The transcriptional activator nuclear factor kappa B (NF-B) is required for the upregulation of a large number of genes in response to inflammation, viral and bacterial infection, and other stress stimuli. Genes that respond to NF-B encode a variety of cytokines, cell adhesion molecules, and acute-phase response proteins as well as apoptotic suppressor and effector proteins. It is believed that this reprogramming of gene expression is essential for cell survival during situations of physiological crisis (61). The activation of NF-B in response to stimulation by the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin 1 beta (IL-1␤) has been extensively studied (17, 30); however, the mechanisms that modulate and eventually limit these responses are still poorly understood (61).We report here that the recently discovered protein kinase inhibitor protein RKIP (Raf kinase inhibitor protein) acts to inhibit NF-B activation. RKIP was first identified as an interacting partner of Raf-1 and shown to function as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by 76). The Raf-1-initiated pathway is comprised of three sequentially acting protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. This basic relationship has now been found to be conserved in several protein kinase pathways. In the Raf-1 pathway the MAPK is ERK1/2 (extracellular signal-regulated kinase 1 and 2), the MAPKK is MEK1 (MAP/ERK kinase 1), and the MAPKKK is Raf-1 itself. Functional studies using both gain-of-function and loss-of-function approaches demonstrated that RKIP disrupts the interaction between 76). Depletion of endogenous RKIP upregulated Raf-1 kinase activity and MAPK signaling, whereas ectopic expression of RKIP suppressed Raf-1 kinase activity and MAPK signaling as well as v-Raf-mediated transformation. Biochemical studies showed that RKIP efficiently dissociated preformed Raf/MEK complexes and behaved kinetically as a competitive inhibitor of MEK phosphorylation. In vivo, the association of endogenous RKIP with Raf-1 correlated inversely with Raf-1 kinase activity during serum stimulation of quiescent cells.Active NF-B is a dimer that can be assembled from several members of the Rel family of transcription factors, and some form of NF-B is expressed in most cell types (61). In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form bound to a family of inhibitory proteins known as IB (inhibitors of B). Activation of NF-B requires the phosphorylation and degradation of IB, which allows the NF-B dimer to translocate into the nucleus. Virtually all of the many stimuli that can activate NF-B cause the phosphorylation of IB on

The very process of deregulated oncogene expression during cancer development also sensitizes cancer cells to apoptotic signals (1-3). Deregulated oncoproteins such as E1a and c-Myc promote apoptosis by activating multiple downstream proapoptotic effector pathways (4, 5). Additional mechanisms of sensitizing cancer cells to apoptosis by an activated oncoprotein have been described (6, 7). For example, E2F sensitized cells to apoptosis through down-regulation of anti-apoptotic signals (7). Here we show that cancer cells can also be sensitized to apoptosis by up-regulating the expression levels of RKIP (Raf kinase inhibitor protein). RKIP was originally identified as an interacting partner of Raf-1 and a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (8). RKIP also inhibits nuclear factor B (NF-B)1 signaling by negatively modulating the activating phosphorylation of IKK␣ and IKK␤ via upstream kinases (9). Although the molecular mechanism by which RKIP inhibits the Raf and NF-B signaling pathways has been partially delineated, little is known about the biological relevance of the inhibition of these pathways by RKIP. In addition to these functions, we presently demonstrate the rapid up-regulation of RKIP during induction of chemotherapy-triggered apoptosis in human prostate and breast cancer cells. However, in DNA-damaging agent-resistant cancer cells, treatment with the drugs does not up-regulate RKIP expression. Ectopic expression of RKIP sensitizes DNA damage agentresistant cells to undergo apoptosis. Down-regulation of RKIP expression confers resistance to 9-nitrocamptothecin (9NC) by releasing its inhibitory constraint on two major survival pathways in cancer cells. Our studies suggest that RKIP represents a novel apoptotic marker in human cancer cells. MATERIALS AND METHODSCell Lines, Plasmid Constructs, and Chemicals-The human breast cell lines 578T and 578Bst were purchased from American Type Culture Collection (Manassas, VA). A human breast cancer MCF7 cell subline resistant to 9NC treatment was a gift from Dr. Ray Frackelton (Brown University). The human prostate cell lines LNCaP, DU145, and PC3 were purchased from American Type Culture Collection. Early (Ͻ30)-or late (Ͼ100)-passage DU145 cells were not used for this study. The 9NC-resistant DU145 cell subline, RC1, was established by continuous exposure of DU145 cells to 9NC (10). All cell lines were grown in conditions suggested by American Type Culture Collection. MCF7 and

Pancreatic cancer is an exceptionally aggressive disease in great need of more effective therapeutic options. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis and there is a gain of stem cell properties during EMT. Here we report increased expression of the putative pancreatic stem cell marker DCAMKL-1 in an established KRAS transgenic mouse model of pancreatic cancer and in human pancreatic adenocarcinoma. Co-localization of DCAMKL-1 with vimentin, a marker of mesenchymal lineage, along with 14-3-3 σ was observed within pre-malignant PanIN lesions that arise in the mouse model. siRNA-mediated knockdown of DCAMKL-1 in human pancreatic cancer cells induced microRNA miR-200a, an EMT inhibitor, along with down-regulation of EMT-associated transcription factors ZEB1, ZEB2, Snail, Slug and Twist. Furthermore, DCAMKL-1 knockdown resulted in downregulation of c-Myc and KRAS through a let-7a microRNA-dependent mechanism, and downregulation of Notch-1 through a miR-144 microRNA-dependent mechanism. These findings illustrate direct regulatory links between DCAMKL-1, microRNAs and EMT in pancreatic cancer. Moreover, they demonstrate a functional role for DCAMKL-1 in pancreatic cancer. Together, our results rationalize DCAMKL-1 as a therapeutic target for eradicating pancreatic cancers.