MCF-10A-NeoST: a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.

PURPOSE: There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. EXPERIMENTAL DESIGN: We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. RESULTS: NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in three-dimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, and EphA2 tyrosine kinases in transformed MCF-10A cells. CONCLUSIONS: MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.

Predicting the sites of metastases from lung cancer using molecular biologic markers.

BACKGROUND: The use of molecular markers in staging non-small cell lung cancer (NSCLC) has been supported in retrospective prognostic models but has not been evaluated in predicting sites of metastases. METHODS: Pathologic specimens were collected from 202 patients after complete resection for stage I NSCLC, who were subsequently found to have no metastases at 5 years (n = 108), isolated brain metastases (n = 25), or other distant metastases (n = 69). A panel of eight molecular markers of metastatic potential was chosen for immunohistochemical analysis of the tumor: p53, erbB2, angiogenesis factor viii, EphA2, E-cadherin, urokinase plasminogen activator (UPA), UPA receptor, and plasminogen activator inhibitor. RESULTS: Patients with isolated brain relapse had significantly higher expression of p53 (p = 0.02) and UPA (p = 0.002). The quantitative expression of E-cadherin was used to predict the site of metastases using recursive partitioning: 0 of 92 patients with E-cadherin expression of 0, 1, or 2 developed isolated cerebral metastases; 0 of 33 patients with E-cadherin expression of 3 with UPA of 1 or 2 and ErbB2 of 0 developed brain metastases. Of the remaining patients at risk (UPA = 3), the risk of isolated cerebral metastases was 21 of 57 patients (37%). CONCLUSIONS: This study demonstrates that molecular markers may predict the site of relapse in early stage NSCLC. If validated in an ongoing prospective study, these results could be used to select patients with isolated brain metastases for adjuvant therapy, such as prophylactic cranial irradiation.

Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.

EphA2 overexpression causes tumorigenesis of mammary epithelial cells.

Elevated levels of protein tyrosine phosphorylation contribute to a malignant phenotype, although the tyrosine kinases that are responsible for this signaling remain largely unknown. Here we report increased levels of the EphA2 (ECK) protein tyrosine kinase in clinical specimens and cell models of breast cancer. We also show that EphA2 overexpression is sufficient to confer malignant transformation and tumorigenic potential on nontransformed (MCF-10A) mammary epithelial cells. The transforming capacity of EphA2 is related to the failure of EphA2 to interact with its cell-attached ligands. Interestingly, stimulation of EphA2 reverses the malignant growth and invasiveness of EphA2-transformed cells. Taken together, these results identify EphA2 as a powerful oncoprotein in breast cancer.

TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1.

TEL is a member of the ETS family of transcription factors that interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute leukemia, and loss of heterozygosity at the TEL locus has been observed in various cancers. Here we show that expression of TEL in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and in normal cultures. Unexpectedly, cells expressing both Ras and TEL grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL bound sequences in the stromelysin-1 promoter and repressed the promoter in transient-expression assays, suggesting that it is a direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain failed to repress stromelysin-1. When BB-94, a matrix metalloproteinase inhibitor, was added to the culture medium of Ras-expressing cells, it caused a cell aggregation phenotype similar to that caused by TEL expression. In addition, TEL inhibited the invasiveness of Ras-transformed cells in vitro and in vivo. Our results suggest that TEL acts as a tumor suppressor, in part, by transcriptional repression of stromelysin-1.

Overexpression of the EphA2 tyrosine kinase in prostate cancer.

BACKGROUND: Molecules that are highly expressed by human prostate cancers may serve as therapeutically relevant targets or tumor markers. Tyrosine kinases are frequently overexpressed in metastatic tumor cells and this prompted us to screen for tyrosine kinases that are overexpressed in prostate cancer cells. METHODS: Expression levels of the EphA2 receptor tyrosine kinase were determined by Western blot analysis in canine and human prostate cancer cell lines and in immortalized and transformed variants of 267B1 prostatic epithelial cells. EphA2 levels in benign human prostate and prostate cancers were also determined in formalin-fixed, paraffin-embedded tissues using immunohistochemical staining. RESULTS: Metastatic prostate cancer cells overexpressed EphA2 by 10-100 fold as compared with non-invasive prostatic epithelial cells. EphA2 immunoreactivity in vivo was also significantly greater in human prostate cancers as compared with benign prostate epithelium. CONCLUSIONS: The EphA2 receptor tyrosine kinase is differentially expressed in human and canine prostate cancer cell lines and overexpressed in human prostate cancers as compared with benign prostate tissues. Metastasis-derived canine prostate carcinoma cell lines overexpress EphA2 and may provide pre-clinical models to further evaluate the role of EphA2 in prostate carcinogenesis. Further investigations are needed to determine the utility of EphA2 as a tumor marker and a novel target in human prostate cancer.

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.

The engagement of beta1 integrins on promonocytic cells promotes phosphorylation of Syk and formation of a protein complex containing Lyn and beta1 integrin.

