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Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors. Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments. Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an "up then down" dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a "persistently up" profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity. Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.
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DNA Topoisomerases Tipo I , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/farmacologia , DNA Topoisomerases Tipo I/uso terapêutico , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética , Topotecan/farmacologia , Topotecan/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Adverse prognosis in Ewing sarcoma (ES) is associated with the presence of metastases, particularly in bone, tumor hypoxia and chromosomal instability (CIN). Yet, a mechanistic link between these factors remains unknown. We demonstrate that in ES, tumor hypoxia selectively exacerbates bone metastasis. This process is triggered by hypoxia-induced stimulation of the neuropeptide Y (NPY)/Y5 receptor (Y5R) pathway, which leads to RhoA over-activation and cytokinesis failure. These mitotic defects result in the formation of polyploid ES cells, the progeny of which exhibit high CIN, an ability to invade and colonize bone, and a resistance to chemotherapy. Blocking Y5R in hypoxic ES tumors prevents polyploidization and bone metastasis. Our findings provide evidence for the role of the hypoxia-inducible NPY/Y5R/RhoA axis in promoting genomic changes and subsequent osseous dissemination in ES, and suggest that targeting this pathway may prevent CIN and disease progression in ES and other cancers rich in NPY and Y5R.
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Neoplasias Ósseas , Sarcoma de Ewing , Neoplasias Ósseas/genética , Instabilidade Cromossômica , Humanos , Hipóxia , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Sarcoma de Ewing/patologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: To determine the relationship between affective measures and cognition before and after non-cardiac surgery in older adults. METHODS: Observational prospective cohort study in 103 surgical patients age ≥ 60 years old. All participants underwent cognitive testing, Center for Epidemiologic Studies-Depression, and State Anxiety Inventory screening before and 6 weeks after surgery. Cognitive test scores were combined by factor analysis into 4 cognitive domains, whose mean was defined as the continuous cognitive index (CCI). Postoperative global cognitive change was defined by CCI change from before to after surgery, with negative CCI change indicating worsened postoperative global cognition and vice versa. RESULTS: Lower global cognition before surgery was associated with greater baseline depression severity (Spearman's r = -0.30, p = 0.002) and baseline anxiety severity (Spearman's r = -0.25, p = 0.010), and these associations were similar following surgery (r = -0.36, p < 0.001; r = -0.26, p = 0.008, respectively). Neither baseline depression or anxiety severity, nor postoperative changes in depression or anxiety severity, were associated with pre- to postoperative global cognitive change. CONCLUSIONS: Greater depression and anxiety severity were each associated with poorer cognitive performance both before and after surgery in older adults. Yet, neither baseline depression or anxiety symptoms, nor postoperative change in these symptoms, were associated with postoperative cognitive change.
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Cognição , Depressão , Idoso , Ansiedade/epidemiologia , Depressão/epidemiologia , Humanos , Testes Neuropsicológicos , Estudos ProspectivosRESUMO
INTRODUCTION: Constitutive activation of NF-κB has been implicated as being contributive to cancer cell growth, drug resistance, and tumor recurrence in many cancers including breast cancer. Activation of NF-κB leads to nuclear translocation of RelA, a critical component of the NF-κB transcription factor complex, which subsequently binds to specific DNA sites and activates a multitude of genes involved in diverse cell functions. Studies show that triple-negative breast cancer (TNBC) cells possess constitutively active NF-κB and concomitantly have higher levels of nuclear localization of RelA than cytoplasmic RelA. This feature is considered to be associated with the response to chemotherapy. However, currently, there is no specific inhibitor to block nuclear translocation of RelA. METHODS: A structure-based approach was used to develop a small-molecule inhibitor of RelA nuclear translocation. The interaction between this molecule and RelA was verified biophysically through isothermal titration calorimetry and microscale thermophoresis. TNBC cell lines MDA-MB-231 and MDA-MB-468 and a human TNBC xenograft model were used to verify in vitro and in vivo efficacy of the small molecule, respectively. RESULTS: We found that the small molecule, CRL1101, bound specifically to RelA as indicated by the biophysical assays. Further, CRL1101 blocked RelA nuclear translocation in breast cancer cells in vitro, and markedly reduced breast tumor growth in a triple-negative breast cancer xenograft model. CONCLUSION: Our study demonstrates that CRL1101 may lead to new NF-κB-targeted therapeutics for TNBC. Further, blocking of nuclear translocation of shuttling transcription factors may be a useful general strategy in cancer drug development.
