ABSTRACT
Spatially annotated single-cell datasets provide unprecedented opportunities to dissect cell-cell communication in development and disease. Heterotypic signaling includes interactions between different cell types and is well established in tissue development and spatial organization. Epithelial organization requires several different programs that are tightly regulated. Planar cell polarity is the organization of epithelial cells along the planar axis orthogonal to the apical-basal axis. In this study, we investigate planar cell polarity factors and explore the implications of developmental regulators as malignant drivers. Utilizing cancer systems biology analysis, we derive gene expression network for WNT-ligands (WNT) and their cognate frizzled (FZD) receptors in skin cutaneous melanoma. The profiles supported by unsupervised clustering of multiple-sequence alignments identify ligand-independent signaling and implications for metastatic progression based on the underpinning developmental spatial program. Omics studies and spatial biology connect developmental programs with oncological events and explain key spatial features of metastatic aggressiveness. Dysregulation of prominent planar cell polarity factors such specific representative of the WNT and FZD families in malignant melanoma recapitulates the development program of normal melanocytes but in an uncontrolled and disorganized fashion.
ABSTRACT
Spatially annotated single-cell datasets provide unprecedented opportunities to dissect cell-cell communication in development and disease. Heterotypic signaling includes interactions between different cell types and is well established in tissue development and spatial organization. Epithelial organization requires several different programs that are tightly regulated. Planar cell polarity (PCP) is the organization of epithelial cells along the planar axis, orthogonal to the apical-basal axis. Here, we investigate PCP factors and explore the implications of developmental regulators as malignant drivers. Utilizing cancer systems biology analysis, we derive a gene expression network for WNT-ligands (WNT) and their cognate frizzled (FZD) receptors in skin cutaneous melanoma. The profiles supported by unsupervised clustering of multiple-sequence alignments identify ligand-independent signaling and implications for metastatic progression based on the underpinning developmental spatial program. Omics studies and spatial biology connect developmental programs with oncological events and explain key spatial features of metastatic aggressiveness. Dysregulation of prominent PCP factors such as specific representatives of the WNT and FZD families in malignant melanoma recapitulates the development program of normal melanocytes but in an uncontrolled and disorganized fashion.
ABSTRACT
Lineage-specific differentiation programs are activated by epigenetic changes in chromatin structure. Melanin-producing melanocytes maintain a gene expression program ensuring appropriate enzymatic conversion of metabolites into the pigment, melanin, and transfer to surrounding cells. During neuroectodermal development, SMARCA4 (BRG1), the catalytic subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes, is essential for lineage specification. SMARCA4 is also required for development of multipotent neural crest precursors into melanoblasts, which differentiate into pigment-producing melanocytes. In addition to the catalytic domain, SMARCA4 and several SWI/SNF subunits contain bromodomains which are amenable to pharmacological inhibition. We investigated the effects of pharmacological inhibitors of SWI/SNF bromodomains on melanocyte differentiation. Strikingly, treatment of murine melanoblasts and human neonatal epidermal melanocytes with selected bromodomain inhibitors abrogated melanin synthesis and visible pigmentation. Using functional genomics, iBRD9, a small molecule selective for the bromodomain of BRD9 was found to repress pigmentation-specific gene expression. Depletion of BRD9 confirmed a requirement for expression of pigmentation genes in the differentiation program from melanoblasts into pigmented melanocytes and in melanoma cells. Chromatin immunoprecipitation assays showed that iBRD9 disrupts the occupancy of BRD9 and the catalytic subunit SMARCA4 at melanocyte-specific loci. These data indicate that BRD9 promotes melanocyte pigmentation whereas pharmacological inhibition of BRD9 is repressive.
