Mitogen-activated protein kinase phosphatase (MKP)1 -1 is a dual specificity phosphatase that is overexpressed in many human tumors and protects cells from apoptosis by the anticancer agent cisplatin (1), UV irradiation (2), and proteasome inhibitors (3). Over the past five years, reports have become more numerous describing high levels of MKP-1 in human tumors. For example, high levels of MKP-1 have been found in prostate (4), gastric (5), breast (6), and pancreatic cancer (7). In ovarian cancer samples, MKP-1 expression was correlated with decreased progression-free survival (8). High levels of MKP-1 expression were also found in the early phases of prostate, colon, and bladder carcinogenesis (9). Evidence that MKP-1 may actually support the transformed phenotype comes from a recent study by Liao et al. (7) who showed that PANC-1 human pancreatic cancer cells stably transfected with a full-length MKP-1 antisense construct had longer doubling times, decreased ability to form colonies in soft agar, and were unable to form tumors in nude mice. The precise mechanism by which loss of MKP-1 expression affects tumorigenicity, however, remains elusive. Several reports have implicated JNK/SAPK and p38 as the primary mediators of MKP-1 mediated cytoprotection (1-2). On the other hand, the data presented by Liao et al. (7) argue that the primary mechanism by which MKP-1 supports the transformed phenotype is mediated by ERK, but not JNK, as suppression of MKP-1 expression by antisense did not affect basal levels of phospho-JNK or phospho-p38, but instead increased basal ERK phosphorylation and prolonged ERK phosphorylation after epidermal growth factor stimulation in PANC-1 cells.The availability of a cell-active selective MKP-1 inhibitor would be a valuable tool for dissecting the complex regulatory processes involved in the attenuation of ERK, JNK, and p38 activation and for defining the contributions of MKP-1 and its cellular targets to the maintenance of the transformed phenotype. In light of recent findings, inhibitors of MKP-1 might also find applications as novel target-based antineoplastic therapies, either alone or in combination with clinically used antineoplastic agents (1-3).The search for MKP-1 inhibitors has been challenging for several reasons. In contrast to MKP-3, whose crystal structure has been reported and found to possess high structural similarity to the related VHR in its catalytic domain (10), no structural information is available for MKP-1. This may in part be due to difficulties in producing large amounts of recombinant enzyme and the need of MKPs to interact with their physiolog-

Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinonebased, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC 50 of 13 Mmol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxeltreated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a welldefined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression. [Mol Cancer Ther 2008;7(2):330 -40]

Kaposi's sarcoma (KS) associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) K13 is a potent activator of the NF-κB pathway. Here we demonstrate that infection with KHSV and ectopic expression of K13, but not its NF-κB-defective mutant, suppressed the expression of CXCR4. Suppression of CXCR4 by KSHV and K13 was associated with upregulated expression of miR-146a, a microRNA that is known to bind to the 3′ untranslated region of CXCR4 mRNA. Reporter studies identified two NF-κB sites in the promoter of miR-146a that were essential for its activation by K13. Accordingly, ectopic expression of K13, but not its NF-κB-defective mutant or other vFLIPs, strongly stimulated the miR-146a promoter activity, which could be blocked by specific genetic and pharmacological inhibitors of the NF-κB pathway. Finally, expression of CXCR4 was downregulated in clinical samples of KS and this was accompanied by increased expression of miR-146a. Our results demonstrate that K13-induced NF-κB activity suppresses CXCR4 via upregulation of miR-146a. Downregulation of CXCR4 expression by K13 may contribute to KS development by promoting premature release of KSHV-infected endothelial progenitors into the circulation.