nutlin-3a and Skin-Neoplasms

Targeting the MAPK pathway by combined inhibition of BRAF and MEK has increased overall survival in advanced BRAF-mutant melanoma in both therapeutic and adjuvant clinical settings. However, a significant proportion of tumors develop acquired resistance, leading to treatment failure. We have previously shown p63 to be an important inhibitor of p53-induced apoptosis in melanoma following genotoxic drug exposure. Here, we investigated the role of p63 in acquired resistance to MAPK inhibition and show that p63 isoforms are upregulated in melanoma cell lines chronically exposed to BRAF and MEK inhibition, with consequent increased resistance to apoptosis. This p63 upregulation was the result of its reduced degradation by the E3 ubiquitin ligase FBXW7. FBXW7 was itself regulated by MDM2, and in therapy-resistant melanoma cell lines, nuclear accumulation of MDM2 caused downregulation of FBXW7 and consequent upregulation of p63. Consistent with this, both FBXW7-inactivating mutations and MDM2 upregulation were found in melanoma clinical samples. Treatment of MAPK inhibitor-resistant melanoma cells with MDM2 inhibitor Nutlin-3A restored FBXW7 expression and p63 degradation in a dose-dependent manner and sensitized these cells to apoptosis. Collectively, these data provide a compelling rationale for future investigation of Nutlin-3A as an approach to abrogate acquired resistance of melanoma to MAPK inhibitor targeted therapy. SIGNIFICANCE: Upregulation of p63, an unreported mechanism of MAPK inhibitor resistance in melanoma, can be abrogated by treatment with the MDM2 inhibitor Nutlin-3A, which may serve as a strategy to overcome resistance.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; F-Box-WD Repeat-Containing Protein 7; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Mutation; Piperazines; Protein Kinase Inhibitors; Proteolysis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-mdm2; Skin; Skin Neoplasms; Transcription Factors; Tumor Suppressor Proteins; Up-Regulation; Young Adult

Cutaneous melanoma, the most aggressive form of skin cancer, is characterized by activating BRAF mutations. Despite the initial success of selective BRAF inhibitors, only few patients exhibited complete responses, whereas many showed disease progression. Melanoma is one of the few types of cancer in which p53 is not frequently mutated, but p53 inactivation can be indirectly achieved by a stable activation of MDM2 induced by a deletion in CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) locus, encoding for p16

Topics: Apoptosis; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Melanoma; Melanoma, Cutaneous Malignant; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Pyrroles; Skin Neoplasms; Transcription, Genetic; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53

Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2-p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells.. A panel of three p53. GSK2830371, at doses (⩽10 μM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53. GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.

Topics: Aminopyridines; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dipeptides; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Melanoma; Melanoma, Cutaneous Malignant; Mutagenesis, Site-Directed; para-Aminobenzoates; Phosphorylation; Piperazines; Protein Binding; Protein Phosphatase 2C; Protein Stability; Proto-Oncogene Proteins c-mdm2; Pyrrolidines; Skin Neoplasms; Tumor Suppressor Protein p53

Nutlin3, a non-genotoxic agonist of p53, is currently in phase II clinical trials for cancer treatment. However, its effects on normal tissues and cell types remain largely to be determined. Drugs that can selectively target cancer cells as well as cooperate with the p53 pathway are thus greatly needed. Iron-superoxide dismutase (Fe-SOD) is a potential candidate as it selectively targets cancer cells by eliminating the abnormally high levels of reactive oxygen species (ROS) in cancer cells; it also inhibits cancer cell growth by induction of p27. Here, we show evidence that modulating redox and ROS homeostasis cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo. Co-treatment of Fe-SOD and Nutlin3 showed synergistic inhibition on cancer cells in vitro, and the induction of p27 appeared to be involved. No effects were observed on normal cells. In addition, such co-treatment further exhibited synergistic inhibition on tumor growth in vivo in a murine B16 xenograft model, while the individual treatments only achieved very limited inhibition. Thus, Fe-SOD cooperated with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo.

