems are highly prevalent in younger siblings of children with autism spectrum disorder (HR-sibs), yet little is known about early predictors. There is growing evidence that motor and language development are linked and this connection might be mediated by joint attention. Developmental changes in motor abilities change how children interact with objects and people (e.g., by showing), which may influence language development. This association has however not yet been studied in HR-sibs. The interrelationship between motor, joint attention and language skills was explored in younger siblings of typically developing children (LR-sibs, N = 31) and HR-sibs (N = 32). In both groups, motor skills (composite of fine and gross motor skills) at 10 months influenced receptive and expressive language at 36 months directly and indirectly through joint attention at 14 months. Group status moderated this direct and indirect effect with mainly significant effects in HR-sibs. This indicates that lower motor skills can have cascading effects on joint attention and language in HR-sibs. Consequently, assessment of early motor skills in HR-sibs might hold promise for early identification of motor difficulties but can also be indicative of language difficulties later in life, especially when difficulties with joint attention are also present.

Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis.

Deleted in colon cancer (DCC) and UNC5 function as netrin dependence receptors by inducing apoptosis in the absence of their ligand and accordingly were recently designated as putative conditional tumor suppressors. Herein, we determined whether netrin-1 and its receptors are implicated in cancer cell invasion and tumor progression. Expression of DCC, UNC5 and adenosine A2B-receptors (A2B-Rs) was investigated by reverse transcription polymerase chain reaction in human colon cancer cells. The impact of DCC restitution and netrin-1 was evaluated on collagen type I invasion, tumor growth and metastasis in nude mice, cancer cell survival and gene expression profiling. Flow cytometry, poly(ADP-ribose)polymerase-1 and caspase-8 activation were used to evaluate the impact of DCC on cell death. Both netrin-1 and A2B-R activation induced the invasive phenotype through the Rho-Rho kinase axis in DCC-deficient human colorectal cancer cells. Restitution of wild-type DCC blocked invasion induced by netrin-1, A2B-R agonist and other agents. Ectopic expression of netrin-1 led to increased growth of human colon tumor xenografts in athymic mice. Conversely, introduction of wt-DCC in kidney MDCKts.src-ggl cells strongly inhibited metastasis in lymph nodes and lungs and increased sensitivity to apoptosis in hypoxia. DNA microarrays revealed that netrin and DCC had common and divergent impacts on gene expression linked to cell cycle, survival, surface signaling and adhesion. Our findings underscore that netrin is a potent invasion and tumor growth-promoting agent and that DCC is a metastasis suppressor gene targeting both proinvasive and survival pathways in a cumulative manner.

Edelfosine protects precultured heart fragments against the invasion of malignant cells through altered sialylation.

1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe)-treated precultured heart fragments (PHF) are resistant to the invasion of malignant cells. Previous studies have demonstrated that this effect is due to the alterations of the N-linked glycoproteins in PHF after 48-h ET-18-OMe treatment. Moreover, the observed effect was still present seven days after ET-18-OMe was omitted. The present study reveals that approximately 13.4% of the administered ET-18-OMe was taken up by PHF and about 75% of the initial uptake was still present after ET-18-OMe was removed. In addition, we found significant changes in the sialic acid content and sialyltransferase activities in both conditions. Overall, these results clearly demonstrate that the uptake and retention of ET-18-OMe are responsible for the resistance to the invasion of malignant cells due to the altered sialylation of the cell surface glycoproteins in PHF.

Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells.

TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma-carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.

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.

Molecular characterization of cell substratum attachments in human glial tumors relates to prognostic features.

