Cell adhesion molecule uvomorulin expression in human breast cancer cell lines : relationship to morphology and invasive capacities

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/ VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and β-catenin, localized increased levels of vimentin and redistribution of α6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.

MTI-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression

Membrane type 1 metalloprotease (MT1-MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro-MMP2. We investigated the effects of MT1-MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1-MMP or MMP-2. MT1-MMP and MMP-2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1-MMP 1) were able to activate endogenous or exogenous pro-MMP-2, 2) displayed an enhanced in vitro invasiveness through matrigel-coated filters independent of MMP-2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutanously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1-MMP regardless of MMP-2 expression levels, suggesting that the production of MMP-2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1-MMP-producing cells was associated with an up-regulation of VEGF expression. These results emphasize the importance of MT1-MMP during tumor angiogenesis and open new opportunities for the development of antiangiogenic strategies combining inhibitors of MT1-MMP and VEGF antagonists. - Sounni, N. E., Devy, L., Hajitou, A., Frankenne, F., Munaut, C., Gilles, C., Deroanne, C., Thompson, E. W., Foidart, J. M., Noel, A. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression.

High level of MT-MMP expression is associated with invasiveness of cervical cancer cells

MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-β, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymphnode metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice

Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Expression of β1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity

Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 μg/ml) of anti-β1 integrin antibody. The amounts of total cellular β1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of β1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more β1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of β1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells.

Multiplexed tandem polymerase chain reaction identifies strong expression of oestrogen receptor and Her-2 from single, formalin-fixed, paraffin-embedded breast cancer sections

Aim To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section. Methods Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10μm FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results. Results MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR. Conclusion MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Vimentin expression in cervical carcinomas : association with invasive and migratory potential

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines. In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB- 231, MDA-MB-436, BT549, MCF-7(AOR), Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

Gelatinase A (MMP-2) activation by skin fibroblasts : dependence on MT1-MMP expression and fibrillar collagen form

The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 μg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Engineering salinity tolerance in rice by exogenous expression of cell death regulators

Rice, an important crop that feeds more than half of the world's population is very sensitive to salinity stress – a growing problem affecting crop production globally. This PhD study addressed this problem by manipulating the programmed cell death pathways in rice resulting in significant enhancement of salinity stress tolerance. The impact of this work is that farmers would be in a position to grow rice containing such a trait in environments where salinisation of the soil exists, thereby addressing food security needs.