Topics: Animals; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drug Synergism; Female; Hep G2 Cells; Humans; Imidazoles; Liposomes; MCF-7 Cells; Melanoma, Experimental; Mice; Neoplasms; Piperazines; Reactive Oxygen Species; Skin Neoplasms; Superoxide Dismutase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Oncogenesis reflects an orchestrated interaction between misguided growth signals. Although much effort has been launched to pharmacologically disable activated oncogenes, one sidelined approach is the restoration of tumor suppressive signals. As TP53 is often structurally preserved, but functionally crippled, by CDKN2A/ARF loss in melanoma, rescue of p53 function represents an attractive point of vulnerability in melanoma. In this study, we showed that both p53 protein and activity levels in melanoma cells were strongly induced by nutlin-3, a canonical HDM2 antagonist. Among a test panel of 51 cell lines, there was a marked reduction in melanoma viability that was directly linked to TP53 status. Moreover, we also found that the melanoma growth suppression mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase inhibition was potentiated by HDM2 antagonism. These results provide fundamental insights into the intact p53 circuitry, which can be restored through small molecule inhibitors and potentially deployed for therapeutic gain.

Topics: Apoptosis; Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Humans; Imidazoles; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Piperazines; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-mdm2; Skin Neoplasms; Tumor Suppressor Protein p53

Malignant cells express ligands for the natural killer cell immunoreceptor NKG2D, which sensitizes to early recognition and elimination by cytotoxic lymphocytes and provides an innate barrier against tumor development. However, the mechanisms that control NKG2D ligand (NKG2DL) expression in tumor cells remain unknown. We recently identified the NKG2DL ULBP2 as strong prognostic marker in human malignant melanoma. Here, we provide evidence that the tumor-suppressive microRNAs (miRNA) miR-34a and miR-34c control ULBP2 expression. Reporter gene analyses revealed that both miRNAs directly targeted the 3'-untranslated region of ULBP2 mRNA and that levels of miR-34a inversely correlated with expression of ULBP2 surface molecules. Accordingly, treatment of cancer cells with miRNA inhibitors led to upregulation of ULBP2, whereas miR-34 mimics led to downregulation of ULBP2, diminishing tumor cell recognition by NK cells. Treatment with the small molecule inhibitor Nutlin-3a also decreased ULBP2 levels in a p53-dependent manner, which was due to a p53-mediated increase in cellular miR-34 levels. Taken together, our study shows that tumor-suppressive miR-34a and miR-34c act as ULBP2 repressors. These findings also implicate p53 in ULBP2 regulation, emphasizing the role of the specific NKG2DL in tumor immune surveillance.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Down-Regulation; GPI-Linked Proteins; HCT116 Cells; HEK293 Cells; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Melanoma; MicroRNAs; NK Cell Lectin-Like Receptor Subfamily K; Piperazines; RNA, Messenger; Skin Neoplasms; Transfection; Tumor Suppressor Protein p53

P53 is rarely mutated in cutaneous T-cell lymphoma (CTCL) and is therefore a promising target for innovative therapeutic approaches. Nutlin-3a is an inhibitor of MDM2 (human homolog of murine double minute 2), which disrupts its interaction with p53, leading to the stabilization and activation of p53. To investigate the potential therapeutic use of nutlin-3a in CTCL, we screened CTCL lines Hut-78, SeAx, MyLa2000, Mac1, and Mac2a by measuring p53 levels after nutlin-3a treatment. In MyLa2000, Mac1, and Mac2a, we observed the increase in p53, indicating the fully functional p53. In the remaining cell lines, P53 mutation analysis identified a homozygous nonsense mutation (R196Stop in Hut-78) and a homozygous missense mutation (G245S in SeAx). In MyLa2000, Mac1, and Mac2a carrying wild-type P53, nutlin-3a induced apoptosis and senescence demonstrated by permanent G0/G1 cell-cycle block and expression of the senescence-associated β-galactosidase. This effect was abolished in cells in which p53 was silenced by small interfering RNA. Sézary cells lack functional p53 and were resistant to nutlin-3a. However, nutlin-3a potentiated the efficacy of conventional chemotherapeutics not only in cells with intact p53 but also in Hut-78, SeAx, and Sézary cells. Thus, targeting p53 by nutlin-3a may constitute a therapeutic approach in CTCL because of increased apoptosis and senescence of tumor cells.

