RESUMEN
As many as 30% of non-small cell lung cancer (NSCLC) patients harbor oncogenic KRAS mutations, which lead to extensive remodeling of the tumor immune microenvironment. Though co-mutations in several genes have prognostic relevance in KRAS-mutated patients, their effect on tumor immunogenicity are poorly understood. In the present study, a total of 189 NSCLC patients underwent a standardized analysis including immunohistochemistry, whole exome DNA sequencing, and whole transcriptome RNA sequencing. Patients with activating KRAS mutations demonstrated a significant increase in PD-L1 expression and CD8+ T-cell infiltration. Both were increased in the presence of a co-occurring TP53 mutation and lost with STK11 co-mutation. Subsequent genomic analysis demonstrated that KRAS/TP53 co-mutated tumors had a significant decrease in the expression of glycolysis-associated genes, and increase in several genes involved in lipid metabolism, notably Lipoprotein Lipase (LPL), Low Density Lipoprotein Receptor (LDLR), and LDLRAD4. Conversely, in the immune-excluded KRAS/STK11 co-mutated group, we observed diminished lipid metabolism and no change in anaerobic glycolysis. Interestingly, in patients with low expression of LPL, LDLR, or LDLRAD4, KRAS mutations had no effect on tumor immunogenicity. However, in patients with robust expression of these genes, KRAS mutations were associated with increased immunogenicity and associated with improved overall survival. Our data further suggest that the loss of STK11 may function as a metabolic switch, suppressing lipid metabolism in favor of glycolysis, thereby negating KRAS-induced immunogenicity. Hence, this concept warrants continued exploration, both as a predictive biomarker and potential target for therapy in patients receiving ICI-based immunotherapy.
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There is currently no effective treatment for pancreatic ductal adenocarcinoma (PDAC). While palliative chemotherapy offers a survival benefit to most patients, nearly all will eventually progress on treatment and long-term survivability remains poor. Given the lack of subsequent line treatment options, in this study, we sought to identify novel strategies to prevent, delay, or overcome resistance to gemcitabine, one of the most widely used medications in PDAC. Using a combination of single-cell RNA sequencing and high-throughput proteomic analysis, we identified a subset of gemcitabine-resistant tumor cells enriched for calcium/calmodulin signaling. Pharmacologic inhibition of calcium-dependent calmodulin activation led to the rapid loss of drug-resistant phenotypes in vitro, which additional single-cell RNA sequencing identified was due to impaired activation of the RAS/ERK signaling pathway. Consistent with these observations, calcium chelation or depletion of calcium in the culture media also impaired ERK activation in gemcitabine-resistant cells, and restored therapeutic responses to gemcitabine in vitro. We observed similar results using calcium channel blockers (CCBs) such as amlodipine, which inhibited prosurvival ERK signaling in vitro and markedly enhanced therapeutic responses to gemcitabine in both orthotopic xenografts and transgenic models of PDAC. Combined, these results offer insight into a potential means of gemcitabine resistance and suggest that select CCBs may provide a clinical benefit to PDAC patients receiving gemcitabine-based chemotherapy.
Asunto(s)
Antineoplásicos , Neoplasias Pancreáticas , Amlodipino/farmacología , Amlodipino/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Calmodulina , Desoxicitidina/análogos & derivados , Humanos , Neoplasias Pancreáticas/patología , Estados Unidos , Gemcitabina , Neoplasias PancreáticasRESUMEN
Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3ß to facilitate GSK3ß phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3ß targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.
