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Background: Aberrant activation of mTORC1 is clearly defined in TSC, causing uncontrolled cell growth. While mTORC1 inhibitors show efficacy to stabilise tumour growth in TSC, they are not fully curative. Disease facets of TSC that are not restored with mTOR inhibitors might involve NF-κB. The study aimed to characterise NF-κB in the context of TSC. Results: Enrichment of NF-κB-regulated genes was observed in TSC patient tumours, SEN/SEGAs, cortical tubers and a TSC tumour-derived cell line (621 - 101). Highlighting an inflammatory component of TSC, TSC cell models showed an elevated level of NF-κB and STAT3 activation. Herein, we report a dysregulated inflammatory phenotype of TSC2-deficient cells where NF-κB promotes autocrine signalling involving IL-6. Of importance, mTORC1 inhibition does not block this inflammatory signal to promote STAT3, while NF-κB inhibition was much more effective. Combined mTORC1 and NF-κB inhibition was potent at preventing anchorage-independent growth of TSC2-deficient cells, and unlike mTORC1 inhibition alone was sufficient to prevent colony regrowth after cessation of treatment. Conclusion: This study reveals autocrine signalling crosstalk between NF-κB and STAT3 in TSC cell models. Furthermore, the data presented indicate that NF-κB pathway inhibitors could be a viable adjunct therapy with the current mTOR inhibitors to treat TSC.
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Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population.
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Kinase domains are highly conserved within protein kinases in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. The serine/threonine kinases ULK1 and ULK2 are highly conserved with N- and C-terminal domains, phosphate-binding P-loops, αC-helix, regulatory and catalytic spines, and activation loop DFG and APE motifs. Here, we performed molecular dynamics (MD) simulations to understand better the potency and selectivity of the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the P-loop are also associated with ULK-101 inhibitor binding and exit from ULK2. Our data further suggests that ULK-101 could induce a folded P-loop conformation and hydrophobic pocket reflected in its nanomolar potency and kinome selectivity.
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Cancer cells utilize variable metabolic programs in order to maintain homeostasis in response to environmental challenges. To interrogate cancer cell reliance on glycolytic programs under different nutrient availabilities, we analyzed a gene panel containing all glycolytic genes as well as pathways associated with glycolysis. Using this gene panel, we analyzed the impact of an siRNA library on cellular viability in cells containing only glucose or only pyruvate as the major bioenergetic nutrient source. From these panels, we aimed to identify genes that elicited conserved and glycolysis-dependent changes in cellular bioenergetics across glycolysis-promoting and OXPHOS-promoting conditions. To further characterize gene sets within this panel and identify similarities and differences amongst glycolytic tumor RNA-seq profiles across a pan-cancer cohort, we then used unsupervised statistical classification of RNA-seq profiles for glycolytic cancers and non-glycolytic cancer types. Here, Kidney renal clear cell carcinoma (KIRC); Head and Neck squamous cell carcinoma (HNSC); and Lung squamous cell carcinoma (LUSC) defined the glycolytic cancer group, while Prostate adenocarcinoma (PRAD), Thyroid carcinoma (THCA), and Thymoma (THYM) defined the non-glycolytic cancer group. These groups were defined based on glycolysis scoring from previous studies, where KIRC, HNSC, and LUSC had the highest glycolysis scores, meanwhile, PRAD, THCA, and THYM had the lowest. Collectively, these results aimed to identify multi-omic profiles across cancer types with demonstrated variably glycolytic rates. Our analyses provide further support for strategies aiming to classify tumors by metabolic phenotypes in order to therapeutically target tumor-specific vulnerabilities.
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The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.
