Margarete Schön

Inhibition of Platelet GPIbα and Promotion of Melanoma Metastasis

Platelet glycoprotein Ibalpha (GPIb alpha) is part of the receptor complex GPIb-V-IX, which has a critical role in hemostasis, especially through interactions with the subendothelial von Willebrand factor. As there is accumulating evidence for a contribution of platelet receptors to hematogenous tumor metastasis, GPIb alpha is an interesting molecule to study in this context. We have investigated the effect of GPIb alpha inhibition by monovalent Fab fragments on experimental pulmonary metastasis in a syngeneic mouse model using C57BL/6 mice and B16F10 melanoma cells. The early fate of green fluorescent protein (GFP)-transfected melanoma cells under GPIb alpha blockade was also assessed, as was the effect of GPIb alpha inhibition on pulmonary metastasis in mice lacking P-selectin. Surprisingly and, to our knowledge previously unreported, GPIb alpha inhibition led to a significant increase in pulmonary metastasis, and assessment of the early fate of circulating GFP-labeled B16F10 showed improved survival and pulmonary arrest of tumor cells shortly after GPIb alpha inhibition, indicating that inhibition of a platelet protein can, in some cases, promote metastasis of a malignant tumor. In contrast, GPIb alpha blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIb alpha in the initial, P-selectin-dependent steps of metastasis. These findings suggest that GPIb alpha contributes to the control of tumor metastasis, in addition to its role in hemostasis.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

Significance Our results imply a direct impact of the p38–MAP kinase-activated protein kinase 2 (MK2) kinase pathway on the cellular response to replicative stress. In this situation, MK2 activity determines the decision between replication fork stalling and translesion synthesis. In the absence of MK2 activity, even the otherwise essential checkpoint kinase Chk1 becomes dispensable for S phase progression and cell survival. Moreover, MK2 represents a determinant of cancer cell sensitivity toward nucleoside analogue treatment. DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.

NF-κB Inhibition through Proteasome Inhibition or IKKβ 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.

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

Abstract:u2002 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 IKKβ by small‐molecular compounds. While the IKKα inhibitor did not affect doxorubicin‐induced NF‐κB activation, this process was completely abrogated when the IKKβ inhibitor, KINK‐1, was used. Moreover, inhibition of IKKβ, 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 IKKβ, but not IKKα, by small molecules can antagonize this activity and, thus, increase chemosensitivity.

Inositol-C2-PAF down-regulates components of the antigen presentation machinery in a 2D-model of epidermal inflammation

In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis.

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

Abstract:u2002 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 24u2003h (sixfold increase) and returned to baseline within 72u2003h. 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.

A pro-apoptotic function of iASPP by stabilizing p300 and CBP through inhibition of BRMS1 E3 ubiquitin ligase activity

The p53 family and its cofactors are potent inducers of apoptosis and form a barrier to cancer. Here, we investigated the impact of the supposedly inhibitory member of the apoptosis-stimulating protein of p53, iASPP, on the activity of the p53 homolog TAp73, and its cofactors p300 and CBP. We found that iASPP interacted with and stabilized the histone acetyltransferase p300 and its homolog CBP upon cisplatin treatment. Vice versa, iASPP depletion by shRNA resulted in decreased amounts of p300 and CBP, impaired binding of p300 and TAp73 to target site promoters, reduced induction of pro-apoptotic TAp73 target genes, and impaired apoptosis. Mechanistically, we observed that the p300-regulatory E3 ubiquitin ligase BRMS1 could rescue the degradation of p300 and CBP in cisplatin-treated, iASPP-depleted cells. This argues that iASPP stabilizes p300 and CBP by interfering with their BRMS1-mediated ubiquitination, thereby contributing to apoptotic susceptibility. In line, iASPP overexpression partially abolished the interaction of BRMS1 and CBP upon DNA damage. Reduced levels of iASPP mRNA and protein as well as CBP protein were observed in human melanoma compared with normal skin tissue and benign melanocytic nevi. In line with our findings, iASPP overexpression or knockdown of BRMS1 each augmented p300/CBP levels in melanoma cell lines, thereby enhancing apoptosis upon DNA damage. Taken together, destabilization of p300/CBP by downregulation of iASPP expression levels appears to represent a molecular mechanism that contributes to chemoresistance in melanoma cells.

Inositoylated platelet-activating factor (Ino-C2-PAF) modulates dynamic lymphocyte-endothelial cell interactions and alleviates psoriasis-like skin inflammation in two complementary mouse models.

Psoriasis, a tumor necrosis factor alpha (TNFα)-governed inflammatory disorder with prominent dysregulation of cutaneous vascular functions, has evolved into a model disorder for studying anti-inflammatory therapies. We present experimental in vitro and in vivo data on 1-O-octadecyl-2-O-(2-(myo-inositolyl)-ethyl)-sn-glycero-3-(R/S)-phosphatidyl-choline (Ino-C2-PAF), the lead compound of a class of synthetic glycosylated phospholipids, in anti-inflammatory therapy. Ino-C2-PAF strongly induced apoptosis only in TNFα-stimulated, but not in untreated human vascular endothelial cells. Moreover, TNFα-induced endothelial adhesion molecules that mediated the rolling and firm adhesion of leukocytes (vascular cell adhesion protein-1 (VCAM-1), E-selectin, and ICAM-1) were selectively downregulated by Ino-C2-PAF. Similarly, expression of L-selectin, VCAM-1 receptor α4β1 integrin , and lymphocyte function-associated antigen-1 on human peripheral blood mononuclear cells was reduced without induction of apoptosis. Functionally, these changes were accompanied by significant impairment of rolling and adhesion of human peripheral blood lymphocytes on TNFα-activated endothelial cells in a dynamic flow chamber system. When the therapeutic potential of Ino-C2-PAF was assessed in two complementary mouse models of psoriasis, K5.hTGFβ1 transgenic and JunB/c-Jun-deficient mice, Ino-C2-PAF led to significant alleviation of the clinical symptoms and normalized the pathological cutaneous changes including vascularization. There were no overt adverse effects. These findings suggested that Ino-C2-PAF is a potential candidate in the therapy of inflammatory skin diseases that include abnormal vascular functions.

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.

Treatment with diphenyl–pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death

Significance People with Parkinson’s disease, the second most common neurodegenerative disorder, have a lower risk and decreased incidence of cancer with the one exception being melanoma. The fact that, compared with other malignancies, melanoma occurs more frequently in patients with Parkinson’s disease and vice versa and that there is an association between a history of melanoma and an increased prevalence of prodromal markers of Parkinson’s disease prompted us to explore the possibility of an inverse biological link between these two diseases. The findings of our study suggest that α-synuclein, one of the key regulators in Parkinson’s disease, although toxic to dopaminergic neurons, is protective for advanced melanoma cells. Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson’s disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson’s disease-related proteins—α-synuclein, LRRK2, and Parkin—α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell’s survival.