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BACKGROUND: Postoperative cognitive dysfunction (POCD), a syndrome of cognitive deficits occurring 1-12 months after surgery primarily in older patients, is associated with poor postoperative outcomes. POCD is hypothesized to result from neuroinflammation; however, the pathways involved remain unclear. Unbiased proteomic analyses have been used to identify neuroinflammatory pathways in multiple neurologic diseases and syndromes but have not yet been applied to POCD. OBJECTIVE: To utilize unbiased mass spectrometry-based proteomics to identify potential neuroinflammatory pathways underlying POCD. METHODS: Unbiased LC-MS/MS proteomics was performed on immunodepleted cerebrospinal fluid (CSF) samples obtained before, 24 hours after, and 6 weeks after major non-cardiac surgery in older adults who did (nâ=â8) or did not develop POCD (nâ=â6). Linear mixed models were used to select peptides and proteins with intensity differences for pathway analysis. RESULTS: Mass spectrometry quantified 8,258 peptides from 1,222 proteins in >â50%of patient samples at all three time points. Twelve peptides from 11 proteins showed differences in expression over time between patients with versus withoutPOCD (qâ<â0.05), including proteins previously implicated in neurodegenerative disease pathophysiology. Additionally, 283 peptides from 182 proteins were identified with trend-level differences (qâ<â0.25) in expression over time between these groups. Among these, pathway analysis revealed that 50 were from 17 proteins mapping to complement and coagulation pathways (qâ=â2.44*10-13). CONCLUSION: These data demonstrate the feasibility of performing unbiased mass spectrometry on perioperative CSF samples to identify pathways associated with POCD. Additionally, they provide hypothesis-generating evidence for CSF complement and coagulation pathway changes in patients with POCD.
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Biomarcadores/líquido cefalorraquidiano , Complicações Cognitivas Pós-Operatórias/líquido cefalorraquidiano , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Masculino , Proteoma/análise , Espectrometria de Massas em TandemRESUMO
BACKGROUNDWe hypothesized that obesity-associated hepatosteatosis is a pathophysiological chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were used to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepatosteatosis versus healthy controls.METHODSCustom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepatosteatosis under investigational new drug guidelines. Fluorescently labeled α-tocopherol was custom-synthesized for cell studies.RESULTSIn healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 3-4 hours; d3- and d6-α-tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1 hour, indicating a key role of the liver in uptake and re-release. Compared with healthy subjects who received 2 mg, subjects with hepatosteatosis had similar d6-α-tocopherol entry rates into liver but reduced initial release rates (P < 0.001). Similarly, pharmacokinetics parameters were reduced in hepatosteatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reductions in kinetics and pharmacokinetics parameters in hepatosteatosis subjects who received 2 mg were echoed by similar reductions in healthy subjects when comparing 5- and 2-mg doses. In vitro, fluorescent-labeled α-tocopherol localized to lipid in fat-loaded hepatocytes, indicating sequestration.CONCLUSIONSThe unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepatosteatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepatosteatosis may similarly alter hepatic physiology of other fat-soluble vitamins.TRIAL REGISTRATIONClinicalTrials.gov, NCT00862433.FUNDINGNational Institute of Diabetes and Digestive and Kidney Diseases and NIH grants DK053213-13, DK067494, and DK081761.
