PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

Histological and functional characterization of 3D human skin models mimicking the inflammatory skin diseases psoriasis and atopic dermatitis.

Three-dimensional (3D) human skin equivalents have emerged as valuable tools in skin research, replacing animal experimentation and precluding the need for patient biopsies. In this study, we advanced 3D skin equivalents to model the inflammatory skin diseases atopic dermatitis and psoriasis by cytokine stimulation, and were successful in integrating TH1 T cells into skin models to develop an immunocompetent 3D psoriasis model. We performed in-depth histological and functional characterization of 3D skin equivalents and validated them in terms of tissue architecture, pathological changes, expression of antimicrobial peptides and Staphylococcus aureus colonization using 3D reconstruction by multiphoton microscopy and phenotyping by highly multiplexed 'co-detection by indexing' (CODEX) microscopy. We show that our skin equivalents have a structural architecture with a well-developed dermis and epidermis, thus resembling human skin. In addition, the skin models of atopic dermatitis and psoriasis show several phenotypic features of inflammatory skin disease, including disturbed epidermal differentiation and alterations in the expression of epidermal barrier genes and antimicrobial peptides, and can be reliably used to test novel treatment strategies. Therefore, these 3D equivalents will be a valuable tool in experimental dermatological research.

PARP Inhibitors Effectively Reduce MAPK Inhibitor Resistant Melanoma Cell Growth and Synergize with MAPK Inhibitors through a Synthetic Lethal Interaction In Vitro and In Vivo.

The efficacy of targeting the MAPK signaling pathway in patients with melanoma is limited by the rapid development of resistance mechanisms that result in disease relapse. In this article, we focus on targeting the DNA repair pathway as an antimelanoma therapy, especially in MAPK inhibitor resistant melanoma cells using PARP inhibitors. We found that MAPK inhibitor resistant melanoma cells are particularly sensitive to PARP inhibitor treatment due to a lower basal expression of the DNA damage sensor ataxia-telangiectasia mutated (ATM). As a consequence, MAPK inhibitor resistant melanoma cells have decreased homologous recombination repair activity leading to a reduced repair of double-strand breaks caused by the PARP inhibitors. We validated the clinical relevance of our findings by ATM expression analysis in biopsies from patients with melanoma before and after development of resistance to MAPK inhibitors. Furthermore, we show that inhibition of the MAPK pathway induces a homologous recombination repair deficient phenotype in melanoma cells irrespective of their MAPK inhibitor sensitivity status. MAPK inhibition results in a synthetic lethal interaction of a combinatorial treatment with PARP inhibitors, which significantly reduces melanoma cell growth in vitro and in vivo. In conclusion, this study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression. Significance: We show that MAPK inhibitor resistant melanoma cells exhibit low ATM expression increasing their sensitivity toward PARP inhibitors and that a combination of MAPK/PARP inhibitors act synthetically lethal in melanoma cells. Our study shows that PARP inhibitor treatment is a valuable therapy option for patients with melanoma, either as a single treatment or as a combination with MAPK inhibitors depending on ATM expression, which could serve as a novel biomarker for treatment response.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion.

BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

Tumour Progression Stage-Dependent Secretion of YB-1 Stimulates Melanoma Cell Migration and Invasion.

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

Targeting Rad51 as a strategy for the treatment of melanoma cells resistant to MAPK pathway inhibition.

Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.

Staphylococcus aureus Skin Colonization Is Enhanced by the Interaction of Neutrophil Extracellular Traps with Keratinocytes.

Staphylococcus aureus is a facultative pathogen found on skin and nasal surfaces. It is usually absent from the skin of healthy humans but frequently colonizes the skin of patients with atopic dermatitis. Here, we investigate the functional role of neutrophils in the initial steps of S. aureus skin colonization and how skin commensals modulate the S. aureus-induced recruitment of neutrophils to the skin. Using an epicutaneous mouse skin colonization model, we show that skin inflammation induced by tape-stripping leads to a rapid recruitment of neutrophils, which correlates with enhanced S. aureus skin colonization. Interestingly, the depletion of neutrophils in vivo reduces S. aureus colonization, and in vitro coculture of primary human keratinocytes with neutrophils promotes S. aureus adherence. We demonstrate that the interaction of neutrophil extracellular traps with keratinocytes are responsible for the increased S. aureus skin colonization. Finally, we show that S. epidermidis as part of the skin microbiota can reduce the neutrophil recruitment induced by S. aureus infection. These data suggest that microbiota-mediated skin protection against S. aureus is dampened in an inflammatory environment in which neutrophil extracellular traps released by infiltrating neutrophils unexpectedly contribute to enhanced S. aureus skin colonization.

