The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

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.

Transcriptional activation of Odf2/Cenexin by cell cycle arrest and the stress activated signaling pathway (JNK pathway).

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.

Doxorubicin-induced activation of NF-κB in melanoma cells is abrogated by inhibition of IKKß, but not by a novel IKKα inhibitor.

Drug resistance is arguably the most important challenge in cancer therapy. Here, doxorubicin induced profound of NF-κB activation in melanoma cells with a maximum (3.5-fold) at concentrations relevant in vivo. This was followed by transcriptional induction of several gene products involved in tumor progression. A novel IKKα inhibitor (BAY32-5915) was identified and characterized, and doxorubicin-induced NF-κB activation was assessed following inhibition of IKKα or IKKß by small-molecular compounds. While the IKKα inhibitor did not affect doxorubicin-induced NF-κB activation, this process was completely abrogated when the IKKß inhibitor, KINK-1, was used. Moreover, inhibition of IKKß, but not IKKα, 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-κB activation, blocking IKKß, but not IKKα, by small molecules can antagonize this activity and, thus, increase chemosensitivity.

Stimulation of pulmonary immune responses by the TLR2/6 agonist MALP-2 and effect on melanoma metastasis to the lung.

Given that metastasized melanoma is a fatal disease in most cases, it is tempting to develop strategies to a priori prevent metastasis. We have stimulated the pulmonary innate immune system by macrophage-activating lipopeptide-2 (MALP-2), a specific agonist at Toll-like receptor (TLR) 2/6, and investigated its impact on experimental melanoma metastasis. In C57BL/6 mice, intratracheal application of MALP-2 induced a profound influx of neutrophils and macrophages into the lung, which peaked after 24 h (sixfold increase) and returned to baseline within 72 h. Further analysis revealed that MALP-2 also markedly induced VCAM-1 expression on pulmonary blood vessels. In vitro experiments demonstrated that this adhesion molecule mediates binding of B16F10 melanoma cells. Furthermore, in vivo or in vitro treatment with MALP-2 did not significantly affect the ability of immune cells to lyse melanoma cells. As a consequence, notwithstanding the profound pulmonary immune response induction and in contrast to conclusions drawn from some previous publications, the net extent of experimental metastasis did not change significantly, regardless of the application regimen of MALP-2 prior to, concomitant with or after tumor cell inoculation. Melanoma cells stably transfected with green fluorescent protein allowed tracking of early events after tumor cell dissemination and showed that MALP-2-mediated TLR2/6 activation did not interfere with pulmonary melanoma cell arrest. Likewise, boosting the immune induction after establishment of metastases did not change the clinical outcome. These unexpected results vividly counsel caution regarding predictions of immunomodulating therapies, as multiple intertwined effects may influence the net outcome.

NF-kappaB inhibition through proteasome inhibition or IKKbeta blockade increases the susceptibility of melanoma cells to cytostatic treatment through distinct pathways.

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.