Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Induction of tumorigenicity by galectin-3 in a nontumorigenic human breast-carcinoma cell-line

The human galectin-3 is a galactoside-binding protein of 31 kDa which functions as a receptor for glycoproteins containing poly N-acetyllactosamine side chains and as a substrate for matrix metalloproteinases-2 and -9. We studied its expression by flow cytofluorimetry, Western, Northern and Southern analyses, in five cultured human breast carcinoma cell lines previously characterized as non-tumorigenic, poorly metastatic or metastatic in nude mice. The expression of galectin-3 correlated with the reported tumorigenicity of the cells. The introduction of recombinant galectin-3 into the null expressing non-tumorigenic BT-549 cells resulted in the acquisition of anchorage-independent growth properties in alland tumorigenicity in 3/4 sense transfected cell crones. The data indicate a relationship between galectin-3 expression and malignancy of human breast carcinoma cell lines.

GATA3 : a promising marker for metastatic breast carcinoma in serous effusion specimens

Background The utility of GATA3 as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. Methods Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from breast (30), female genital tract (13), gastrointestinal tract (7), lung adenocarcinoma (9), pancreas (1), kidney (1) and bladder (1). The breast carcinoma cases included 15 ductal carcinomas, 8 lobular carcinomas and in 7 the histology sub-type was not available. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and GCDFP-15 to compare sensitivity with GATA3. Results Positive nuclear staining for GATA3 was present in 90% (27/30) of metastatic breast carcinoma specimens. All non-breast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases showed weak positivity of benign lymphoid cells. Staining results were unambiguous as all positive cases showed intense nuclear staining in >50% of tumor cells. Mammaglobin (57%; 17/30) and GCDFP-15 (33%; 10/30) were less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only one additional case to those stained with GATA3. Conclusions GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration.

The inter-relationships between ovarian-independent growth, tumorigenicity, invasiveness and antioestrogen resistance in the malignant progression of human breast cancer

Among the processes contributing to the progressive acquisition of the highly malignant phenotype in breast cancer are ovarian-independent growth, antioestrogen resistance and increased metastatic potential. We have previously observed that increased invasiveness and development of ovarian-independent growth occur independently. In an attempt to define the inter-relationships between these processes further, we have compared the phenotypes of ovarian-independent, invasive and antioestrogen-resistant sublines of the ovarian-dependent human breast cancer cell line MCF-7. Cells acquiring ovarian-independent growth can retain sensitivity to anti-oestrogens. One clone of MCF-7 cells selected for stable antioestrogen resistance has become non-tumorigenic but its invasive potential remains unaltered. Thus, acquisitions of some characteristics of the progressed phenotype can occur independently. This phenomenon of independent parameters in phenotypic progression could partly explain the considerable intra- and intertumour heterogeneity characteristic of breast tumours.

Progression of human breast cancer cells from hormone-dependent to hormone-independent growth both in vitro and in vivo

We have isolated a series of sublines of the hormone-dependent MCF-7 human breast cancer cell line after selection both in vivo and in vitro for growth in the presence of subphysiological concentrations of estrogens. These sublines represent a model system for study of the processes leading to hormonal autonomy. The cells form growing tumors in ovariectomized athymic nude mice in the absence of estrogen supplementation but retain some responsivity to estrogen as determined by stimulation of the rate of tumor growth in vivo and by induction of progesterone receptor. An ovarian-independent but hormone-responsive phenotype may occur early in the natural progression to hormone-independent and unresponsive growth in breast cancer. We observed no change in the affinity or decrease in the level of expression of estrogen receptors and progesterone receptors among the sublines and the parental cells. Epidermal growth factor receptors are not overexpressed in ovarian-independent cells. Thus, altered hormone receptor expression may be a late event in the acquisition of a hormone-independent and unresponsive phenotype. Sublines isolated by in vivo but not in vitro selection are more invasive than the parental cells both in vivo and across an artificial basement membrane in vitro. Thus, as yet unknown tumor-host interactions may be important in the development of an invasive phenotype. Furthermore, acquisition of the ovarian-independent and invasive phenotypes can occur independently.

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.

A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer

Epithelial-to-mesenchymal transition (EMT) processes endow epithelial cells with enhanced migratory/invasive properties and are therefore likely to contribute to tumor invasion and metastatic spread. Because of the difficulty in following EMT processes in human tumors, we have developed and characterized an animal model with transplantable human breast tumor cells (MDA-MB-468) uniquely showing spontaneous EMT events to occur. Using vimentin as a marker of EMT, heterogeneity was revealed in the primary MDA-MB-468 xenografts with vimentin-negative and vimentin-positive areas, as also observed on clinical human invasive breast tumor specimens. Reverse transcriptase-PCR after microdissection of these populations from the xenografts revealed EMT traits in the vimentin-positive zones characterized by enhanced 'mesenchymal gene' expression (Snail, Slug and fibroblast-specific protein-1) and diminished expression of epithelial molecules (E-cadherin, ZO-3 and JAM-A). Circulating tumor cells (CTCs) were detected in the blood as soon as 8 days after s.c. injection, and lung metastases developed in all animals injected as examined by in vivo imaging analyses and histology. High levels of vimentin RNA were detected in CTCs by reverse transcriptase-quantitative PCR as well as, to a lesser extent, Snail and Slug RNA. Von Willebrand Factor/vimentin double immunostainings further showed that tumor cells in vascular tumoral emboli all expressed vimentin. Tumoral emboli in the lungs also expressed vimentin whereas macrometastases displayed heterogenous vimentin expression, as seen in the primary xenografts. In conclusion, our data uniquely demonstrate in an in vivo context that EMT occurs in the primary tumors, and associates with an enhanced ability to intravasate and generate CTCs. They further suggest that mesenchymal-to-epithelial phenomena occur in secondary organs, facilitating the metastatic growth.

Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44HI/CD24lO/-stem cell phenotype in human breast cancer

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44highCD24low. Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.

Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focusses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

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.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by αVβ3 integrin in human breast cancer cells

The influence of αVβ3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing β3 integrin status. Overexpression of β3 integrin caused increased cell surface expression of αV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. β3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, αVβ3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of β3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with β3 integrin expression. Although our studies confirm important biological effects of αVβ3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, β3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by αVβ3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.