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Tuberous Sclerosis Complex (TSC) is caused by loss of function variants in either TSC1 or TSC2 and is characterized by broad phenotypic heterogeneity. Currently, there is limited knowledge regarding the role of the mitochondrial genome (mtDNA) in TSC pathogenesis. In this study, we aimed to determine the prevalence and spectrum of germline and somatic mtDNA variants in TSC and identify potential disease modifiers. Analysis of mtDNA amplicon massively parallel sequencing (aMPS) data, off-target mtDNA from whole-exome sequencing (WES), and/or qPCR, revealed mtDNA alterations in 270 diverse tissues (139 TSC-associated tumors and 131 normal tissue samples) from 199 patients and six healthy individuals. Correlation of clinical features to mtDNA variants and haplogroup analysis was done in 102 buccal swabs (age: 20-71 years). No correlation was found between clinical features and either mtDNA variants or haplogroups. No pathogenic variants were identified in the buccal swab samples. Using in silico analysis, we identified three predicted pathogenic variants in tumor samples: MT-ND4 (m.11742G>A, p. Cys328Tyr, VAF: 43%, kidney angiomyolipoma), MT-CYB (m.14775T>C, p. Leu10Pro, VAF: 43%, LAM abdominal tumor) and MT-CYB (m.15555C>T, p. Pro270Leu, VAF: 7%, renal cell carcinoma). Large deletions of the mitochondrial genome were not detected. Analysis of tumors from 23 patients with corresponding normal tissue did not reveal any recurrent tumor-associated somatic variants. The mtDNA/gDNA ratio between tumors and corresponding normal tissue was also unchanged. Overall, our findings demonstrate that the mitochondrial genome is highly stable across tissues and within TSC-associated tumors.
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In cancer, autophagy is upregulated to promote cell survival and tumor growth during times of nutrient stress and can confer resistance to drug treatments. Several major signaling networks control autophagy induction, including the p53 tumor suppressor pathway. In response to DNA damage and other cellular stresses, p53 is stabilized and activated, while HDM2 binds to and ubiquitinates p53 for proteasome degradation. Thus blocking the HDM2-p53 interaction is a promising therapeutic strategy in cancer; however, the potential survival advantage conferred by autophagy induction may limit therapeutic efficacy. In this study, we leveraged an HDM2 inhibitor to identify kinases required for p53-dependent autophagy. Interestingly, we discovered that p53-dependent autophagy requires several kinases, including the myotonic dystrophy protein kinase-like alpha (MRCKα). MRCKα is a CDC42 effector reported to activate actin-myosin cytoskeletal reorganization. Overall, this study provides evidence linking MRCKα to autophagy and reveals additional insights into the role of kinases in p53-dependent autophagy.
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Tumorigenesis relies on the ability of cancer cells to obtain necessary nutrients and fulfill increased energy demands associated with rapid proliferation. However, as a result of increased metabolite consumption and poor vascularization, most cancer cells must survive in a nutrient poor and high cellular stress microenvironment. Cancer cells undergo metabolic reprogramming to evade cell death and ensure proliferation; in particular, cancer cells utilize the catabolic process of autophagy. Autophagy creates an intracellular pool of metabolites by sequestering cytosolic macromolecules in double-membrane vesicles targeted for lysosomal degradation. During times of environmental stress and nutrient starvation, autophagy is upregulated through the dynamic interactions between two nutrient sensing proteins, AMP activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), in cooperation with Unc-51 like autophagy activating kinase 1 (ULK1). In this way, a lack of metabolic nutrients plays a critical role in inducing autophagy, while the products of autophagy also serve as readily available fuel for the cell. In this chapter, we describe methods to visualize and quantify autophagy using a fluorescent sensor of autophagic membranes. Thus, the impact of specific nutrients on autophagy can be measured using live-cell fluorescent microscopy.
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Técnicas de Cultura de Células/métodos , Microscopia Intravital/métodos , Metabolômica/métodos , Nutrientes/análise , Animais , Autofagossomos/metabolismo , Autofagia/fisiologia , Carcinogênese/patologia , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral , Meios de Cultura/química , Proteínas de Fluorescência Verde/química , Humanos , Microscopia Intravital/instrumentação , Metabolômica/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Nutrientes/metabolismo , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodosRESUMO
In response to stress, cancer cells generate nutrients and energy through a cellular recycling process called autophagy, which can promote survival and tumor progression. Accordingly, autophagy inhibition has emerged as a potential cancer treatment strategy. Inhibitors targeting ULK1, an essential and early autophagy regulator, have provided proof of concept for targeting this kinase to inhibit autophagy; however, these are limited individually in their potency, selectivity, or cellular activity. In this study, we report two small molecule ULK1 inhibitors, ULK-100 and ULK-101, and establish superior potency and selectivity over a noteworthy published inhibitor. Moreover, we show that ULK-101 suppresses autophagy induction and autophagic flux in response to different stimuli. Finally, we use ULK-101 to demonstrate that ULK1 inhibition sensitizes KRAS mutant lung cancer cells to nutrient stress. ULK-101 represents a powerful molecular tool to study the role of autophagy in cancer cells and to evaluate the therapeutic potential of autophagy inhibition.
