RESUMEN
Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.
Asunto(s)
Senescencia Celular , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Proteínas Proto-Oncogénicas B-rafRESUMEN
Poly(amidoamine) dendrimer (PAMAM) is well-known for its high efficiency as a drug delivery vehicle. However, the intrinsic cytotoxicity and lack of a detectable signal to facilitate tracking have impeded its practical applications. Herein, we have developed a novel label-free fluorescent and biocompatible PAMAM derivative by simple surface modification of PAMAM using acetaldehyde. The modified PAMAM possessed a strong green fluorescence, which was generated by the C=N bonds of the resulting Schiff Bases via n-π* transition, while the intrinsic cytotoxicity of PAMAM was simultaneously ameliorated. Through further PEGylation, the fluorescent PAMAM demonstrated excellent intracellular tracking in human melanoma SKMEL28 cells. In addition, our PEGylated fluorescent PAMAM derivative achieved enhanced loading and delivery efficiency of the anticancer drug doxorubicin (DOX) compared to the original PAMAM. Importantly, the accelerated kinetics of DOX-encapsulated fluorescent PAMAM nanocomposites in an acidic environment facilitated intracellular drug release, which demonstrated comparable cytotoxicity to that of the free-form doxorubicin hydrochloride (DOX·HCl) against melanoma cells. Overall, our label free fluorescent PAMAM derivative offers a new opportunity of traceable and controlled delivery for DOX and other drugs of potential clinical importance.
Asunto(s)
Antineoplásicos/administración & dosificación , Dendrímeros/química , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Nanocompuestos/química , Poliaminas/química , Acetaldehído/química , Antineoplásicos/química , Línea Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Nanocompuestos/toxicidad , Polietilenglicoles/química , Bases de Schiff/químicaRESUMEN
Resistance to immune checkpoint inhibitor therapies in melanoma is common and remains an intractable clinical challenge. In this study, we comprehensively profile immune checkpoint inhibitor resistance mechanisms in short-term tumor cell lines and matched tumor samples from melanoma patients progressing on immune checkpoint inhibitors. Combining genome, transcriptome, and high dimensional flow cytometric profiling with functional analysis, we identify three distinct programs of immunotherapy resistance. Here we show that resistance programs include (1) the loss of wild-type antigen expression, resulting from tumor-intrinsic IFNγ signaling and melanoma de-differentiation, (2) the disruption of antigen presentation via multiple independent mechanisms affecting MHC expression, and (3) immune cell exclusion associated with PTEN loss. The dominant role of compromised antigen production and presentation in melanoma resistance to immune checkpoint inhibition highlights the importance of treatment salvage strategies aimed at the restoration of MHC expression, stimulation of innate immunity, and re-expression of wild-type differentiation antigens.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Melanoma , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Transcriptoma , Inmunoterapia/métodos , Inmunidad InnataRESUMEN
Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.
Asunto(s)
Inhibidores de Proteínas Quinasas , Neoplasias de la Úvea , Línea Celular Tumoral , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/uso terapéutico , Humanos , Melanoma , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositoles/uso terapéutico , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patologíaRESUMEN
Immunotherapy has transformed the management of patients with advanced melanoma, with five-year overall survival rates reaching 52% for combination immunotherapies blocking the cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) and programmed cell death-1 (PD1) immune axes. Yet, our understanding of local and systemic determinants of immunotherapy response and resistance is restrained by the paucity of preclinical models, particularly those for anti-PD1 monotherapy. We have therefore generated a novel murine model of melanoma by integrating key immunotherapy response biomarkers into the model development workflow. The resulting YUMM3.3UVRc34 (BrafV600E; Cdkn2a-/-) model demonstrated high mutation burden and response to interferon (IFN)γ, including induced expression of antigen-presenting molecule MHC-I and the principal PD1 ligand PD-L1, consistent with phenotypes of human melanoma biopsies from patients subsequently responding to anti-PD1 monotherapy. Syngeneic immunosufficient mice bearing YUMM3.3UVRc34 tumors demonstrated durable responses to anti-PD1, anti-CTLA4, or combined treatment. Immunotherapy responses were associated with early on-treatment changes in the tumor microenvironment and circulating T-cell subsets, and systemic immunological memory underlying protection from tumor recurrence. Local and systemic immunological landscapes associated with immunotherapy response in the YUMM3.3UVRc34 melanoma model recapitulate immunotherapy responses observed in melanoma patients and identify discrete immunological mechanisms underlying the durability of responses to anti-PD1 and anti-CTLA4 treatments.
RESUMEN
Inherited mutations affecting the INK4a/ARF locus (CDKN2A) are associated with melanoma susceptibility in 40% of multiple case melanoma families. Over 60 different germline INK4a/ARF mutations have been detected in more than 190 families worldwide. The majority of these alterations are missense mutations affecting p16(INK4a), and only 25% of these have been functionally assessed. There is therefore a need for an accurate and rapid assay to determine the functional significance of p16(INK4a) mutations. We reviewed the performance of several in vivo functional assays that measure critical aspects of p16(INK4a) function, including subcellular location, CDK binding and cell cycle inhibition. In this report the function of 28 p16(INK4a) variants, many associated with melanoma susceptibility were compared. We show that assessment of CDK4 binding and subcellular localization can accurately and rapidly determine the functional significance of melanoma-associated p16(INK4a) mutations. p16(INK4a)-CDK6 binding affinity was unhelpful, as no disease-associated mutation showed reduced CDK6 affinity while maintaining the ability to bind CDK4. Likewise, in silico analyses did not contribute substantially, with only 12 of 25 melanoma-associated missense variants consistently predicted as deleterious. The ability to determine variant functional activity accurately would identify disease-associated mutations and facilitate effective genetic counselling of individuals at high risk of melanoma.