Lugdunin amplifies innate immune responses in the skin in synergy with host- and microbiota-derived factors.

Recently our groups discovered lugdunin, a new cyclic peptide antibiotic that inhibits Staphylococcus aureus epithelial colonization in humans and rodents. In this work, we analyzed its immuno-modulatory and antimicrobial potential as a single agent or in combination with other microbiota- or host-derived factors. We show that pretreatment of primary human keratinocytes or mouse skin with lugdunin in combination with microbiota-derived factors results in a significant reduction of S. aureus colonization. Moreover, lugdunin increases expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin, and results in the recruitment of monocytes and neutrophils in vivo, both by a TLR/MyD88-dependent mechanism. Interestingly, S. aureus elimination by lugdunin is additionally achieved by synergistic antimicrobial activity with LL-37 and dermcidin-derived peptides. In summary, our results indicate that lugdunin provides multi-level protection against S. aureus and may thus become a promising treatment option for S. aureus skin infections in the future.

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.

Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase.

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.

Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression.

BACKGROUND: We previously identified CK1α as a novel tumor suppressor in melanoma and reported that the loss of CK1α leads to increased proliferation and invasive growth of melanoma cells by strong activation of the Wnt/ß-catenin signaling pathway. METHODS: In this study we analyzed expression and the functional effects of the dominantly expressed CK1- isoforms α, δ and ε in melanoma cells by quantitative real-time PCR, western blot and immunohistochemistry. We down-regulated CK1 kinase activity with isoform specific siRNAs and small molecule inhibitors. Vice versa we overexpressed the CK1 isoforms α, δ and ε using viral vectors and tested the biological effects on melanoma cell proliferation, migration and invasion. RESULTS: We show that protein expression of all three CK1-isoforms is downregulated in metastatic melanoma cells compared to benign melanocytic cells. Furthermore, the CK1δ and ε isoforms are able to negatively regulate expression of each other, whereas CK1α expression is independently regulated in melanoma cells. Inhibition of the expression and activity of CK1δ or CK1ε by specific inhibitors or siRNAs had no significant effect on the growth and survival of metastatic melanoma cells. Moreover, the over-expression of CK1δ or CK1ε in melanoma cells failed to induce cell death and cell cycle arrest although p53 signaling was activated. This is in contrast to the effects of CK1α where up-regulated expression induces cell death and apoptosis in metastatic melanoma cells. CONCLUSION: These data indicate that CK1α has a dominant and non-redundant function in melanoma cells and that the CK1δ and ε isoforms are not substantially involved in melanoma progression.

PTEN regulates IGF-1R-mediated therapy resistance in melanoma.

Inhibition of the mitogen-activated protein kinase (MAPK) pathway is a major advance in the treatment of metastatic melanoma. However, its therapeutic success is limited by the rapid emergence of drug resistance. The insulin-like growth factor-1 receptor (IGF-1R) is overexpressed in melanomas developing resistance toward the BRAF(V) (600) inhibitor vemurafenib. Here, we show that hyperactivation of BRAF enhances IGF-1R expression. In addition, the phosphatase activity of PTEN as well as heterocellular contact to stromal cells increases IGF-1R expression in melanoma cells and enhances resistance to vemurafenib. Interestingly, PTEN-negative melanoma cells escape IGF-1R blockade by decreased expression of the receptor, implicating that only in melanoma patients with PTEN-positive tumors treatment with IGF-1R inhibitors would be a suitable strategy to combat therapy resistance. Our data emphasize the crosstalk and therapeutic relevance of microenvironmental and tumor cell-autonomous mechanisms in regulating IGF-1R expression and by this sensitivity toward targeted therapies.

MAPK and PI3K/AKT mediated YB-1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop.