Topics: Apoptosis; Cell Cycle Checkpoints; Cellular Senescence; Codon, Nonsense; Humans; Imidazoles; Lymphoma, T-Cell, Cutaneous; Mutation, Missense; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group with Sézary syndrome (SS) as one of the most aggressive variants. Recently, we identified a loss of E2A as a recurrent event in SS, which enhanced proliferation via upregulation of the proto-oncogene MYC. MYC-induced transformation usually requires deleterious alterations of key apoptotic genes including p53; however, p53 functionality and mutation status in SS are unclear.. We investigated functionality of p53 signalling by pharmacological treatment with the MDM2 antagonist nutlin-3, which might result in p53 activation. Furthermore, we analysed the TP53 mutation status in CTCL cell lines and highly purified tumour cells from patients with SS by mRNA and DNA sequencing.. We analysed the apoptosis induction due to nutlin-3 treatment in various SS cell lines and primary patient samples by annexin V/propidium iodide staining. Induction of p53 target genes was analysed by immunoblotting, and TP53 was sequenced at the mRNA and DNA level.. We identified various TP53 mutations and an impaired p53 signalling in the vast majority of the investigated cell lines and primary SS cells.. In accordance with the importance of MYC deregulation in SS, p53 signalling is frequently nonfunctional in SS. However, although most likely ineffective as exclusive treatment in SS, it remains possible that pharmacological p53 activation could be beneficial in combination with other approaches including classical chemotherapeutics.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Genes, p53; Humans; Imidazoles; Immunoblotting; Loss of Heterozygosity; Mutation; Piperazines; Proto-Oncogene Mas; Proto-Oncogene Proteins c-mdm2; Sequence Analysis, DNA; Sequence Analysis, RNA; Sezary Syndrome; Signal Transduction; Skin Neoplasms; Tumor Suppressor Protein p53

Nutlin-3a increases p53 levels after UVB radiation, which could result in a decrease in DNA damage and thus lead to a lower risk of non-melanoma skin cancer. Especially, organ transplant recipients might derive benefit from such a topical formulation with an active ingredient to prevent DNA damage.. To investigate whether topical nutlin-3a can decrease photocarcinogenesis induced by simulated solar radiation.. 72 hairless C3.Cg/TifBomTac mice were treated 3 days/week topically with 100 μl nutlin-3a (9 mM) [Groups 1 and 3 (120 days)) or 100 μl vehicle (Group 2). Three hours later, all mice were exposed to simulated solar radiation (a radiometric equivalent of three standard erythema dose units).. The median time to tumours did not differ between the mice treated with nutlin-3a and with the vehicle. The median time to the first and second tumours did not differ between 'nutlin-3a-120 days' and vehicle-treated mice, but there was a small significant difference in the median time to the third tumour (211 vs. 196 days, P = 0.043). However, after Bonferroni correction, there was no difference at all.. Nutlin-3a had no reductive effect on photocarcinogenesis and we do not believe in nutlin-3a as a potential drug against DNA damage in a topical formulation for organ transplant patients.

Topics: Administration, Topical; Animals; Cell Transformation, Neoplastic; DNA Damage; Female; Humans; Imidazoles; Mice; Mice, Hairless; Piperazines; Skin Neoplasms; Sunlight; Ultraviolet Rays

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the ‘high-p53’Mdm4+/− mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Clone Cells; Disease Models, Animal; Disease Progression; Humans; Imidazoles; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Nevus; Pigmentation; Piperazines; Proto-Oncogene Proteins; Skin Neoplasms; Staining and Labeling; Survival Analysis; Transcription, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases