Isogenic human mammary epithelial cell lines: novel tools for target identification and validation. Comprehensive characterization of an isogenic human mammary epithelial cell model provides evidence for epithelial-mesenchymal transition.

Tumorigenesis is a multi-step process involving several consecutive genetic alterations resulting in loss of genomic stability and deregulated signal transduction pathways. To study these deregulated processes in vitro, typically established cancer cell lines derived from primary tumors, ascites, or from metastases are used. However, these cancer cell lines reflect only late stages of the tumorigenic process. To better understand the consequences of the sequential genetic alterations in an in vitro model system, we applied consecutive immortalization and transformation of primary human mammary epithelial cells (HMECs) combining shRNA-mediated knockdown of tumor suppressor genes and overexpression of oncogenes. Thereby, we developed a panel of isogenic HMEC-derived cell lines reflecting the multi-step process of tumorigenesis. The immortalized cell lines have a normal epithelial morphology and proliferate indefinitely and anchorage-dependently. In contrast, the transformed cells exhibit mesenchymal-like morphological changes and strong colony-forming activity in soft agar. SNP array analysis showed that none of the cell lines displayed gross chromosomal aberrations in 80 % of the chromosomes. However, massive changes were observed in some chromosomes of the transformed cells indicating that the transformed phenotype is characterized by chromosomal alterations. The isogenic immortalized and transformed cells described here provide a powerful tool for the in vitro validation of target genes for cancer therapy.

RNA interference targeting protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta suppresses glioblastoma growth in vitro and in vivo.

The protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) are overexpressed in human glioblastomas. Both molecules are involved in neuronal cell migration during CNS development. In addition, PTN can induce glioma cell migration which is at least in part mediated through binding to PTPzeta/RPTPbeta. To study the relevance of this ligand-receptor pair for glioma growth in vitro and in vivo, we transfected the human glioblastoma cell line U251-MG with small interfering RNA (siRNA) directed against PTPzeta/RPTPbeta. Stable siRNA transfection resulted in strong down-regulation of PTPzeta/RPTPbeta expression. When injected subcutaneously into nude mice, clones that expressed normal levels of PTPzeta/RPTPbeta (PTPzeta + clones) formed exponentially growing tumours, whereas tumour growth was almost completely abrogated for clones that expressed reduced PTPzeta/RPTPbeta levels (PTPzeta - clones). Similar results were obtained using an orthotopic intracerebral model. Proliferation of PTPzeta - cells in vitro was significantly reduced compared with that of control clones. Matrix-immobilized PTN stimulated the proliferation of PTPzeta + cells but not of PTPzeta - cells. Haptotactic migration induced by PTN was reduced for PTPzeta - clones compared with control clones. Our findings suggest that antagonization of PTPzeta/RPTPbeta expression can inhibit glioma growth in vivo and may thus represent a potentially promising treatment strategy.

Contactin is expressed in human astrocytic gliomas and mediates repulsive effects.

Contactin is a cell surface adhesion molecule that is normally expressed by neurons and oligodendrocytes. Particularly high levels of contactin are present during brain development. Using subtractive cloning, we identified contactin transcripts as overexpressed in glioblastomas compared with normal brain. We confirmed contactin overexpression in glioblastomas at the protein level, and localized contactin to the surface of glial fibrillary acidic protein (GFAP)-expressing glioblastoma cells. In contrast, normal astrocytes did not express contactin. Analyzing different types of astrocytic tumors, we detected an association between increasing malignancy grade and contactin expression. Functionally, contactin had repellent effects on glioma cells in vitro, as demonstrated by adhesion and migration assays. Overexpression of contactin by transfection into glioblastoma cells did not alter the proliferation rate or adhesion to various extracellular matrix proteins as well as adhesion to cells expressing the specific contactin ligand the protein tyrosine phosphatase zeta (PTPzeta). Our findings suggest that contactin has repellent effects on glioma cells to which it is presented as a ligand, but it does not alter the proliferative or adhesive capacities of cells that overexpress the molecule. The repulsive properties of contactin may be a key factor in glioma disaggregation, and may contribute to the diffuse infiltration pattern characteristic of glioma cells in human brain.

Expression and function of the receptor protein tyrosine phosphatase zeta and its ligand pleiotrophin in human astrocytomas.

