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.

Interactions between receptor tyrosine kinases of the Eph family and their ligands, ephrins, are implicated in establishment of organ boundaries and repulsive guidance of cell migration during development, but the mechanisms by which this is achieved are unclear. Here we show that activation of endogenous EphA2 kinase induces an inactive conformation of integrins and inhibits cell spreading, migration and integrin-mediated adhesion. Moreover, EphA2 is constitutively associated with focal-adhesion kinase (FAK) in resting cells. Within one minute after stimulation of EphA2 with its ligand, ephrin-A1, the protein tyrosine phosphatase SHP2 is recruited to EphA2; this is followed by dephosphorylation of FAK and paxillin, and dissociation of the FAK-EphA2 complex. We conclude that Eph kinases mediate some of their functions by negatively regulating integrins and FAK.

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Abstract. Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-ceU 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 B-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including [3-catenin and p120 Cas. Whereas in the normal cells [3-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 [3-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated [3-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 13-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 [3-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

Purpose: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study.Experimental Design: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables.Results: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P ‫؍‬ 0.02) and advanced stage of disease (P ‫؍‬ 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P ‫؍‬ 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival.Conclusions: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.

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. Prostate 41:275–280, 1999. © 1999 Wiley‐Liss, Inc.

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.Mutated forms of the three ras genes (H-, K-, and N-ras) are associated with 30% of all human cancers and encode potent transforming and oncogenic mutant proteins (1). Normal Ras proteins function as GDP/GTP-regulated molecular switches (2). Guanine nucleotide exchange factors (SOS and RasGRF/ CDC25) promote formation of the active, GTP-bound state (2-4), whereas GTPase activating proteins (p120-and NF1-GTPase activating proteins) promote formation of inactive, GDP-bound Ras (5). Mutated Ras proteins contain single amino acid substitutions (at residues 12, 13, or 61) that render the proteins insensitive to GTPase activating protein stimulation and, consequently, persist as constitutively activated proteins. Ras proteins serve as key intermediate relay switches in diverse signaling pathways that control cell growth and differentiation (6 -8). Consequently, mutated Ras proteins cause constitutive, ligand-independent activation of these pathways, thereby promoting to the aberrant growth of tumor cells.Ras proteins are prototypes for a large superfamily of Rasrelated proteins (Ͼ60 mammalian members) that function as GDP/GTP-regulated molecular switches (2, 6, 9). However, despite their strong amino acid sequence identity with Ras proteins (30 -55%), the majority of these small GTPases lack the potent transforming potential of Ras proteins. Exceptions include TC21/R-Ras2 (10, 11), R-Ras (12, 13), RhoA (14 -18), RhoB (19), and Rac1 (17,20), where constitutively activated versions of these Ras-related proteins can cause tumorigenic transformation of NIH 3T3 cells. The transforming activities of TC21 and R-Ras reflect the fact that these two Ras-...