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Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.
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Endocitose , Células Epiteliais/enzimologia , Complexo de Golgi/enzimologia , Túbulos Renais Proximais/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/deficiência , Lisossomos/enzimologia , Autofagia , Linhagem Celular , Proliferação de Células , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Genótipo , Complexo de Golgi/patologia , Humanos , Túbulos Renais Proximais/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Lisossomos/patologia , Proteínas Sensíveis a N-Etilmaleimida/genética , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Fenótipo , Transporte Proteico , ProteóliseRESUMO
CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.
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Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Dimetil Sulfóxido/farmacologia , Etoposídeo/farmacologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Mitose/efeitos dos fármacos , Piperidonas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/farmacologia , Pirróis/farmacologia , Transdução de SinaisRESUMO
Preclinical compounds tested in animal models often demonstrate limited efficacy when transitioned into patients. As a result, individuals are assigned to treatment regimens that may be ineffective at treating their disease. The development of more clinically relevant models, such as patient-derived xenografts (PDXs), will (1) more completely mimic the human condition and (2) more accurately predict tumor responses to previously untested therapeutics.PDX models are clinically relevant as tumor tissue is implanted directly from human donor to the mouse recipient. Therefore, these models prevent cell population selection, intentional or unintentional, as the human tissue adapts to an in vitro, two-dimensional environment prior to implantation into a three-dimensional in vivo murine host. Often, cell heterogeneity and tumor architecture can be maintained from human to the PDX model in the mouse. This protocol describes the engraftment and propagation processes for establishing colorectal (CRC) PDX models in mice, using tumor tissue from human subjects.
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Colo/patologia , Neoplasias Colorretais/patologia , Reto/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentaçãoRESUMO
Fer kinase, a protein involved in the regulation of cell-cell adhesion and proliferation, has been shown to be required during invertebrate development and has been implicated in leukemia, gastric cancer, and liver cancer. However, in vivo roles for Fer during vertebrate development have remained elusive. In this study, we bridge the gap between the invertebrate and vertebrate realms by showing that Fer kinase is required during zebrafish embryogenesis for normal hematopoiesis and vascular organization with distinct kinase dependent and independent functions. In situ hybridization, quantitative PCR and fluorescence activated cell sorting (FACS) analyses revealed an increase in both erythrocyte numbers and gene expression patterns as well as a decrease in the organization of vasculature endothelial cells. Furthermore, rescue experiments have shown that the regulation of hematopoietic proliferation is dependent on Fer kinase activity, while vascular organizing events only require Fer in a kinase-independent manner. Our data suggest a model in which separate kinase dependent and independent functions of Fer act in conjunction with Notch activity in a divergent manner for hematopoietic determination and vascular tissue organization.
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Phosphoinositide 3-kinase (PI3K) family members are involved in diverse cellular fates including cell growth, proliferation, and survival. While many molecular details are known about the Class I and III PI3Ks, less is known about the Class II PI3Ks. To explore the function of all eight PI3K isoforms in autophagy, we knock down each gene individually and measure autophagy. We find a significant decrease in autophagy following siRNA-mediated PIK3C2A (encoding the Class 2 PI3K, PI3K-C2α) knockdown. This defective autophagy is rescued by exogenous PI3K-C2α, but not kinase-dead PI3K-C2α. Using confocal microscopy, we probe for markers of endocytosis and autophagy, revealing that PI3K-C2α colocalizes with markers of endocytosis. Though endocytic uptake is intact, as demonstrated by transferrin labeling, PIK3C2A knockdown results in vesicle accumulation at the recycling endosome. We isolate distinct membrane sources and observe that PI3K-C2α interacts with markers of endocytosis and autophagy, notably ATG9. Knockdown of either PIK3C2A or ATG9A/B, but not PI3KC3, results in an accumulation of transferrin-positive clathrin coated vesicles and RAB11-positive vesicles at the recycling endosome. Taken together, these results support a role for PI3K-C2α in the proper maturation of endosomes, and suggest that PI3K-C2α may be a critical node connecting the endocytic and autophagic pathways.
