RESUMEN
Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.
RESUMEN
Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/ß-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/ß-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/ß-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. SIGNIFICANCE: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/ß-catenin pathway, which can be targeted therapeutically.
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Carcinoma Neuroendocrino/etiología , Proteína Proto-Oncogénica N-Myc/metabolismo , Neoplasias de la Próstata/etiología , Vía de Señalización Wnt , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Animales , Carbazoles/uso terapéutico , Carcinoma Neuroendocrino/tratamiento farmacológico , Carcinoma Neuroendocrino/metabolismo , Carcinoma Neuroendocrino/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Humanos , Masculino , Ratones , Mutación , Proteína Proto-Oncogénica N-Myc/genética , Células Madre Neoplásicas , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/etiología , Neuroblastoma/patología , Piperidinas/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Secuenciación del Exoma , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Small molecules that directly target MYC and are also well tolerated in vivo will provide invaluable chemical probes and potential anti-cancer therapeutic agents. We developed a series of small-molecule MYC inhibitors that engage MYC inside cells, disrupt MYC/MAX dimers, and impair MYC-driven gene expression. The compounds enhance MYC phosphorylation on threonine-58, consequently increasing proteasome-mediated MYC degradation. The initial lead, MYC inhibitor 361 (MYCi361), suppressed in vivo tumor growth in mice, increased tumor immune cell infiltration, upregulated PD-L1 on tumors, and sensitized tumors to anti-PD1 immunotherapy. However, 361 demonstrated a narrow therapeutic index. An improved analog, MYCi975 showed better tolerability. These findings suggest the potential of small-molecule MYC inhibitors as chemical probes and possible anti-cancer therapeutic agents.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-H1/farmacología , Descubrimiento de Drogas/métodos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Antígeno B7-H1/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Estudios de Factibilidad , Femenino , Humanos , Masculino , Ratones , Neoplasias/inmunología , Neoplasias/patología , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Treonina/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Recurrence following androgen-deprivation therapy is associated with adverse clinical outcomes in prostate cancer, but the cellular origins and molecular mechanisms underlying this process are poorly defined. We previously identified a population of castration-resistant luminal progenitor cells expressing Bmi1 in the normal mouse prostate that can serve as a cancer cell-of-origin. Here, we investigate the potential of Bmi1-expressing tumor cells that survive castration to initiate recurrence in vivo. METHODS: We employed lineage retracing in Bmi1-CreER; R26R-confetti; Ptenf/f transgenic mice to mark and follow the fate of emerging recurrent tumor clones after castration. A tissue recombination strategy was used to rescue transgenic mouse prostates by regeneration as grafts in immunodeficient hosts. We also used a small molecule Bmi1 inhibitor, PTC-209, to directly test the role of Bmi1 in recurrence. RESULTS: Transgenic prostate tumors (n = 17) regressed upon castration but uniformly recurred within 3 months. Residual regressed tumor lesions exhibited a transient luminal-to-basal phenotypic switch and marked cellular heterogeneity. Additionally, in these lesions, a subpopulation of Bmi1-expressing castration-resistant tumor cells overexpressed the stem cell reprogramming factor Sox2 (mean [SD] = 41.1 [3.8]%, n = 10, P < .001). Bmi1+Sox2+ cells were quiescent (BrdU+Bmi1+Sox2+ at 3.4 [1.5]% vs BrdU+Bmi1+Sox2- at 18.8 [3.4]%, n = 10, P = .009), consistent with a cancer stem cell phenotype. By lineage retracing, we established that recurrence emerges from the Bmi1+ tumor cells in regressed tumors. Furthermore, treatment with the small molecule Bmi1 inhibitor PTC-209 reduced Bmi1+Sox2+ cells (6.1 [1.4]% PTC-209 vs 38.8 [2.3]% vehicle, n = 10, P < .001) and potently suppressed recurrence (retraced clone size = 2.6 [0.5] PTC-209 vs 15.7 [5.9] vehicle, n = 12, P = .04). CONCLUSIONS: These results illustrate the utility of lineage retracing to define the cellular origins of recurrent prostate cancer and identify Bmi1+Sox2+ cells as a source of recurrence that could be targeted therapeutically.
Asunto(s)
Linaje de la Célula , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/patología , Complejo Represivo Polycomb 1/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Factores de Transcripción SOXB1/metabolismo , Animales , Proliferación Celular , Humanos , Masculino , Ratones , Ratones Transgénicos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Factores de Transcripción SOXB1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immune checkpoint inhibitors have not been effective for immunologically "cold" tumors, such as prostate cancer, which contain scarce tumor infiltrating lymphocytes. We hypothesized that select tissue-specific and immunostimulatory bacteria can potentiate these immunotherapies. Here we show that a patient-derived prostate-specific microbe, CP1, in combination with anti-PD-1 immunotherapy, increases survival and decreases tumor burden in orthotopic MYC- and PTEN-mutant prostate cancer models. CP1 administered intra-urethrally specifically homes to and colonizes tumors without causing any systemic toxicities. CP1 increases immunogenic cell death of cancer cells, T cell cytotoxicity, and tumor infiltration by activated CD8 T cells, Th17 T cells, mature dendritic cells, M1 macrophages, and NK cells. CP1 also decreases intra-tumoral regulatory T cells and VEGF. Mechanistically, blocking CP1-recruited T cells from infiltrating the tumor inhibits its therapeutic efficacy. CP1 is an immunotherapeutic tool demonstrating how a tissue-specific microbe can increase tumor immunogenicity and sensitize an otherwise resistant cancer type to immunotherapy.
Asunto(s)
Inmunoterapia/métodos , Neoplasias de la Próstata/terapia , Prostatitis/microbiología , Escherichia coli Uropatógena/inmunología , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/inmunología , Células HEK293 , Humanos , Inyecciones Intralesiones , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Próstata/inmunología , Próstata/microbiología , Próstata/patología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Prostatitis/inmunología , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Resultado del Tratamiento , Microambiente Tumoral/inmunología , Escherichia coli Uropatógena/aislamiento & purificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.