Glioma cell attachments to substratum play crucial roles in the invasion by glioma cells of normal brain tissue. These attachments are mediated through interactions between extracellular matrix (ECM) components, integrins, focal adhesion-linked molecules, and the actin cytoskeleton. In the present study, we investigate the molecular elements involved in cell substratum attachments in human glial tumors and their potential relationships to prognostic features. We used 10 human glioma cell lines, for which we characterized glial differentiation by means of quantitative RT-PCR for nestin, vimentin, and GFAP mRNA. We quantitatively determined the amounts of laminin, fibronectin, vitronectin, and thrombospondin secreted by these glioma cell lines in vitro, as well as the amount of each of the eight beta integrin subunits and the adhesion complex-related molecules, including talin, vinculin, profilin, zyxin, alpha-actinin, paxillin, and VASP. After quantification of the levels of migration and invasion of these 10 cell lines in vitro and, through grafts into the brains of nude mice, of their biological aggressiveness in vivo, it appeared that the levels of the beta 5 integrin subunit and alpha-actinin were directly related to biological aggressiveness. These experimental data were clinically confirmed because increasing immunohistochemical amounts of the beta 5 integrin subunit and alpha-actinin were directly related to dismal prognoses in the case of astrocytic tumors. In addition, we show that the beta 4 integrin subunit are expressed significantly more in oligodendrogliomas than in astrocytic tumors. A potential role for the beta 8 integrin subunit in glioma cell substratum attachments is also emphasized.

alphaT-catenin: a novel tissue-specific beta-catenin-binding protein mediating strong cell-cell adhesion.

Cadherins are major cell-cell adhesion proteins whose cytoplasmic domains bind to catenin proteins. Strong intercellular adhesion depends on linkage of the cadherin/catenin complex to the actin cytoskeleton via alpha-catenin. To date, it is not clear how different cell types achieve the variable strength of cell-cell adhesion clearly needed in a multicellular organism. Here, we report the cloning and molecular characterization of alphaT(testis)-catenin, a novel human cDNA encoding a protein with homology to both human alphaE(epithelial)-catenin and alphaN(neural)-catenin. Although originally discovered in testis, alphaT-catenin is expressed in other tissues, the highest levels being observed in heart. Immunohistochemical analysis showed human alphaT-catenin localization at intercalated discs of cardiomyocytes and in peritubular myoid cells of testis. In cells transfected with alphaT-catenin cDNA, interaction with beta-catenin was demonstrated by co-immunoprecipitation. Transfection of alpha-catenin-deficient colon carcinoma cells recruited E-cadherin and beta-catenin to cell-cell contacts and functional cadherin-mediated cell-cell adhesion was restored in this way. Moreover, compaction of these cells was at least as prominent as in the case of cells expressing endogenous alphaE-catenin. We propose that alphaT-catenin is necessary for the formation of stretch-resistant cell-cell adhesion complexes, in particular, muscle cells.

The role of bile acids in carcinogenesis.

Bile acids play a role in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. Some bile acids stimulate growth of normal colonic and adenoma cells, but not of colorectal cancer cells. Moreover, bile acids stimulate invasion of colorectal cancer cells, at least in vitro. One possible mechanism of action is bile acid-induced DNA binding and transactivation of the activator protein-1 (AP-1) by co-operate activation of extracellular signal-regulated kinases (ERKs) and PKC signaling. In the present paper, we review the mechanisms by which bile acids influence carcinogenesis.

Suppression of cellular invasion by activated G-protein subunits Galphao, Galphai1, Galphai2, and Galphai3 and sequestration of Gbetagamma.

It was shown previously that platelet-activating factor receptors (PAF-Rs) inhibit invasiveness of colonic and kidney epithelial cells induced by the src and Met oncogenes via a pertussis toxin-sensitive mechanism. Therefore, Madin-Darby canine kidney (MDCKts.src) cells were stably transfected with constitutively activated forms of Galphao, Galphai1, Galphai2, Galphai3 (AGalphao/i), two Gbetagamma sequestering proteins [C-terminal end of beta-adrenergic receptor kinase (ct-betaARK) and the Galphat subunit of retinal G-protein transducin], and Gbeta1-Ggamma2 subunits alone or in combination. Cellular invasion induced by src, Met, and leptin was abrogated by the AGalphao/i, ct-betaARK, and Galphat-positive clones, but was induced by coexpression of Gbeta1gamma2. In contrast, invasion stimulated by the trefoil factors (TFFs) pS2 and intestinal trefoil factor in MDCKts.src cells or human colonic epithelial cells PCmsrc and HCT8/S11 was insensitive to PAF, AGalphao, AGalphai1, and AGalphai2, but was abolished by AGalphai3 and the protease-activated receptor-1 (PAR-1) agonist thrombin receptor-activating peptide. Depletion of free Gbetagamma heterodimers by ct-betaARK resulted in a remarkable decrease of cellular adhesion and spreading on collagen matrix. Our data demonstrate the following: 1) PAF-Rs impair cellular invasion induced by src, Met, and leptin via the activation of Galphao and Galphai1 to -3; 2) invasion induced by TFFs is selectively inhibited by PAR-1 receptors and Galphai3 activation; and 3) Gbetagamma dimers are required as positive effectors of invasion pathways induced by oncogenes and epigenetic factors. Thus, redistribution of Galphao/Galphai and Gbeta/gamma heterotrimeric G-proteins by PAF-R and PAR-1 exert differential functions on positive and negative signaling pathways involved in cellular invasion and may serve as potential targets for anticancer therapy.

