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BACKGROUND: Epigenetic changes link medical, social, and environmental factors with cardiovascular and kidney disease and, more recently, with cancer. The mechanistic link between metabolic health and epigenetic changes is only starting to be investigated. In our in vitro and in vivo studies, we performed a broad analysis of the link between hyperinsulinemia and chromatin acetylation; our top "hit" was chromatin opening at H3K9ac. METHODS: Building on our published preclinical studies, here, we performed a detailed analysis of the link between insulin resistance, chromatin acetylation, and inflammation using an initial test set of 28 women and validation sets of 245, 22, and 53 women. RESULTS: ChIP-seq identified chromatin acetylation and opening at the genes coding for TNFα and IL6 in insulin-resistant women. Pathway analysis identified inflammatory response genes, NFκB/TNFα-signaling, reactome cytokine signaling, innate immunity, and senescence. Consistent with this finding, flow cytometry identified increased senescent circulating peripheral T-cells. DNA methylation analysis identified evidence of accelerated aging in insulin-resistant vs. metabolically healthy women. CONCLUSIONS: This study shows that insulin-resistant women have increased chromatin acetylation/opening, inflammation, and, perhaps, accelerated aging. Given the role that inflammation plays in cancer initiation and progression, these studies provide a potential mechanistic link between insulin resistance and cancer.
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Trastuzumab therapy in HER2+ breast cancer patients has mixed success owing to acquired resistance to therapy. A detailed understanding of downstream molecular cascades resulting from trastuzumab resistance is yet to emerge. In this study, we investigate the cellular mechanisms underlying acquired resistance using trastuzumab-sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG across time. We probe early receptor organization through microscopy and signaling events through multiomics measurements and assess the bioenergetic state through mitochondrial measurements. Integrative analyses of our measurements reveal significant alterations in EGF-treated BT474 HER2 membrane dynamics and robust downstream activation of PI3K/AKT/mTORC1 signaling. EGF-treated BT474R shows a sustained interferon-independent activation of the IRF1/STAT1 cascade, potentially contributing to trastuzumab resistance. Both cell lines exhibit temporally divergent metabolic demands and HIF1A-mediated stress responses. BT474R demonstrates inherently increased mitochondrial activity. HRG treatment in BT474R leads to a pronounced reduction in AR expression, affecting downstream lipid metabolism with implications for treatment response. Our results provide novel insights into mechanistic changes underlying ligand treatment in BT474 and BT474R and emphasize the pivotal role of endogenous ligands. These results can serve as a framework for furthering the understanding of trastuzumab resistance, with therapeutic implications for women with acquired resistance.
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BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high mortality rate due to a lack of therapeutic targets. Many TNBC cells are reliant on extracellular arginine for survival and express high levels of binding immunoglobin protein (BiP), a marker of metastasis and endoplasmic reticulum (ER) stress response. METHODS: In this study, the effect of arginine shortage on BiP expression in the TNBC cell line MDA-MB-231 was evaluated. Two stable cell lines were generated in MDA-MB-231 cells: the first expressed wild-type BiP, and the second expressed a mutated BiP free of the two arginine pause-site codons, CCU and CGU, termed G-BiP. RESULTS: The results showed that arginine shortage induced a non-canonical ER stress response by inhibiting BiP translation via ribosome pausing. Overexpression of G-BiP in MDA-MB-231 cells promoted cell resistance to arginine shortage compared to cells overexpressing wild-type BiP. Additionally, limiting arginine led to decreased levels of the spliced XBP1 in the G-BiP overexpressing cells, potentially contributing to their improved survival compared to the parental WT BiP overexpressing cells. CONCLUSION: In conclusion, these findings suggest that the downregulation of BiP disrupts proteostasis during arginine shortage-induced non-canonical ER stress and plays a key role in cell growth inhibition, indicating BiP as a target of codon-specific ribosome pausing upon arginine shortage.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Transporte , Arginina/metabolismo , Ribossomos , Linhagem Celular TumoralRESUMO
Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.