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Biomarcadores/metabolismo , Endossomos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Vesículas Transportadoras/metabolismo , Autofagia , Linhagem Celular , Proliferação de Células , Endocitose , Técnicas de Silenciamento de Genes , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Among breast cancers, the triple-negative breast cancer (TNBC) subtype has the worst prognosis with no approved targeted therapies and only standard chemotherapy as the backbone of systemic therapy. Unique metabolic changes in cancer progression provide innovative therapeutic opportunities. The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), and MET receptor are highly expressed in TNBC, making both promising therapeutic targets. RTK signaling profoundly alters cellular metabolism by increasing glucose consumption and subsequently diverting glucose carbon sources into metabolic pathways necessary to support the tumorigenesis. Therefore, detailed metabolic profiles of TNBC subtypes and their response to tyrosine kinase inhibitors may identify therapeutic sensitivities. METHODS: We quantified the metabolic profiles of TNBC cell lines representing multiple TNBC subtypes using gas chromatography mass spectrometry. In addition, we subjected MDA-MB-231, MDA-MB-468, Hs578T, and HCC70 cell lines to metabolic flux analysis of basal and maximal glycolytic and mitochondrial oxidative rates. Metabolic pool size and flux measurements were performed in the presence and absence of the MET inhibitor, INC280/capmatinib, and the EGFR inhibitor, erlotinib. Further, the sensitivities of these cells to modulators of core metabolic pathways were determined. In addition, we annotated a rate-limiting metabolic enzymes library and performed a siRNA screen in combination with MET or EGFR inhibitors to validate synergistic effects. RESULTS: TNBC cell line models displayed significant metabolic heterogeneity with respect to basal and maximal metabolic rates and responses to RTK and metabolic pathway inhibitors. Comprehensive systems biology analysis of metabolic perturbations, combined siRNA and tyrosine kinase inhibitor screens identified a core set of TCA cycle and fatty acid pathways whose perturbation sensitizes TNBC cells to small molecule targeting of receptor tyrosine kinases. CONCLUSIONS: Similar to the genomic heterogeneity observed in TNBC, our results reveal metabolic heterogeneity among TNBC subtypes and demonstrate that understanding metabolic profiles and drug responses may prove valuable in targeting TNBC subtypes and identifying therapeutic susceptibilities in TNBC patients. Perturbation of metabolic pathways sensitizes TNBC to inhibition of receptor tyrosine kinases. Such metabolic vulnerabilities offer promise for effective therapeutic targeting for TNBC patients.
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Tuberous sclerosis complex (TSC) is a rare genetic disease causing multisystem growth of benign tumours and other hamartomatous lesions, which leads to diverse and debilitating clinical symptoms. Patients are born with TSC1 or TSC2 mutations, and somatic inactivation of wild-type alleles drives MTOR activation; however, second hits to TSC1/TSC2 are not always observed. Here, we present the genomic landscape of TSC hamartomas. We determine that TSC lesions contain a low somatic mutational burden relative to carcinomas, a subset feature large-scale chromosomal aberrations, and highly conserved molecular signatures for each type exist. Analysis of the molecular signatures coupled with computational approaches reveals unique aspects of cellular heterogeneity and cell origin. Using immune data sets, we identify significant neuroinflammation in TSC-associated brain tumours. Taken together, this molecular catalogue of TSC serves as a resource into the origin of these hamartomas and provides a framework that unifies genomic and transcriptomic dimensions for complex tumours.