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Fígado Gorduroso/tratamento farmacológico , Vitamina E/administração & dosagem , Vitamina E/farmacocinética , Adolescente , Adulto , Linhagem Celular , Feminino , Células Hep G2 , Humanos , Cinética , Lipídeos , Lipoproteínas , Fígado/metabolismo , Obesidade , Adulto Jovem , alfa-Tocoferol/administração & dosagem , alfa-Tocoferol/farmacocinéticaRESUMO
Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the ß2 and ß3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.
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Lipídeos de Membrana/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sequência de Aminoácidos , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
PURPOSE: Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. RESULTS: We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. MATERIALS AND METHODS: We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. CONCLUSIONS: The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro. We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.
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Triple-negative breast cancer (TNBC) has high rates of local recurrence and distant metastasis, partially due to its high invasiveness. The Forkhead box C1 (FOXC1) transcription factor has been shown to be specifically overexpressed in TNBC and associated with poor clinical outcome. How TNBC's high invasiveness is driven by FOXC1 and its downstream targets remains poorly understood. In the present study, pathway-specific PCR array assays revealed that WNT5A and matrix metalloproteinase-7 (MMP7) were upregulated by FOXC1 in TNBC cells. Interestingly, WNT5A mediates the upregulation of MMP7 by FOXC1 and the WNT5A-MMP7 axis is essential for FOXC1-induced invasiveness of TNBC cells in vitro. Xenograft models showed that the lung extravasation and metastasis of FOXC1-overexpressing TNBC cells were attenuated by knocking out WNT5A, but could be restored by MMP7 overexpression. Mechanistically, FOXC1 can bind directly to the WNT5A promoter region to activate its expression. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), coupled with mass spectrometry, identified FOXC1-interacting proteins including a group of heterogeneous nuclear ribonucleoproteins involved in WNT5A transcription induction. Finally, we found that WNT5A activates NF-κB signaling to induce MMP7 expression. Collectively, these data demonstrate a FOXC1-elicited non-canonical WNT5A signaling mechanism comprising NF-κB and MMP7 that is essential for TNBC cell invasiveness, thereby providing implications toward developing an effective therapy for TNBC.
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Fatores de Transcrição Forkhead/fisiologia , Metaloproteinase 7 da Matriz/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Wnt-5a/genética , Animais , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transdução de Sinais/genética , Ativação Transcricional , Proteína Wnt-5a/metabolismoRESUMO
BACKGROUND: The pathogenesis of human basal-like breast cancer (BLBC) is not well understood and patients with BLBC have a poor prognosis. Expression of the epidermal growth factor receptor (EGFR) and nuclear factor-κB (NF-κB) is well-known to be upregulated in BLBC. The forkhead box C1 (FOXC1) transcription factor, an important prognostic biomarker specific for BLBC, has been shown to be induced by EGF and is critical for EGF effects in breast cancer cells. How FOXC1 is transcriptionally activated in BLBC is not clear. METHODS: Luciferase reporter assays were performed to show that NF-κB-p65 enhances FOXC1 promoter activity in BLBC cells (MDA-MB-468). Electrophoretic mobility shift assay, biotinylated oligonucleotide precipitation assay, and chromatin immunoprecipitation assay were used to show that NF-κB interacts and binds to the promoter region of FOXC1. RESULTS: In this study, we demonstrate that NF-κB is a pivotal mediator of the EGF/EGFR regulation of FOXC1 expression by binding to the FOXC1 promoter to activate FOXC1 transcription. Loss or inhibition of NF-κB diminished FOXC1 expression. CONCLUSION: Collectively, our findings reveal a novel EGFR-NF-κB-FOXC1 signaling axis that is critical for BLBC cell function, supporting the notion that intervention in the FOXC1 pathway may provide potential modalities for BLBC treatment.
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Neoplasias da Mama/patologia , Fator de Crescimento Epidérmico/metabolismo , Fatores de Transcrição Forkhead/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Sequência de Bases , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich "lipid rafts." Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes.IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDα protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.