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Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hexoquinasa/metabolismo , Neoplasias/patología , Proteínas de Anclaje a la Quinasa A/metabolismo , Células A549 , Animales , Células CHO , Carcinogénesis/patología , Línea Celular Tumoral , Cricetulus , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Desoxiglucosa/farmacología , Transición Epitelial-Mesenquimal/fisiología , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glicosilación , Células HCT116 , Células HEK293 , Hexoquinasa/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Metástasis de la Neoplasia/patología , Fosforilación/efectos de los fármacos , Ratas , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
Lapachol is a well-studied natural product that has been receiving great interest due to its anticancer properties that target oxidative stress. In the present work, two novel lapachol-containing ruthenium(II) complexes [Ru(Lap)(dppm)(bipy)]PF6 (1) and [Ru(Lap)(dppm)(phen)]PF6 (2) [Lap = lapachol, dppm = 1,1'-bis(diphosphino)methane, bipy = 2,2'-bipyridine, phen = 1,10-phenantroline] were synthesized, fully characterized, and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures, as well as in a 3D model of multicellular spheroids formed from DU-145 human prostate adenocarcinoma cells. Furthermore, the complex (2) suppressed the colony formation, induced G2/M-phase arrest, and downregulated Aurora-B. The mechanism studies suggest that complex (2) stimulate the overproduction of reactive oxygen species (ROS) and triggers caspase-dependent apoptosis as a result of changes in expression of several genes related to cell proliferation and caspase-3 and -9 activation. Interestingly, we found that N-acetyl-L-cysteine, a ROS scavenger, suppressed the generation of intracellular ROS induced by complex (2), and decreased its cytotoxicity, indicating that ROS-mediated DNA damage leads the DU-145 cells into apoptosis. Overall, we highlighted that coordination of lapachol to phosphinic ruthenium(II) compounds considerably improves the antiproliferative activities of resulting complexes granting attractive selectivity to human prostate adenocarcinoma cells. The DNA damage response to ROS seems to be involved in the induction of caspase-mediated cell death that plays an important role in the complexes' cytotoxicity. Upon further investigations, this novel class of lapachol-containing ruthenium(II) complexes might indicate promising chemotherapeutic agents for prostate cancer therapy.
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Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.
Asunto(s)
Resistencia a Antineoplásicos/genética , Tolerancia a Medicamentos/genética , Tolerancia a Medicamentos/fisiología , Neoplasias/genética , Neoplasias/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Combinación de Medicamentos , Descubrimiento de Drogas , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células U937RESUMEN
Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4â¯h of stimulus, with DNA damages persisting in HepG2 cells after 24â¯h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.
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Daño del ADN/efectos de los fármacos , L-Aminoácido Oxidasa/toxicidad , Estrés Oxidativo/efectos de los fármacos , Venenos de Serpiente/toxicidad , Animales , Daño del ADN/fisiología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , L-Aminoácido Oxidasa/aislamiento & purificación , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Estrés Oxidativo/fisiología , Venenos de Serpiente/aislamiento & purificaciónRESUMEN
Erythrosine B (ErB) is a cherry pink food colorant and is widely used in foods, drugs, and cosmetics. Quinoline yellow (QY) is a chinophthalon derivative used in cosmetic compositions for application to the skin, lips, and/or body surface. Previously, ErB and QY synthetic dyes were found to induce DNA damage in HepG2 cells. The aim of this study was to investigate the molecular basis underlying the genotoxicity attributed to ErB and QY using the RT2 Profiler polymerase chain reaction array and by analyzing the expression profile of 84 genes involved in cell cycle arrest, apoptosis, and DNA repair in HepG2 cells. ErB (70 mg/L) significantly decreased the expression of two genes ( FEN1 and REV1) related to DNA base repair. One gene ( LIG1) was downregulated and 20 genes related to ATR/ATM signaling ( ATR, RBBP8, RAD1, CHEK1, CHEK2, TOPB1), nucleotide excision repair ( ERCC1, XPA), base excision repair ( FEN1, MBD4), mismatch repair ( MLH1, MSH3, TP73), double strand break repair ( BLM), other DNA repair genes ( BRIP1, FANCA, GADD45A, REV1), and apoptosis ( BAX, PPP1R15A) were significantly increased after treatment with QY (20 mg/L). In conclusion, our data suggest that the genotoxic mechanism of ErB and QY dyes involves the modulation of genes related to the DNA repair system and cell cycle.
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Colorantes/toxicidad , Reparación del ADN/efectos de los fármacos , Eritrosina/toxicidad , Expresión Génica/efectos de los fármacos , Quinolinas/toxicidad , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , NutrigenómicaRESUMEN
L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1ß, LTB4 and PGE2. In vitro, IL-6 and IL-1ß production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1ß that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.