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Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases ß-catenin-mediated proliferation of multiple TNBC cell lines and TNBC patient-derived xenograft (PDX)-derived cell lines. In vitro, ICG-001 was effective in combination with the conventional cytotoxic chemotherapeutics, cisplatin and doxorubicin, to decrease the proliferation of MDA-MB-231 cells. In contrast, in TNBC PDX-derived cells doxorubicin plus ICG-001 was synergistic, while pairing with cisplatin was not as effective. Mechanistically, cytotoxicity induced by doxorubicin, but not cisplatin, with ICG-001 was associated with increased cleavage of PARP-1 in the PDX cells only. In vivo, MDA-MB-231 and TNBC PDX orthotopic primary tumors initiated de novo simultaneous multi-organ metastases, including bone metastases. WNT monotherapy blocked multi-organ metastases as measured by luciferase imaging and histology. The loss of expression of the WNT10B/ß-catenin direct targets HMGA2, EZH2, AXIN2, MYC, PCNA, CCND1, transcriptionally active ß-catenin, SNAIL and vimentin both in vitro and in vivo in the primary tumors mechanistically explains loss of multi-organ metastases. WNT monotherapy induced VEGFA expression in both tumor model systems, whereas increased CD31 was observed only in the MDA-MB-231 tumors. Moreover, WNT-inhibition sensitized the anticancer response of the TNBC PDX model to doxorubicin, preventing simultaneous metastases to the liver and ovaries, as well as to bone. Our data demonstrate that WNT-inhibition sensitizes TNBC to anthracyclines and treats multi-organ metastases of TNBC.
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The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.
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Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for ß-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for ß-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on ß-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.
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Proteínas Proto-Oncogênicas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Wnt/metabolismo , Acetilação , Alelos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste/biossíntese , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Proteína HMGA2/biossíntese , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Metástase Neoplásica , Pirimidinonas/administração & dosagem , Pirimidinonas/farmacologia , Taxa de Sobrevida , Fator de Transcrição 4 , Neoplasias de Mama Triplo Negativas/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. METHODS: We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. RESULTS: Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. CONCLUSIONS: This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.
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Reprogramação Celular/genética , Vírus da Encefalite Equina Venezuelana/genética , Células Epiteliais/efeitos dos fármacos , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Retiniana/terapia , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Fibroblastos/citologia , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Injeções Intraoculares , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Cultura Primária de Células , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/fisiologia , Pele/citologiaRESUMO
Stem cell technologies, especially patient-specific, induced stem cell pluripotency and directed differentiation, hold great promise for changing the landscape of medical therapies. Proper exploitation of these methods may lead to personalized organ transplants, but to regenerate organs, it is necessary to develop methods for assembling differentiated cells into functional, organ-level tissues. The generation of three-dimensional human tissue models also holds potential for medical advances in disease modeling, as full organ functionality may not be necessary to recapitulate disease pathophysiology. This is specifically true of lung diseases where animal models often do not recapitulate human disease. Here, we present a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor-ß1 to single cell-type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two-dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell-cell contact and recapitulating the lung microenvironment. Our bottom-up approach for synthesizing patient-specific lung tissue in a scalable system allows for the development of relevant human lung disease models with the potential for high throughput drug screening to identify targeted therapies. Stem Cells Translational Medicine 2017;6:622-633.
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Técnicas de Cultura de Células , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Pulmão/patologia , Organoides/patologia , Engenharia Tecidual/métodos , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Pulmão/fisiopatologia , Organoides/efeitos dos fármacos , Fenótipo , Fatores de Tempo , Engenharia Tecidual/instrumentação , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Bronchopulmonary dysplasia (BPD) is a leading complication of premature birth and occurs primarily in infants delivered during the saccular stage of lung development. Histopathology shows decreased alveolarization and a pattern of fibroblast proliferation and differentiation to the myofibroblast phenotype. Little is known about the molecular pathways and cellular mechanisms that define BPD pathophysiology and progression. We have developed a novel three-dimensional human model of the fibroblast activation associated with BPD, and using this model we have identified the Notch pathway as a key driver of fibroblast activation and proliferation in response to changes in oxygen. Fetal lung fibroblasts were cultured on sodium alginate beads to generate lung organoids. After exposure to alternating hypoxia and hyperoxia, the organoids developed a phenotypic response characterized by increased α-smooth muscle actin (α-SMA) expression and other genes known to be upregulated in BPD and also demonstrated increased expression of downstream effectors of the Notch pathway. Inhibition of Notch with a γ-secretase inhibitor prevented the development of the pattern of cellular proliferation and α-SMA expression in our model. Analysis of human autopsy tissue from the lungs of infants who expired with BPD demonstrated evidence of Notch activation within fibrotic areas of the alveolar septae, suggesting that Notch may be a key driver of BPD pathophysiology.
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Displasia Broncopulmonar/patologia , Transdução de Sinais , Alginatos/química , Displasia Broncopulmonar/metabolismo , Técnicas de Cultura de Células , Hipóxia Celular , Células Cultivadas , Meios de Cultura/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Receptores Notch/metabolismoRESUMO
Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal and an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer.
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Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Notch/metabolismo , Células-Tronco/citologia , Traqueia/citologia , Animais , Antioxidantes/metabolismo , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Homeostase , Humanos , Camundongos , Oxirredução , Polidocanol , Polietilenoglicóis/química , Transdução de Sinais , CicatrizaçãoRESUMO
Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.