RESUMEN
Natural Killer (NK) cell responses are regulated by a variety of different surface receptors. While we can determine the overall positive or negative effect of a given receptor on NK cell functions, investigating NK cell regulation in a quantitative way is challenging. To quantitatively investigate individual receptors for their effect on NK cell activation, we chose to functionalize latex beads that have approximately the same size as lymphocytes with defined amounts of specific antibodies directed against distinct activating receptors. This enabled us to investigate NK cell reactivity in a defined, clean, and controllable system. Only CD16 and NKp30 could activate the degranulation of resting human NK cells. CD16, NKG2D, NKp30, NKp44, and NKp46 were able to activate cultured NK cells. NK cell activation resulted in the induction of polyfunctional cells that degranulated and produced IFN-γ and MIP-1ß. Interestingly, polyfunctional NK cells were only induced by triggering ITAM-coupled receptors. NKp44 showed a very sensitive response pattern, where a small increase in receptor stimulation caused maximal NK cell activity. In contrast, stimulation of 2B4 induced very little NK cell degranulation, while providing sufficient signal for NK cell adhesion. Our data demonstrate that activating receptors differ in their effectiveness to stimulate NK cells.
Asunto(s)
Anticuerpos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Anticuerpos/química , Adhesión Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Microesferas , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/genética , Receptor 2 Gatillante de la Citotoxidad Natural/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/genética , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Unión Proteica , Receptores de IgG/genética , Receptores de IgG/inmunología , Transducción de SeñalRESUMEN
Adaptive plasticity to the standard chemotherapeutic temozolomide (TMZ) leads to glioblastoma progression. Here, we examine early stages of this process in patient-derived cellular models, exposing the human lysine-specific demethylase 5B (KDM5B) as a prospective indicator for subclonal expansion. By integration of a reporter, we show its preferential activity in rare, stem-like ALDH1A1+ cells, immediately increasing expression upon TMZ exposure. Naive, genetically unmodified KDM5Bhigh cells phosphorylate AKT (pAKT) and act as slow-cycling persisters under TMZ. Knockdown of KDM5B reverses pAKT levels, simultaneously increasing PTEN expression and TMZ sensitivity. Pharmacological inhibition of PTEN rescues the effect. Interference with KDM5B subsequent to TMZ decreases cellular vitality, and clonal tracing with DNA barcoding demonstrates high individual levels of KDM5B to predict subclonal expansion already before TMZ exposure. Thus, KDM5Bhigh treatment-naive cells preferentially contribute to the dynamics of drug resistance under TMZ. These findings may serve as a cornerstone for future biomarker-assisted clinical trials.
RESUMEN
Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.
Asunto(s)
Antineoplásicos , Apoptosis , Procesamiento Proteico-Postraduccional , Proteómica , Antígenos CD20/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Daño del ADN , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteómica/métodos , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , HumanosRESUMEN
Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for most cancer-related deaths but the cellular changes that enable cancer cells to leave the primary tumor and establish inoperable and lethal metastases remain poorly understood. To uncover genes that are specifically required to sustain metastasis survival or growth, we performed a genome-scale pooled lentiviral-shRNA library screen in cells that represent nonmetastatic and metastatic states of lung adenocarcinoma. Mitochondrial ribosome and mitochondria-associated genes were identified as top gene sets associated with metastasis-specific lethality. Metastasis-derived cell lines in vitro and metastases analyzed ex vivo from an autochthonous lung cancer mouse model had lower mitochondrial membrane potential and reduced mitochondrial functionality than nonmetastatic primary tumors. Electron microscopy of metastases uncovered irregular mitochondria with bridging and loss of normal membrane structure. Consistent with these findings, compounds that inhibit mitochondrial translation or replication had a greater effect on the growth of metastasis-derived cells. Finally, mice with established tumors developed fewer metastases upon treatment with phenformin in vivo. These results suggest that the metastatic cell state in lung adenocarcinoma is associated with a specifically altered mitochondrial functionality that can be therapeutically exploited. SIGNIFICANCE: This study characterizes altered mitochondria functionality of the metastatic cell state in lung cancer and opens new avenues for metastasis-specific therapeutic targeting.
Asunto(s)
Adenocarcinoma/genética , Modelos Animales de Enfermedad , Neoplasias Pulmonares/genética , Mitocondrias/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/metabolismo , Metástasis de la Neoplasia , Interferencia de ARNRESUMEN
Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.
Asunto(s)
Plasticidad de la Célula/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Neoplasias/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Metástasis de la Neoplasia , Células Madre Neoplásicas/patologíaRESUMEN
Lung cancer mortality largely results from metastasis. Despite curative surgery many patients with early-stage non-small cell lung cancer ultimately succumb to metastatic relapse. Current risk reduction strategies based on cytotoxic chemotherapy and radiation have only modest activity. Against this background, we functionally screened for novel metastasis modulators using a barcoded shRNA library and an orthotopic lung cancer model. We identified aryl hydrocarbon receptor (AHR), a sensor of xenobiotic chemicals and transcription factor, as suppressor of lung cancer metastasis. Knockdown of endogenous AHR induces epithelial-mesenchymal transition signatures, increases invasiveness of lung cancer cells in vitro and metastasis formation in vivo. Low intratumoral AHR expression associates with inferior outcome of patients with resected lung adenocarcinomas. Mechanistically, AHR triggers ATF4 signaling and represses matrix metalloproteinase activity, both counteracting metastatic programs. These findings link the xenobiotic defense system with control of lung cancer progression. AHR-regulated pathways are promising targets for innovative anti-metastatic strategies.
RESUMEN
Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence of disseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.