Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP- 2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c- ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT- derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin- positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to he regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA- MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits

We have investigated the gelatinase profiles and invasiveness of clonal tumour sublines derived from a spontaneously arising mammary tumour in a Balb/cfC3H mouse. The 67NR, 66c14 and 4T1.2 sublines have low, intermediate and high metastatic potential respectively. In Boyden chamber studies, Matrigel invasion was seen to be progressively higher in the more metastatic lines 4T1.2>66c14>67NR, consistent with MMP-2 activation potential, MMP-9 secretion, and migration over either type I or IV collagen, which were low in both 67NR and 66c14 cells compared to 4T1.2 cells. These attributes are consistent with those seen in human breast cancer cell lines which appear to have undergone an epithelial-mesenchymal transition (EMT) as indicated by vimentin expression. We were, however, surprised to find vimentin expression, MT1-MMP expression and stellate Matrigel outgrowth in the non-invasive, non-metastatic 67NR cells, indicating that they had undergone an EMT despite not being invasive. We conclude that the EMT is manifested to differing degrees in these three clonal cell lines, and that the 67NR cells have either undergone a partial EMT or have since lost certain important attributes of the EMT-derived phenotype. This model should prove useful in further characterizing the regulation of MT1-MMP mediated MMP-2 activation and delineating the EMT in breast cancer progression.

EMT and MET in carcinoma : clinical observations, regulatory pathways and new models

The articles collected here in this special edition Epithelial-Mesenchymal (EMT) and Mesenchymal-Epithelial Transitions (MET) in Cancer provide a snapshot of the very rapidly progressing cinemascope of the involvement of these transitions in carcinoma progression. Pubmed analysis of EMT and cancer shows an exponential increase in the last few years in the number of papers and reviews published under these terms (Fig. 1). The last few years have seen these articles appearing in high calibre journals including Nature, Nature Cell Biology, Cancer Cell, PNAS, JNCI, JCI, and Cell, signaling the acceptance and quality of work in this field.

The epithelial to mesenchymal transition and metastatic progression in carcinoma

Many data have emerged in support of the concept that the promotion of a migratory phenotype in epithelial cells is the result of an EMT, which leads to the loss of differentiation and to the acquisition of several mesenchymal features. Such an event is likely to occur during the metastatic conversion enabling the metastatic cell to invade the connective tissue and disseminate. Even though it cannot be excluded that some cancer cells might constitutively express a metastatic phenotype, the EMT implicated in the tumor progression process would rather be transient, induced in certain cells of the primary tumor by different factors, and reversed when the cells settle down in secondary organs (as schematically represented in Figure 1). However, it is clear that the phenotype that would emerge from the EMT occurring during tumor progression would also be modulated by the genetic background of the cells and must be to some extent different than the ones observed in normal physiological processes, and must also vary from one tumor to another.

Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity

During invasion and metastasis, cancer cells interact closely with the extracellular matrix molecules by attachment, degradation, and migration. We demonstrated previously the local degradation of fluorescently labeled gelatin matrix by cancer cells at invasive membrane protrusions, called invadopodia. Using the newly developed quantitative fluorescence-activated cell sorting-phagocytosis assay and image analysis of localized degradation of fluorescently labeled matrix, we document here that degradation and site- specific removal of cross-linked gelatin matrix is correlated with the extent of phagocytosis in human breast cancer cells. A higher phagocytic capacity is generally associated with increasing invasiveness, documented in other invasion and motility assays as well. Gelatin phagocytosis is time and cell density dependent, and it is mediated by the actin cytoskeleton. Most of the intracellular gelatin is routed to actively acidified vesicles, as demonstrated by the fluorescent colocalization of gelatin with acidic vesicles, indicating the intracellular degradation of the phagocytosed matrix in lysosomes. We show here that normal intracellular routing is blocked after treatment with acidification inhibitors. In addition, the need for partial proteolytic degradation of the matrix prior to phagocytosis is demonstrated by the inhibition of gelatin phagocytosis with different serine and metalloproteinase inhibitors and its stimulation by conditioned medium containing the matrix metalloproteinases MMP-2 and MMP-9. Our results demonstrate that phagocytosis of extracellular matrix is an inherent feature of breast tumor cells that correlates with and may even directly contribute to their invasive capacity. This assay is useful for screening and evaluating potential anti-invasive agents because it is fast, reproducible, and versatile.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

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.

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.

Advanced mass spectrometry-based multi-omics technologies for exploring the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the primary hepatic malignancies and is the third most common cause of cancer related death worldwide. Although a wealth of knowledge has been gained concerning the initiation and progression of HCC over the last half century, efforts to improve our understanding of its pathogenesis at a molecular level are still greatly needed, to enable clinicians to enhance the standards of the current diagnosis and treatment of HCC. In the post-genome era, advanced mass spectrometry driven multi-omics technologies (e.g., profiling of DNA damage adducts, RNA modification profiling, proteomics, and metabolomics) stand at the interface between chemistry and biology, and have yielded valuable outcomes from the study of a diversity of complicated diseases. Particularly, these technologies are being broadly used to dissect various biological aspects of HCC with the purpose of biomarker discovery, interrogating pathogenesis as well as for therapeutic discovery. This proof of knowledge-based critical review aims at exploring the selected applications of those defined omics technologies in the HCC niche with an emphasis on translational applications driven by advanced mass spectrometry, toward the specific clinical use for HCC patients. This approach will enable the biomedical community, through both basic research and the clinical sciences, to enhance the applicability of mass spectrometry-based omics technologies in dissecting the pathogenesis of HCC and could lead to novel therapeutic discoveries for HCC.