Asunto(s)
Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Mutación , Unión Competitiva , Western Blotting , Ciclo Celular/fisiología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Citoplasma/metabolismo , Humanos , Melanoma/genética , Melanoma/patología , Mutación Missense , Unión Proteica , Eliminación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Nearly all patients with BRAF-mutant melanoma will progress on BRAF inhibitor monotherapy and combination BRAF/MEK inhibitor therapy within the first year of therapy. In the vast majority of progressing melanomas, resistance occurs via the re-activation of MAPK signalling, commonly via alterations in BRAF, NRAS and MEK1/2. A small proportion of resistant melanomas rely on the activation of the compensatory PI3K/AKT signalling cascade, although activation of this pathway does not preclude patient responses to BRAF/MEK inhibition. We now show, that PI3K/AKT signalling via potent oncogenic PIK3CA and AKT3 mutants, is not sufficient to overcome proliferative arrest induced by BRAF/MEK inhibition, but rather enables the survival of a dormant population of MAPK-inhibited melanoma cells. The evolution of resistance in these surviving tumour cells was associated with MAPK re-activation and no longer depended on the initial PI3K/AKT-activating oncogene. This dynamic form of resistance alters signalling dependence and may lead to the evolution of tumour subclones highly resistant to multiple targeted therapies.
RESUMEN
BACKGROUND: MEK1 mutations in melanoma can confer resistance to BRAF inhibitors, although preexisting MEK1(P124) mutations do not preclude clinical responses. We sought to determine whether recurrent, preexisting MEK1(P124) mutations affected clinical outcome in BRAF inhibitor-treated patients with melanoma. METHODS: Data from four published datasets were analyzed to determine whether preexisting MEK1(P124) mutations affect radiologic response or progression-free survival (PFS) in patients with BRAF(V600)-mutant metastatic melanoma treated with vemurafenib or dabrafenib. The effects of MEK1(P124) mutations on MAPK pathway activity and response to BRAF inhibition were also investigated in a series of cell models. RESULTS: In a pooled analysis of 123 patients, the presence of a pretreatment MEK1(P124) mutation (N = 12, 10%) was associated with a poorer RECIST response (33% vs. 72% in MEK1(P124Q/S) vs. MEK1(P124) wild-type, P = 0.018), and a shorter PFS (median 3.1 vs. 4.8 months, P = 0.004). Furthermore, MEK1(P124Q/S) mutations were shown to have independent kinase activity and introduction of these mutations into a BRAF-mutant melanoma cell line diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival in the presence of dabrafenib compared with cells ectopically expressing wild-type MEK1. Consistent with these data, two BRAF-mutant cell lines with endogenous MEK1(P124) mutations showed intermediate sensitivity to dabrafenib, but were highly sensitive to downstream inhibition of MEK or ERK. CONCLUSION: Taken together, our data indicate that preexisting MEK1(P124) mutations are associated with a reduced response to BRAF inhibitor therapy and identify a subset of patients with BRAF-mutant melanoma likely to benefit from combination therapies involving MEK or ERK inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/genética , MAP Quinasa Quinasa 1/genética , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Supervivencia sin Enfermedad , Humanos , Imidazoles/administración & dosificación , Indoles/administración & dosificación , Melanoma/tratamiento farmacológico , Melanoma/patología , Mutación , Oximas/administración & dosificación , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , VemurafenibRESUMEN
The p14(ARF) tumor suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14(ARF) are closely linked with its subcellular distribution. Nucleolar p14(ARF) dampens ribosome biosynthesis and nucleoplasmic forms of p14(ARF) activate the p53 pathway and induce cell cycle arrest. p14(ARF) can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-x(L) and p32 to induce cell death. It has been suggested that the movement of p14(ARF) to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14(ARF) in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14(ARF) act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14(ARF). This allows the interaction of p14(ARF) with mitochondrial proteins, including p32 and enables p53-independent cell death.
Asunto(s)
Mitocondrias/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas Portadoras , Línea Celular Tumoral , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Alterations in the ARF tumor suppressor protein (also known as p14ARF in humans and p19ARF in the mouse) occur frequently in cancer and are associated with susceptibility to melanoma, pancreatic cancer and nervous system tumors. ARF proteins interact with the E2F-1, -2 and -3 transcription activators to inhibit their transcriptional activity and induce their degradation via the 26S proteasome pathway. The impact of ARF on the E2F proteins may provide a mechanism for p53-independent ARF activity on cell cycle progression and tumor susceptibility. In this report we explored the effects of ARF on E2F ubiquitination and degradation in relationship to cell cycle effects and p53 status. We now show that ARF induced the rapid ubiquitination and degradation of E2F-1 only in the presence of functional p53. E2F-1 continued to be ubiquitinated following ARF induction in cycling p53-wild-type, p21-null cells, showing that effects of ARF were not simply a result of p14ARF induced cell-cycle arrest. Importantly, these data establish that the ARF-E2F-1 pathway is an extension of the p53-mdm2-ARF tumor suppressor network and is unlikely to constitute a p53-independent pathway for ARF function.