Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Expression of 67 kDa laminin receptor in human breast cancer cells : regulation by progestins

The level of 67 kDa laminin receptor (67LR) expression on breast and colon tumor cell surfaces was previously shown to be correlated with the capacity of tumor cells to metastasize. In the present work we investigate the effects of progestins and estrogen on the expression of 67LR in two sublines of the T47D human breast cancer cells: weakly tumorigenic, poorly invasive parental T47D cells and a highly tumorigenic, more invasive T47Dco subclone. Inmmunoblotting with an affinity purified antibody directed against a synthetic peptide recognizes the 67LR in these cells. 67LR expression in the T47Dco subclone is 5,5-fold higher than in their parental T47D cells. Treatment of T47D cells with 1 nM of the synthetic progestin R5020 results in a 4-fold increase in 67LR protein expression. Estrogen also induced 67LR expression, but only by 1.5-fold. The progestin-stimulated expression of the 67LR correlates with a 4.3-fold increase in attachment of T47D cells to laminin. A monoclonal antibody, mAb 13, directed against β1 integrin, completely blocks the attachment of T47D cells to fibronectin, only partially inhibits the attachment of T47D cells to laminin, and appears not to affect the progestin-stimulated laminin attachment of T47D cells. A new antiprogestin, ZK 112.993, significantly inhibits both progestin-stimulated 67LR expression and the increased attachment to laminin. These results suggest a possible role for progestin in mediating one of the multiple events thought to be important in metastasis of steroid receptor positive human breast cancer cells.

Epithelial mesenchymal transition traits in human breast cancer cell lines

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

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.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice : real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

Elevated cyclic AMP suppresses ConA-induced MT1-MMP expression in MDA- MB-231 human breast cancer cells

We have previously reported that induction of MMP-2 activation by Concanavalin A (ConA) in MDA-MB-231 human breast cancer cells involves both transcriptional and post-transcriptional mechanisms, and that the continuous presence of ConA is required for MMP-2 activation (Yu et al. Cancer Res, 55, 3272-7, 1995). In an effort to identify signal transduction pathways which may either contribute to or modulate this mechanism, we found that three different cAMP-inducing agents, cholera toxin (CT), forskolin (FSK), and 3- isobutyl-1-methylxanthine (IBMX) partially inhibited ConA-induced MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. Combinations of CT or FSK with IBMX exhibited additive effects on reduction of MT1-MMP mRNA expression and MMP-2 activation. Agents which increase cAMP levels appeared to target transcriptional aspects of ConA induction, reducing MT1-MMP mRNA and protein in parallel with the reduced MMP-2 activation. In the absence of ConA, down-regulation of constitutive production of MT1-MMP mRNA and protein was observed, indicating that cAMP acts independently of ConA. These observations may help to elucidate factors regulating MT1-MMP expression, which may be pivotal to the elaboration of invasive machinery on the cell surface.

Vimentin and epithelial-mesenchymal transition in human breast cancer : observations in vitro and in vivo

Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within these tumours is used for prognosis and selection of therapies, there is a continuing need for additional markers to be identified. A considerable amount of current literature, based predominantly on cell culture systems, suggests that a major mechanism responsible for the progression of breast cancer is due to tumour cells losing their epithelial features and gaining mesenchymal properties. These events are proposed to be very similar to the epithelial-mesenchymal transition (EMT) process that has been well characterised in embryonic development. For the developmental and putative cancer EMT, the cell intermediate filament status changes from a keratin-rich network which connects to adherens junctions and hemidesmosomes, to a vimentin-rich network connecting to focal adhesions. This review summarises observations of vimentin expression in breast cancer model systems, and discusses the potential role of EMT in human breast cancer progression, and the prognostic usefulness of vimentin expression.

Disparate companions : tissue engineering meets cancer research

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Isolation and characterization of tumor cells from the ascites of ovarian cancer patients : molecular phenotype of chemoresistant ovarian tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.