Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

Epithelial expression of FHL2 is negatively associated with metastasis-free and overall survival in colorectal cancer.

BACKGROUND: Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to have a role in cancer progression. It has been shown to be overexpressed in the colorectal cancer (CRC). METHODS: Here, we examined a possible prognostic value of FHL2 in CRC. Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at the time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using small-interfering RNA transfection in hTERT-immortalised myofibroblasts. RESULTS: Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Multivariate Cox analysis shows that expression in the tumour invasion front (P<0.001) as well as in the centre of the tumour (P<0.001) was associated with metachronous metastases independently of the clinicopathological variables; expression in the tumour invasion front was also associated with overall survival independently of the clinicopathological variables (P<0.01). CONCLUSION: Higher FHL2 expression is involved in CRC progression and correlates with the development of metachronous metastases and overall survival, suggesting that FHL2 is an independent adverse prognostic indicator for CRC.

Tumour-associated host cells participating at invasion and metastasis : targets for therapy?

The 100-year old "seed" and "soil" theory explains key features of cancer metastasis: early initiation; late appearance; organ-specificity. The seed is the cancer cell; it undergoes genetic alterations, disturbing the cellular activities that maintain normal tissue organisation and, so, initiating the formation of invasive and metastatic tumours. The soil consists of tumour-associated host cells: endothelial cells and pericytes forming blood-and lymph vessels attracted to the cancer cells by vascular endothelial growth factor (VEGF); nerve cells; fibroblasts converted into myofibroblasts by cancer cell-released transforming growth factor (TGF)-beta; inflammatory cells, attracted by cancer chemokines; osteoclasts activated by metastatic cancer cells in the bone marrow. All these host cells engage in continuous molecular cross talk with the cancer cells, influencing invasion and metastasis. Tumor-associated host cells are themselves invasive and some of them arrive at the site of metastasis ahead of the cancer cells. The high radiosensitivity of the tumor-associated host cells lead us to speculate that radiotherapy may affect invasion and metastasis. Analgesic effects on bone metastasis and prevention of lung metastasis to the brain by ionizing radiation are possibly due to alterations of host cells. We suggest to consider tumour-associated host cells when developping new strategies for cancer radiotherapy, chemotherapy and surgery.

Invasion and MMP expression profile in desmoid tumours.

Desmoid tumours are locally invasive soft tissue tumours in which beta-catenin mediated TCF-dependent transcription is activated. The role of soluble factors secreted by the myofibroblastic desmoid tumour, which could stimulate tumour invasiveness, was investigated. Using collagen gel invasion assays, the presence of factors stimulating invasion in desmoid conditioned media (CM) could be established. Since matrix metalloproteinases (MMPs) have been implicated in the process of tumoral invasion, the expression levels of the MMP family members were evaluated. Quantitative reverse transcription-PCR was used to determine the expression levels of MMP1, MMP2, MMP3, MMP7, MMP11, MMP12, MMP13, MMP14 and the inhibitors TIMP1, TIMP2 and TIMP3. Besides overexpression of MMP7, a known TCF-dependent target gene, a striking upregulation of the expression levels of MMP1, MMP3, MMP11, MMP12 and MMP13 in desmoid tumours, compared to unaffected fibroblasts from the same patients, was found. Treating the CM of desmoids with a synthetic and a physiologic MMP inhibitor reduced the invasion-stimulating capacity of the desmoid CM by approximately 50%. These results suggest the involvement of soluble factors, released by the desmoid cells, in stimulating invasion and implicate the MMPs as facilitators of invasion.

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.

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.

8-Prenylnaringenin, the phytoestrogen in hops and beer, upregulates the function of the E-cadherin/catenin complex in human mammary carcinoma cells.

The E-cadherin/catenin complex is a powerful invasion suppressor in epithelial cells. It is expressed in the human MCF-7 breast cancer cell line family, but functionally defective in the invasive MCF-7/6 variant. Previous experiments have shown that IGF-I, tamoxifen, retinoic acid and tangeretin are able to upregulate the function of this complex in MCF-7/6 cells. We investigated the effect of 8-prenylnaringenin (8-PN), the phytoestrogen present in hops and beer, on aggregation, growth and invasion in MCF-7/6 cells. 8-PN was found to stimulate E-cadherin-dependent aggregation and growth of MCF-7/6 cells in suspension. These effects could be inhibited by the pure anti-estrogen ICI 182,780. 8-PN did not affect invasion of MCF-7/6 cells in the chick heart assay in vitro. In all these aspects 8-PN mimics the effects of 17beta-estradiol on MCF-7/6 cells.

Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer.

PURPOSE: p53 and E-cadherin mutations are associated with a high risk of metastatic potential and local recurrence after colorectal surgery. LoVo, a human colon cancer cell line expressing a wild-type p53 and a normal E-cadherin, was studied. Clone LoVo-XC17 was obtained from LoVo cells transfected with a vector bearing a p53 273his mutation. Clone LoVo-92R4 was obtained from LoVo by culture cells with an E-cadherin down-regulation. LoVo, LoVo-XC17, and LoVo-92R4 were studied for in vivo behavior in a surgical intracolonic graft model. METHODS: Ten nude mice were used per cell line. A colonic tumor was obtained by tumor implantation into the cecal wall. The cecal tumor was resected at Day 15; at this time the volumes of the different tumors were similar. RESULTS: Surgical resection of the LoVo tumor led to 100 percent disease-free animals at one month. Surgical resection of mice grafted with the LoVo-XC17 line did not cure any mice (0/10; P = 0.001). Mice had local recurrences (10/10), mesenteric lymph node metastases (9/10), liver metastases (2/10), and peritoneal carcinomatosis (8/10). Surgical resection of LoVo-92R4 tumors led to cures in 30 percent (3/10), whereas 70 percent had isolated mesenteric lymph node metastases (7/10; P = 0.003). CONCLUSION: In this model surgical tumor resection was consistently effective for colonic tumors with functional p53 and E-cadherin, it was consistently ineffective with tumors displaying a mutated p53, and it was partially effective with E-cadherin-deficient tumors. This study shows that the alteration of a single gene can be associated with a profound alteration of surgical resection benefit.

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.

Defective E-cadherin/catenin complexes in human cancer.

Cancer is caused by a series of genomic changes leading directly or indirectly to disturbance of growth, differentiation and tissue integrity. Genomic, transcriptional or posttranscriptional alterations of E-cadherin/catenin complexes that are implicated in various steps of cancer development comprise mutational inactivation, transcriptional downregulation of E-cadherin sometimes accompanied by upregulation of N-cadherin, proteolysis of E-cadherin and posttranslational stabilisation of beta-catenin and plakoglobin. The E-cadherin/catenin complex serves not only cell-cell adhesion but also transduces signals to the nucleus and to the cytoskeleton, either directly or through its connections with multiple other complexes. We review here the expression of E-cadherin/catenin in human cancers, emphasising methods of observation and prognostic interpretation of results. This is illustrated in thyroid lesions from the benign follicular adenoma to the extremely malignant anaplastic carcinoma. The eye is an organ largely neglected by students of cadherins and catenins. The implication of a variety of members of these molecular families in the embryonic development of the eye strongly suggests that disturbances of cadherin/catenin complexes are crucial also in the development of ocular tumours.

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.