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.

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways.

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 Galphaq (AGalphaq), Galpha12, Galpha13 (AGalpha12/13), 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 TXA2-R-dependent invasion pathway, which is monitored via a Rho- and Galpha12/Galpha13-independent mechanism using the Galphaq/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.

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 beta-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.

Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells.

Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.

Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.

The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E-cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E-cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin-dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.

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.

Cell aggregation assays.

Invasion of carcinoma cells is the result of a disequilibrium between invasion promoter and invasion suppressor gene products (1). The E-cadherin/catenin complex is the most potent invasion suppressor at the cell membrane of epithelioid cells (2).This complex consists of E-cadherin, a transmembrane glycoprotein of 120 kDa, which is linked to the actin cytoskeleton via the catenins (3). Downregulation of the complex is a common feature in invasive carcinoma cells, and has been recognized at several levels, ranging from genomic mutations to functional deficiencies of an apparently intact complex (4). Cell aggregation assays have been set up to test the functionality of the complex in epithelioid tumor cells. Functional integrity of the complex is a prerequisite for cell-cell adhesion between epithelial cells, and measuring cell aggregation in vitro has thus become another elegant tool to study differences between invasive and noninvasive cell types.

Collagen invasion assay.

Invasion occurs when invasion promoter molecules outbalance the function of invasion suppressors (1). Examples of invasion promoters are cell-matrix adhesion molecules, extracellular proteases, and cell motility factors. In normal tissues, positional stability of the cells is maintained through the counteraction of these invasion promoters by invasion suppressors such as enzyme inhibitors and cell-cell adhesion molecules. Within this context, the interaction of the cancer cells with their surrounding extracellular matrix (ECM) is a determining factor. To study this cell-matrix interaction in vitro, several natural ECM types have initially been applied. Bone (2), salt-extracted cartilage (3), and amnion membrane (4) are examples of devitalized substrata that have been launched in the past to discriminate between invasive and noninvasive cells. Lack of homogeneity of these substrata often made interpretation of invasion difficult, and hampered the reproducibility of those assays (5). To overcome these drawbacks, reconstituted and hence more homogeneous ECMs were developed, and proposed as substrata to test invasiveness. Matrigel (6) (as described in Chapter 7 by Hall and Brooks), and employed also in the assay described in Chapter 8 by Hendrix et al.), Humatrix (7) and collagen type I (8) are today frequently used ECMs in invasion assays. It should, however, be noted that, although these preparations may contain cytokines and growth factors, they are unable to react to the confrontation by cancer cells as a living host tissue does.

Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho-, and rac-dependent signaling pathways.