Y-box binding protein 1 (YB-1) is an oncogenic transcription and translation factor and is overexpressed in several types of cancer. Our previous data showed that YB-1 is upregulated and translocated to the nucleus during melanoma progression and that YB-1 is an important transcription factor regulating proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. It has been suggested that YB-1 is activated and translocated to the nucleus after S102-phosphorylation in the DNA binding domain. In this study, we show that activation of YB-1 by S102-phosphorylation and nuclear translocation is increased during melanoma progression using a human tissue microarray with 100 melanocytic lesions. Furthermore, we analysed the mechanisms governing the expression and activity of YB-1 in melanoma cells. We show that the PI3K/AKT and p53 signalling, growth factors and chemotherapeutic agents increase YB-1 promoter activity. This, however, resulted in no or only modest increase in YB-1 protein expression. We show that the MAPK and PI3K/AKT signalling pathways, both activated in melanoma cells, as well as p53 overexpression increase YB-1 S102-phosphorylation, whereas NFκB signalling inhibits phosphorylation. Overexpression of YB-1 in melanoma cells inhibits translation efficiency and by this proliferation and survival of melanoma cells indicating that there is an autoregulatory loop restricting YB-1 protein expression. These data suggest that there is a tightly regulated feedback mechanism regulating YB-1 expression and activation, necessary for proper cell cycle progression of melanoma cells.

ß-Catenin signaling increases during melanoma progression and promotes tumor cell survival and chemoresistance.

Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.

Suppression of casein kinase 1alpha in melanoma cells induces a switch in beta-catenin signaling to promote metastasis.

Casein kinase 1 alpha (CK1alpha) is a multifunctional Ser/Thr kinase that phosphorylates several substrates. Among those is beta-catenin, an important player in cell adhesion and Wnt signaling. Phosphorylation of beta-catenin by CK1alpha at Ser45 is the priming reaction for the proteasomal degradation of beta-catenin. Interestingly, aside from this role in beta-catenin degradation, very little is known about the expression and functional role of CK1alpha in tumor cells. Here, we show that CK1alpha expression in different tumor types is either strongly suppressed or completely lost during tumor progression and that CK1alpha is a key factor determining beta-catenin stability and transcriptional activity in tumor cells. CK1alpha reexpression in metastatic melanoma cells reduces growth in vitro and metastasis formation in vivo, and induces cell cycle arrest and apoptosis, whereas suppression of CK1alpha in primary melanoma cells induces invasive tumor growth. Inactivation of CK1alpha promotes tumor progression by regulating a switch in beta-catenin-mediated signaling. These results show that melanoma cells developed an efficient new mechanism to activate the beta-catenin signaling pathway and define CK1alpha as a novel tumor suppressor.

The increased expression of Y box-binding protein 1 in melanoma stimulates proliferation and tumor invasion, antagonizes apoptosis and enhances chemoresistance.

In previous studies we identified the transcription/translation factor Y-box-binding protein (YB-1) as a gene that is upregulated in primary melanoma and melanoma metastases when compared to benign melanocytic nevi. To analyze whether YB-1 expression correlates with melanoma progression in vitro and in vivo, we performed expression analysis on melanoma cell lines representing different stages of melanoma progression and on tissues of melanocytic nevi, primary melanoma and melanoma metastases. Our data indicate that compared to benign melanocytes YB-1 expression is increased in melanoma cells in vitro and in vivo and that YB-1 is translocated into the nucleus in invasive and metastatic melanoma cells. To reveal the functional role of YB-1 in melanoma progression we achieved a stable downregulation of YB-1 using shRNA in metastatic melanoma cells. Interestingly, YB-1 downregulation resulted in a pronounced reduced rate of proliferation and an increased rate of apoptotic cell death. In addition, migration and invasion of melanoma cells in monolayer and in a three-dimensional skin reconstruct in vitro was significantly reduced. These effects were accompanied by downregulation of genes involved in proliferation, survival and migration/invasion of melanoma cells such as MMP-2, bcl-2, Cyclin D1, p53 and p16INK4A. Furthermore, melanoma cells with a reduced YB-1 expression showed a decreased resistance to the chemotherapeutic agents cisplatin and etoposide. These data suggest that YB-1 is involved in malignant transformation of melanocytes and contributes to the stimulation of proliferation, tumor invasion, survival and chemoresistance. Thus, YB-1 may be a promising molecular target in melanoma therapy.