RESUMO
The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.
Assuntos
Dano ao DNA , Receptor Tipo 1 de Melanocortina/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/efeitos da radiação , Fosforilação/efeitos da radiação , Pigmentação/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/efeitos da radiação , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
The epidermis increases pigmentation and epidermal thickness in response to ultraviolet exposure to protect against UV-associated carcinogenesis; however, the contribution of epidermal thickness has been debated. In a humanized skin mouse model that maintains interfollicular epidermal melanocytes, we found that forskolin, a small molecule that directly activates adenylyl cyclase and promotes cAMP generation, up-regulated epidermal eumelanin accumulation in fair-skinned melanocortin-1-receptor (Mc1r)-defective animals. Forskolin-induced pigmentation was associated with a reproducible expansion of epidermal thickness irrespective of melanization or the presence of epidermal melanocytes. Rather, forskolin-enhanced epidermal thickening was mediated through increased keratinocyte proliferation, indirectly through secreted factor(s) from cutaneous fibroblasts. We identified keratinocyte growth factor (Kgf) as a forskolin-induced fibroblast-derived cytokine that promoted keratinocyte proliferation, as forskolin induced Kgf expression both in the skin and in primary fibroblasts. Lastly, we found that even in the absence of pigmentation, forskolin-induced epidermal thickening significantly diminished the amount of UV-A and UV-B that passed through whole skin and reduced the amount of UV-B-associated epidermal sunburn cells. These findings suggest the possibility of pharmacologic-induced epidermal thickening as a novel UV-protective therapeutic intervention, particularly for individuals with defects in pigmentation and adaptive melanization.
Assuntos
AMP Cíclico/metabolismo , Pigmentos Biológicos/metabolismo , Pele/lesões , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Colforsina/farmacologia , Modelos Animais de Doenças , Epiderme/efeitos dos fármacos , Epiderme/lesões , Epiderme/metabolismo , Epiderme/patologia , Epiderme/efeitos da radiação , Humanos , Queratina-14/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/efeitos dos fármacos , Pele/patologia , Pele/efeitos da radiação , Pigmentação da Pele/efeitos dos fármacos , Pigmentação da Pele/efeitos da radiaçãoRESUMO
BACKGROUND: A functioning ubiquitin proteasome system (UPS) is essential for a number of diverse cellular processes and maintenance of overall cellular homeostasis. The ability of proteasome inhibitors, such as Velcade, to promote extrinsic apoptotic effects illustrates the importance of the ubiquitin proteasome system in the regulation of death receptor signaling. Here, we set out to define the UPS machinery, particularly the E3 ubiquitin ligases, that repress apoptosis through the extrinsic pathway. A cell-based genome-wide E3 ligase siRNA screen was established to monitor caspase-8 activity following the addition of TRAIL. RESULTS: Data from the high-throughput screen revealed that targeting the RING-finger containing E3 ligase Siah2 as well as the signaling platform molecule POSH (SH3RF1) conferred robust caspase-8 activation in response to TRAIL stimulus. Silencing Siah2 or POSH in prostate cancer cells led to increased caspase activity and apoptosis in response to both TRAIL and Fas ligand. The E3 activity of Siah2 was responsible for mediating apoptosis resistance; while POSH protein levels were critical for maintaining viability. Further characterization of Siah2 revealed it to function downstream of early death receptor events in the apoptotic pathway. The observed apoptosis resistance provides one biological explanation for the induction of Siah2 and POSH reported in lung and prostate cancer, respectively. Expanding on an initial yeast-two-hybrid screen we have confirmed a physical interaction between E3 ligases Siah2 and POSH. Utilizing a yeast-two-hybrid mapping approach we have defined the spacer region of POSH, more specifically the RPxAxVxP motif encompassing amino acids 601-607, to be the site of Siah2 binding. CONCLUSIONS: The data presented here define POSH and Siah2 as important mediators of death receptor mediated apoptosis and suggest targeting the interaction of these two E3 ligases is a promising novel cancer therapeutic strategy.