Leptin plays a key role regulating food intake, body weight and fat mass. These critical parameters are associated with an increased risk for digestive and mammary gland cancer in the Western population. Here we determined whether leptin contributes to the invasive phenotype of colonic and kidney epithelial cells at various stages of the neoplastic progression. First, leptin potently (EC50 = 10-30 ng/ml) induces invasion of collagen gels by premalignant familial adenomatous colonic cells PC/AA/C1 and nontumorigenic MDCK kidney epithelial cells, their src-transformed counterparts, and the human adenocarcinoma colonic cells LoVo and HCT-8/S11. Leptin and its Ob-Rb receptors were consistently identified by RT-PCR and immunoblotting in these cell lines, as well as in human colonic epithelial crypts, polyps, colonic tumor resections, and adjacent mucosa. Leptin-induced invasion was effectively blocked by pharmacological inhibitors of several downstream signaling pathways involved in cell transformation, namely, JAK2 tyrosine kinase (AG490), phosphoinositide PI3'-kinase (wortmannin and LY294002), mTOR kinase (rapamycin), and protein kinases C (GF109203X, Gö6976). Accordingly, leptin induces transient elevation of the PI3'-kinase lipid products in JAK2 immunoprecipitates prepared from parental MDCK cells. The leptin effect on invasion was potentiated by the activated form of the small GTPase RhoA and was abrogated by dominant negative mutants of RhoA, Rac1, and the p110alpha of PI3'-K. Our data indicate that leptin may exert a local and beneficial effect on migration of normal colonic epithelial cells and reparation of the inflamed or wounded digestive mucosa. We also emphasize a new role for leptin, linking the nutritional and body fat status to digestive cancer susceptibility by stimulating the invasive capacity of colonic epithelial cells at early stages of neoplasia. This finding has potential clinical implications for colon cancer progression and management of obesity.

Evidence that Smad2 is a tumor suppressor implicated in the control of cellular invasion.

The Smad2 protein plays an essential role in the transforming growth factor-beta (TGF-beta) signaling pathway. This pathway mediates growth inhibitory signals from the cell surface to the nucleus. Although Smad2 protein is significantly mutated in human cancers, there is no definitive evidence implicating Smad2 as a tumor-suppressor gene. Here we show that overexpression of the tumor-derived missense mutation Smad2.D450E, an unphosphorylable form of Smad2 found in colorectal and lung cancers, did not abolish the TGF-beta-mediated growth arrest, suggesting that resistance to the growth-inhibiting effects of TGF-beta exhibited by human tumors cannot be linked to the inactivation of Smad2 protein. In contrast, overexpression of Smad2.D450E induces cellular invasion, and this effect was enhanced by TGF-beta. A similar invasive phenotype was obtained in cells expressing another inactivating mutation in Smad2 (Smad2.P445H) found in colorectal cancer. These findings indicate that genetic defects in Smad2 are sufficient to confer the invasion-promoting effect of TGF-beta and reveal that TGF-beta acts through Smad2 to induce cellular invasion by a novel mechanism that is independent of Smad2 phosphorylation by the activated TGF-beta type I receptor.

Sialylation of E-cadherin does not change the spontaneous or ET-18-OMe-mediated aggregation of MCF-7 human breast cancer cells.

We have investigated the role of sialylation on cell-cell adhesion mediated by E-cadherin. Two MCF-7 human breast cancer cell variants were studied: MCF-7/AZ cells showed a spontaneous cell-cell adhesion in the fast and slow aggregation assay. whereas the adhesion deficient MCF-7/6 cell variant failed to form larger aggregates, suggesting that E-cadherin was not functional under the conditions of both assays. We measured the sialyltransferase activities using Galbeta1-3GalNAcalpha-O-benzyl and Galbeta1-4GlcNAcalpha-O-benzyl as acceptor substrates as well as mRNA levels of four sialyltransferases, ST3Gal I, ST3Gal III, ST3Gal IV, ST6Gal I, using multiplex RT-PCR in MCF-7 cell variants. The alpha2-6 and alpha2-3 sialylation of E-cadherin was investigated by immuno-blot using Sambucus nigra agglutinin and Maackia amurensis agglutinin. Compared to the adhesion-proficient MCF-7/AZ cells, the adhesion-deficient MCF-7/6 cell line apparently lacks ST6Gal I mRNA, has a lower ST3Gal I mRNA, a lower ST3Gal I sialyltransferase activity, and no alpha2-3 linked sialic acid moieties on E-cadherin. The potential anti-cancer drug 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe, 48 h, 25 microg/ml) belonging to the class of alkyllysophospholipids restored the E-cadherin function in the adhesion-deficient MCF-7/6 cells as evidenced by an increased aggregation. ET-18-OMe caused loss of ST6Gal I mRNA in MCF-7/AZ cells but no changes of sialyltransferase activities or sialic acid moieties on E-cadherin could be observed. We conclude that Ca2+-dependent, E-cadherin-specific homotypic adhesion of MCF-7/AZ or MCF-7/6 cells treated with ET-18-OMe was not affected by sialylation of E-cadherin.