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Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Carcinoma/genética , Carcinoma/metabolismo , Genômica , Humanos , Mutação , Esclerose Tuberosa/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
DBF4-dependent kinase (DDK) is a two-subunit kinase required for initiating DNA replication at individual origins and is composed of CDC7 kinase and its regulatory subunit DBF4. Both subunits are highly expressed in many diverse tumor cell lines and primary tumors, and this is correlated with poor prognosis. Inhibiting DDK causes apoptosis of tumor cells, but not normal cells, through a largely unknown mechanism. Firstly, to understand why DDK is often overexpressed in tumors, we identified gene expression signatures that correlate with DDK high- and DDK low-expressing lung adenocarcinomas. We found that increased DDK expression is highly correlated with inactivation of RB1-E2F and p53 tumor suppressor pathways. Both CDC7 and DBF4 promoters bind E2F, suggesting that increased E2F activity in RB1 mutant cancers promotes increased DDK expression. Surprisingly, increased DDK expression levels are also correlated with both increased chemoresistance and genome-wide mutation frequencies. Our data further suggest that high DDK levels directly promote elevated mutation frequencies. Secondly, we performed an RNAi screen to investigate how DDK inhibition causes apoptosis of tumor cells. We identified 23 kinases and phosphatases required for apoptosis when DDK is inhibited. These hits include checkpoint genes, G2/M cell cycle regulators, and known tumor suppressors leading to the hypothesis that inhibiting mitotic progression can protect against DDKi-induced apoptosis. Characterization of one novel hit, the LATS2 tumor suppressor, suggests that it promotes apoptosis independently of the upstream MST1/2 kinases in the Hippo signaling pathway.
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Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a Retinoblastoma/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The search for a single silver bullet for the treatment of cancer has now been overshadowed by the identification of multiple therapeutic targets unique to each malignancy and even to each patient. In recent years, autophagy has emerged as one such therapeutic target. In response to both therapeutic and oncogenic stress, cancer cells upregulate and demonstrate an increased dependence upon this intracellular recycling process. Particularly in malignancies that currently lack targeted therapeutic options, autophagy inhibitors are the next hopeful prospects for the treatment of this disease. In this review, we discuss the rapid evolution of autophagy inhibitors from early lysosomotropic agents to next-generation lysosome-targeted drugs and beyond.
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Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antimaláricos/química , Antineoplásicos/química , Humanos , Lisossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Next-generation sequencing (NGS) capabilities can affect therapeutic decisions in patients with complex, advanced, or refractory cancer. We report the feasibility of a tumor sequencing advisory board at a regional cancer center. Specimens were analyzed for approximately 2800 mutations in 50 genes. Outcomes of interest included tumor sequencing advisory board function and processes, timely discussion of results, and proportion of reports having potentially actionable mutations. NGS results were successfully generated for 15 patients, with median time from tissue processing to reporting of 11.6 days (range, 5 to 21 days), and presented at a biweekly multidisciplinary tumor sequencing advisory board. Attendance averaged 19 participants (range, 12 to 24) at 20 days after patient enrollment (range, 10 to 30 days). Twenty-seven (range, 1 to 4 per patient) potentially actionable mutations were detected in 11 of 15 patients: TP53 (n = 6), KRAS (n = 4), MET (n = 3), APC (n = 3), CDKN2A (n = 2), PTEN (n = 2), PIK3CA, FLT3, NRAS, VHL, BRAF, SMAD4, and ATM. The Hotspot Panel is now offered as a clinically available test at our institution. NGS results can be obtained by in-house high-throughput sequencing and reviewed in a multidisciplinary tumor sequencing advisory board in a clinically relevant manner. The essential components of a center for personalized cancer care can support clinical decisions outside the university.
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Mutação/genética , Neoplasias/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Medicina de Precisão/métodosRESUMO
Tuberous sclerosis complex (TSC) is a genetic autosomal dominant disorder characterized by benign tumor-like lesions, called hamartomas, in multiple organ systems, including the brain, skin, heart, kidneys, and lung. These hamartomas cause a diverse set of clinical problems based on their location and often result in epilepsy, learning difficulties, and behavioral problems. TSC is caused by mutations within the TSC1 or TSC2 genes that inactivate the genes' tumor-suppressive function and drive hamartomatous cell growth. In normal cells, TSC1 and TSC2 integrate growth signals and nutrient inputs to downregulate signaling to mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine kinase that controls cell growth and cell survival. The molecular connection between TSC and mTOR led to the clinical use of allosteric mTOR inhibitors (sirolimus and everolimus) for the treatment of TSC. Everolimus is approved for subependymal giant cell astrocytomas and renal angiomyolipomas in patients with TSC. Sirolimus, though not approved for TSC, has undergone considerable investigation to treat various aspects of the disease. Everolimus and sirolimus selectively inhibit mTOR signaling with similar molecular mechanisms, but with distinct clinical profiles. This review differentiates mTOR inhibitors in TSC while describing the molecular mechanisms, pathogenic mutations, and clinical trial outcomes for managing TSC.