Metastasized melanoma is almost universally resistant to chemotherapy. Given that constitutive or drug-induced upregulation of NF-kappaB activity is associated with this chemoresistance, NF-kappaB inhibition may increase the susceptibility to antitumoral therapy. On the cellular level, two principles of NF-kappaB inhibition, proteasome inhibition by bortezomib and IkappaB kinase-beta (IKKbeta) inhibition by the kinase inhibitor of NF-kappaB-1 (KINK-1), significantly increased the antitumoral efficacy of camptothecin. When combined with camptothecin, either of the two NF-kappaB-inhibiting principles synergistically influenced progression-related in vitro functions, including cell growth, apoptosis, and invasion through an artificial basement membrane. In addition, when C57BL/6 mice were intravenously injected with B16F10 melanoma cells, the combination of cytostatic treatment with either of the NF-kappaB-inhibiting compounds revealed significantly reduced pulmonary metastasis compared to either treatment alone. However, on the molecular level, nuclear translocation of p65, cell cycle analysis, and expression of NF-kappaB-dependent gene products disclosed distinctly different molecular mechanisms, resulting in the same functional effect. That proteasome inhibition and IKKbeta inhibition affect distinct molecular pathways downstream of NF-kappaB, both leading to increased chemosensitivity, is previously unreported. Thus, it is conceivable that switching the two principles of NF-kappaB inhibition, once resistance to one of the agents occurs, will improve future treatment regimens.

The centrosome/basal body protein ODF2/Cenexin is necessary for the formation of the primary cilium. Primary cilia are essential organelles that sense and transduce environmental signals. Primary cilia are therefore critical for embryonic and postnatal development as well as for tissue homeostasis in adulthood. Impaired function of primary cilia causes severe human diseases. ODF2 deficiency prevents formation of the primary cilium and is embryonically lethal. To explore the regulation of primary cilia formation we analyzed the promoter region of Odf2 and its transcriptional activity. In cycling cells, Odf2 transcription is depressed but becomes up-regulated in quiescent cells. Low transcriptional activity is mediated by sequences located upstream from the basal promoter, and neither transcription factors with predicted binding sites in the Odf2 promoter nor Rfx3 or Foxj, which are known to control ciliary gene expression, could activate Odf2 transcription. However, co-expression of either C/EBPα, c-Jun or c-Jun and its regulator MEKK1 enhances Odf2 transcription in cycling cells. Our results provide the first analysis of transcriptional regulation of a ciliary gene. Furthermore, we suggest that transcription of even more ciliary genes is largely inhibited in cycling cells but could be activated by cell cycle arrest and by the stress signaling JNK pathway.

Developing effective therapies against multiple myeloma (MM) is an unresolved challenge. Phosphatidylinositol-3-kinase (PI3K) activation may be associated with tumor progression and drug resistance, and inhibiting PI3K can induce apoptosis in MM cells. Thus, targeting of PI3K is predicted to increase the susceptibility of MM to anticancer therapy. The lead compound of a novel class of PI3K inhibitors, BAY80-6946 (IC50=0.5 nM against PI3K-α), was highly efficacious in four different MM cell lines, where it induced significant antitumoral effects in a dose-dependent manner. The compound inhibited cell cycle progression and increased apoptosis (P<0.001 compared with controls). Moreover, it abrogated the stimulation conferred by insulin-like growth-factor-1, a mechanism relevant for MM progression. These cellular effects were paralleled by decreased Akt phosphorylation, the main downstream target of PI3K. Likewise, profound antitumoral activity was observed ex vivo, as BAY80-6946 significantly inhibited proliferation of freshly isolated myeloma cells from three patients (P<0.001 compared with vehicle). In addition, BAY80-6946 showed convincing in vivo activity against the human AMO-1 and MOLP-8 myeloma cell lines in a preclinical murine xenograft model, where treatment with 6 mg/kg every other day for 2 weeks reduced the cell numbers by 87.0% and 69.3%, respectively (P<0.001 compared with vehicle), without overt toxicity in treated animals.

tissue samples. Thus, we found a strong correlation between the different preservation methods with correlation coefficients ranging from 0.91-0.95 (P < 0.001) (Fig. 1a-c). We observed that there was a weak pattern of clusters with higher correlation coefficients within each preservation method than within matched samples (Fig 2). This might indicate that the preservation method to some degree may affect the miRNA expression level and causes us to suggest that only a single preservation method should be considered for a certain experimental set-up. Even though more samples preferable should be run, the fact that the correlations also were observed when using alternative platforms as panel, and individual qRT-PCR confirms the consistency of the results and supports the robustness of miRNA detection in human skin. Recently, we showed that miRNAs obtained from FFPE tissue samples have a high diagnostic potential for skin diseases as we demonstrated that miRNAs can discriminate Cutaneous T-cell lymphomas (CTCL) from benign inflammatory skin diseases (24).Thus, miRNAs offer an appropriate and flexible approach in clinical practices and may hold great promise for biomarker and novel target discovery for skin diseases in the future. AcknowledgementsWe thank S. Boesen, M. M. Petersen and T. Gejsing for excellent technical assistance and N. Bangsgaard for help with psoriatic skin sample collection. This work was supported by The Danish Advanced Technology Foundation (http://www.mirskin.dk) and The Danish Agency for Science, Technology and Innovation. Author contributionsAll authors performed the research, analysed the data and wrote the article. J. R. Zibert, N. Ødum, M. A. Røpke and L. Skov designed the research study. 301 small-molecular compounds. While the IKKa inhibitor did not affect doxorubicin-induced NF-jB activation, this process was completely abrogated when the IKKb inhibitor, KINK-1, was used. Moreover, inhibition of IKKb, but not IKKa, led to significantly increased apoptosis in response to doxorubicin. Our results indicate that the net outcome of chemotherapy is difficult to predict and may even involve mechanisms conferring chemoresistance. In case of doxorubicin-induced NF-jB activation, blocking IKKb, but not IKKa, by small molecules can antagonize this activity and, thus, increase chemosensitivity. Conflict of interests Key words: chemoresistance -IKK -melanoma -NF-jB -small molecule Accepted for publication 3 January 2012Melanoma is an instructive model for drug resistance, because it is almost universally resistant to chemotherapeutics. Tumors may be intrinsically resistant or acquire resistance during therapy (1,2). NF-jB activation and subsequent transcription of many target genes have been implicated in tumor progression (3,4). The canonical NF-jB activation pathway employs the IjB kinase complex (IKK), consisting of the subunits IKKa, IKKb and IKKc ⁄ -NEMO (3). The non-canonical pathway involves NF-jB-inducing kinase (NIK), which phosphorylates an IKKa homodimer independent of the IKK complex (5...

Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.