Assuntos
Apoptose , Proteínas Nucleares/metabolismo , Receptores de Morte Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Caspase 8/metabolismo , Sobrevivência Celular , Inativação Gênica , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Molecular targeting of apoptotic signaling pathways has been extensively studied in recent years and directed towards the development of effective therapeutic modalities for treating advanced androgen-independent prostate tumors. The majority of therapeutic agents act through intrinsic or mitochondrial pathways to induce programmed cell death. The induction of apoptosis through endoplasmic reticulum (ER) stress pathways may provide an alternative to treat patients. The functional interaction between the BCL-2 family members and regulation of calcium homeostasis in the ER provides a critical link to the life or death outcome of the cell. Apoptosis induction mediated by ER stress-inducing agents is just beginning to be exploited for therapeutic targeting of prostate tumors. Insightful dissection of recently discovered apoptotic signaling pathways that function through the endoplasmic reticulum may identify novel molecules that could effectively target both androgen-dependent and androgen-independent prostate tumors. In this review, we focus on linking ER stress-induced apoptosis to therapeutic targeting of prostate tumors and dissect its cross-talk with the intrinsic and extrinsic apoptotic pathways.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Transdução de Sinais , Estresse Fisiológico/fisiopatologia , Cálcio/metabolismo , Homeostase , Humanos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Inducing apoptosis via the extrinsic death receptor pathway is an attractive anti-cancer treatment strategy, however, numerous cancer cells exhibit significant resistance to death ligand stimuli. Here, we investigated the anti-neoplastic capability of proteasome inhibition, through the administration of Velcade, to synergize with a death receptor agonist in vivo. The death ligand-resistant LNCaP prostate xenograft model was utilized. Tumors were established and mice were treated with Velcade, TRAIL (TNF-Related Apoptosis Inducing Ligand) or the combined regimen. Only mice treated with a combination of Velcade and TRAIL was tumor growth inhibited with a corresponding loss of the hemorrhagic phenotype, decreased tumor cell proliferation and increased tumor cell apoptosis. Next, to determine if the extrinsic pathway is critical for mediating the anti-tumor efficacy that can be achieved in some cell types with Velcade treatment alone, the death receptor sensitive PC-3 xenograft model was used. PC-3 tumors exhibited a 54% decrease in tumor volume in response to Velcade, while c-FLIP overexpressing PC-3 xenografts were resistant to the treatment. These findings suggest that the extrinsic apoptotic pathway can mediate the anti-tumor effects of Velcade and support the therapeutic use of proteasome inhibition in combination with a death receptor stimulus in the treatment of prostate cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Divisão Celular/efeitos dos fármacos , Neoplasias da Próstata/patologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Ácidos Borônicos/uso terapêutico , Bortezomib , Linhagem Celular Tumoral , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Pirazinas/uso terapêutico , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêuticoRESUMO
BACKGROUND: Proteasome inhibition through the administration of Velcade is a viable chemotherapeutic strategy that is approved to treat multiple myeloma and is being evaluated for efficacy against prostate cancer. Currently, the apoptotic pathways that contribute to this anticancer response are poorly understood. Our goal is to test the extent to which proteasome inhibition modulates apoptosis through death receptor pathways. METHODS: Several prostate cancer cell lines and primary prostate epithelial cells (PrECs) were used as models. The death receptor pathway was activated by the expression of Fas ligand (FasL) or addition of TNF-related apoptosis-inducing ligand (TRAIL) in the presence or absence of proteasome inhibitors. The apoptotic response was quantified by annexin V, TUNEL and nuclear condensation assays. Western blot analysis was conducted to quantify protein levels and enzyme assays were used to measure caspase activity. RESULTS: Proteasome inhibition markedly sensitized prostate cancer cells to apoptosis initiated by Fas ligand (FasL) or TRAIL. In the presence of either death ligand, procaspase-8 processing occurred, but led to minimal amounts of active caspase-8. The addition of Velcade, however, led to robust active caspase-8 protein abundance and activity. In the presence of Velcade the caspase-8 p18 subunit half-life increased from 22 min to over 2 hr. CONCLUSIONS: These findings demonstrate that proteasome inhibition can sensitize cells to apoptosis elicited by tumor necrosis factor ligands and retarding caspase-8 degradation provides one explanation for this activity. This study suggests that the clinical efficacy of Velcade may result, at least in part, from the activity of death ligands.