Using subtractive cloning combined with cDNA array analysis, we previously identified the genes encoding for the protein tyrosine phosphatase zeta/receptor-type protein tyrosine phosphatase beta (PTPzeta/RPTPbeta) and its ligand pleiotrophin (PTN) as overexpressed in human glioblastomas compared to normal brain. Both molecules have been implicated in neuronal migration during central nervous system development, and PTN is known to be involved in tumor growth and angiogenesis. We confirm overexpression of both molecules at the protein level in astrocytic gliomas of different malignancy grades. PTPzeta/RPTPbeta immunoreactivity was associated with increasing malignancy grade and localized predominantly to the tumor cells. PTN immunoreactivity as determined by ELISA and immunohistochemistry analysis was increased in low-grade astrocytomas compared to normal brain. Further increase in malignant gliomas was marginal, and thus no correlation with malignancy grade or microvessel density was present. However, PTN levels were significantly associated with those of fibroblast growth factor-2, suggesting co-regulation of both factors. Functionally, PTN induced weak chemotactic and strong haptotactic migration of glioblastoma and cerebral microvascular endothelial cells. Haptotaxis of glioblastoma cells towards PTN was specifically inhibited by an anti-PTPzeta/RPTPbeta antibody. Our findings suggest that upregulated expression of PTN and PTPzeta/RPTPbeta in human astrocytic tumor cells can create an autocrine loop that is important for glioma cell migration. Although PTN is a secreted growth factor, it appears to exert its mitogenic effects mostly in a matrix-immobilized form, serving as a substrate for migrating tumor cells.

A role for receptor tyrosine phosphatase zeta in glioma cell migration.

Glioblastomas (GBM) are the most frequent and malignant human brain tumor type. Typically striking in adulthood, tumor progression is rapid, relentless, and ultimately leads to the patient's death within a year of diagnosis. The identification of transcriptionally regulated genes can lead to the discovery of targets for antibody or small-molecule-mediated therapy, as well as diagnostic markers. We prepared cDNA arrays that are specifically enriched for genes expressed in human brain tumors and profiled gene expression patterns in 14 individual tumor samples. Out of 25,000 clones arrayed, greater than 200 genes were found transcriptionally induced in glioblastomas compared to normal human brain tissue including the receptor tyrosine phosphatasezeta (RPTPzeta) and one of its ligands, pleiotrophin (Ptn). We confirmed by Northern blot analysis and immunohistochemistry that RPTPzeta is enriched in tumor samples. Knockdown of RPTPzeta by RNA interference studies established a functional role of RPTPzeta in cell migration. Our results suggest a novel function for RPTPzeta in regulating glioblastoma cell motility and point to the therapeutic utility of RPTPzeta as a target for antibody-mediated therapy of brain tumors.

Glioblastoma and cerebral microvascular endothelial cell migration in response to tumor-associated growth factors.

OBJECTIVE: Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another. METHODS: Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles. RESULTS: SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold). CONCLUSION: The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

Levels of soluble vascular endothelial growth factor (VEGF) receptor 1 in astrocytic tumors and its relation to malignancy, vascularity, and VEGF-A.

PURPOSE: Vascular endothelial growth factor (VEGF)-A isa key mediator of angiogenesis in malignant gliomas. Soluble VEGF receptor 1 (sVEGFR-1) can complex VEGF-A and reduce its bioavailability. In several animal models sVEGFR-1 inhibited angiogenesis and tumor growth. We analyzed the levels of endogenous sVEGFR-1 in gliomas of different malignancy grades in relation to tumor vascularity and VEGF-A. EXPERIMENTAL DESIGN: The concentration of sVEGFR-1 was determined by ELISA in 104 gliomas and normal brain. Levels of sVEGFR-1 were compared with malignancy grade, microvessel density, and VEGF-A concentration. Effects of sVEGFR-1 on glioma extract-induced endothelial cell chemotaxis were analyzed in vitro. RESULTS: The concentration of sVEGFR-1 correlated with the malignancy grade and was 12-fold higher in glioblastomas than in diffuse astrocytomas (P < 0.001), with intermediate levels for anaplastic astrocytomas. VEGF-A levels were 30-fold higher (P < 0.001) in glioblastomas than in diffuse astrocytomas. The sVEGFR-1:VEGF-A ratio was 0.27 in glioblastomas and 0.70 in diffuse astrocytomas. Both sVEGFR-1 and VEGF-A correlated with microvessel density (P < 0.001) and with each other (P < 0.001); sVEGFR-1 and VEGF-A also correlated with each other when only glioblastomas were analyzed (P = 0.001). In vitro, recombinant sVEGFR-1 inhibited endothelial cell chemotaxis induced by tumor extracts. CONCLUSIONS: Although absolute levels of sVEGFR-1 are increased in the more malignant gliomas, the sVEGFR-1:VEGF-A ratio is decreased 2.6-fold in glioblastomas compared with diffuse astrocytomas, suggesting that the ensuing increased bioavailability of VEGF-A favors angiogenesis. The inhibition of tumor extract-induced endothelial chemotaxis by sVEGFR-1 suggests that sVEGFR-1 could be useful as an angiogenesis inhibitor in the specific context of human gliomas.