The protein-tyrosine kinase Syk participates in signal transduction pathways downstream from multiple immune recognition receptors. Recent evidence indicates that Syk is also functionally coupled to cell surface integrins, which mediate interactions between the actin cytoskeleton and extracellular matrix proteins. The interactions of undifferentiated, promonocytic HL60 or U937 cells with fibronectin or anti-beta1 integrin antibodies leads to an apparent activation and tyrosine phosphorylation of Syk that is independent of tight cellular adhesion and spreading. In response to fibronectin or anti-beta1 integrin antibodies, beta1 integrins become associated with a complex of proteins that include the Lyn protein tyrosine kinase and endogenous kinase substrates of 29 and 75-80 kDa. Lyn becomes transiently activated following integrin engagement and co-localizes with the actin cytoskeleton. These studies suggest a major role for Lyn in coupling beta1 integrins to the activation of Syk.

Identification of tyrosine phosphorylated adhesion proteins in human cancer cells.

Tyrosine phosphorylation is a form of signal transduction that regulates cell growth, differentiation, migration, and survival. This knowledge has promoted much interest in the role of tyrosine kinases and phosphatases in regulating cell behavior during development and tumorigenesis. However, it is generally less well appreciated that tyrosine phosphorylated proteins are enriched within sites of cell adhesion, particularly in transformed cells. To identify these, we developed a panel of monoclonal antibodies specific for tyrosine phosphorylated proteins in breast cancer cells, using extensive modifications of existing technologies for immunization, somatic fusion, and antibody screening. Mice were immunized with a complex mixture of phosphotyrosine containing proteins using the newly developed RIMMS method. By increasing the sensitivity of antigen recognition, we isolated reagents specific for a wide diversity of tyrosine phosphorylated adhesion proteins in breast cancer cells.

Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells.

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell-cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

The Ras-related protein Rheb is farnesylated and antagonizes Ras signaling and transformation.

Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation.

E-cadherin engagement stimulates tyrosine phosphorylation.

Cadherins are cell adhesion molecules concentrated at intercellular adherens junctions, where they form a multiprotein complex with cytoplasmic catenins. Although cell-cell interactions affect many aspects of cell behavior, little is known about signaling pathways triggered by cadherin engagement. We show here that E-cadherin-mediated cell-cell adhesion leads to a rapid increase in tyrosine phosphorylation at sites of cell-cell contact and that this stimulation of tyrosine phosphorylation can be mimicked by aggregation of E-cadherin with antibodies. The proteins that become phosphorylated are distinct from those previously shown to be tyrosine phosphorylated in response to integrin-mediated adhesion and include ras-GAP. We also find that E-cadherin-mediated tyrosine phosphorylation is not required for the assembly of adherens-type junctions.

Overexpression of the Ras-related TC21/R-Ras2 protein may contribute to the development of human breast cancers.

Although experimental studies suggest that aberrant Ras function can promote the malignant progression of human breast epithelial cells, the occurrence of mutated ras genes in breast tumors is infrequent. One possible explanation for this apparent paradox is that aberrant function of the Ras-related protein TC21/R-Ras2, which causes malignant transformation of NIH 3T3 cells via upregulation of the Ras signal transduction pathway, may contribute to breast tumor development in the absence of Ras mutations. To address this possibility, we utilized two complementary approaches. First, we determined that aberrant TC21 function caused transformation of the MCF-10A human breast epithelial cell line. TC21-transformed MCF-10A cells exhibited altered cellular morphology associated with a disruption of cell-cell adherens junctions, formed colonies in soft agar, and showed enhanced motility in vitro. These alterations were similar to, but more dramatic than, those observed with oncogenic Ras-transformed MCF-10A cells. Furthermore, overexpression of normal TC21, but not Ras, also caused transformation of these cells. Second, we observed that TC21 protein expression was greatly elevated in 7 of 9 breast tumor lines when compared to untransformed MCF-10A cells. Taken together, these results support the possibility that overexpression of TC21 may contribute to aberrant growth properties of breast carcinoma cells.

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

The protein tyrosine kinase p56lck regulates cell adhesion mediated by CD4 and major histocompatibility complex class II proteins.

The CD4 protein is expressed on a subset of human T lymphocytes that recognize antigen in the context of major histocompatibility complex (MHC) class II molecules. Using Chinese hamster ovary (CHO) cells expressing human CD4, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In T lymphocytes, CD4 can also function as a signaling molecule, presumably through its intracellular association with p56lck, a member of the src family of protein tyrosine kinases. In the present report, we show that p56lck can affect cell adhesion mediated by CD4 and MHC class II molecules. The expression of wild-type p56lck in CHO-CD4 cells augments the binding of MHC class II+ B cells, whereas the expression of a mutant p56lck protein with elevated tyrosine kinase activity results in decreased binding of MHC class II+ B cells. Using site-specific mutants of p56lck, we demonstrate that the both the enzymatic activity of p56lck and its association with CD4 are required for this effect on CD4/MHC class II adhesion. Further, the binding of MHC class II+ B cells induces CD4 at the cell surface to become organized into structures resembling adhesions-type junctions. Both wild-type and mutant forms of p56lck influence CD4-mediated adhesion by regulating the formation of these structures. The wild-type lck protein enhances CD4/MHC class II adhesion by augmenting the formation of CD4-associated adherens junctions whereas the elevated tyrosine kinase activity of the mutant p56lck decreases CD4-mediated cell adhesion by preventing the formation of these structures.