Subject(s)
Melanins , Pigmentation Disorders , Infant, Newborn , Humans , Mice , Animals , Melanins/metabolism , Melanocytes/metabolism , Cell Differentiation , Epigenesis, Genetic , Pigmentation Disorders/metabolism , Pigmentation , DNA Helicases/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolismABSTRACT
BACKGROUND: Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. RESULTS: Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. CONCLUSION: These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Differentiation , Melanocytes/metabolism , Transcription Factors/metabolism , Cell Cycle Proteins/genetics , Cells, Cultured , HEK293 Cells , Humans , Melanins/biosynthesis , Melanins/genetics , Melanocytes/cytology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Promoter Regions, Genetic , Protein Binding , Transcription Factors/geneticsABSTRACT
PURPOSE OF REVIEW: We critically evaluate the future potential of machine learning (ML), deep learning (DL), and artificial intelligence (AI) in precision medicine. The goal of this work is to show progress in ML in digital health, to exemplify future needs and trends, and to identify any essential prerequisites of AI and ML for precision health. RECENT FINDINGS: High-throughput technologies are delivering growing volumes of biomedical data, such as large-scale genome-wide sequencing assays; libraries of medical images; or drug perturbation screens of healthy, developing, and diseased tissue. Multi-omics data in biomedicine is deep and complex, offering an opportunity for data-driven insights and automated disease classification. Learning from these data will open our understanding and definition of healthy baselines and disease signatures. State-of-the-art applications of deep neural networks include digital image recognition, single-cell clustering, and virtual drug screens, demonstrating breadths and power of ML in biomedicine. SUMMARY: Significantly, AI and systems biology have embraced big data challenges and may enable novel biotechnology-derived therapies to facilitate the implementation of precision medicine approaches.
ABSTRACT
Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.
Subject(s)
Antigens, Neoplasm/metabolism , Idiopathic Pulmonary Fibrosis/diagnosis , Integrins/metabolism , Neoplasms/diagnosis , Crystallography, X-Ray , Healthy Volunteers , Humans , Magnetic Resonance Imaging , Positron-Emission TomographyABSTRACT
Dysregulation of signaling networks controlling self-renewal and migration of developmental cell lineages is closely linked to the proliferative and invasive properties of tumors. Identification of such signaling pathways and their critical regulators is vital for successful design of effective targeted therapies against neoplastic tissue growth. The neurotrophin receptor (CD271/NGFR/p75NTR) is a key regulator of the melanocytic cell lineage through its ability to mediate cell growth, survival, and differentiation. Using clinical melanoma samples, normal melanocytes and global gene expression profiling we have investigated the role of CD271 in rewiring signal transduction networks of melanoma cells during neoplastic transformation. Our analysis demonstrates that depending on the cell fate of tumor initiation vs normal development, elevated levels of CD271 can serve as a switch between proliferation/survival and differentiation/cell death. Two divergent arms of neurotrophin signaling hold the balance between positive regulators of tumor growth controlled by E2F, MYC, SREBP1 and AKT3 pathways on the one hand, and differentiation, senescence, and apoptosis controlled by TRAF6/IRAK-dependent activation of AP1 and TP53 mediated processes on the other hand. A molecular network map revealed in this study uncovers CD271 as a context-specific molecular switch between normal development and malignant transformation.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Melanocytes/metabolism , Melanoma/metabolism , Neoplasm Proteins/physiology , Nerve Tissue Proteins/physiology , Receptors, Nerve Growth Factor/physiology , Cell Survival , Cell Transformation, Neoplastic , DNA Repair , Disease Progression , Gene Regulatory Networks , Humans , Melanocytes/cytology , Real-Time Polymerase Chain Reaction , Signal Transduction , TranscriptomeABSTRACT
Aberrant glutamatergic signaling has been implicated in altered metabolic activity in many cancer types, including malignant melanoma. Previously, we have illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. In this study, we showed that autocrine stimulation constitutively activates the GRM1 receptor and its downstream mitogenic signaling. GRM1-activated (GRM1+) melanomas exhibited significantly increased expression of glutaminase (GLS), which catalyzes the first step in the conversion of glutamine to glutamate. In cultured GRM1+ melanoma cell lines, CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, suppressed cell proliferation, while riluzole, an inhibitor of glutamate release, promoted apoptotic cell death in vitro and in vivo. Combined treatment with CB-839 and riluzole treatment proved to be superior to single-agent treatment, restricting glutamate bioavailability and leading to effective suppression of tumor cell proliferation in vitro and tumor progression in vivo. Hyperactivation of GRM1 in malignant melanoma is an oncogenic driver, which acts independently of canonical melanoma proto-oncogenes, BRAF or NRAS. Overall, these results indicate that expression of GRM1 promotes a metabolic phenotype that supports increased glutamate production and autocrine glutamatergic signaling, which can be pharmacologically targeted by decreasing glutamate bioavailability and the GLS-dependent glutamine to glutamate conversion. SIGNIFICANCE: These findings demonstrate that targeting glutaminolytic glutamate bioavailability is an effective therapeutic strategy for GRM1-activated tumors.
Subject(s)
Benzeneacetamides/pharmacology , Glutamic Acid/metabolism , Glutaminase/antagonists & inhibitors , Melanoma/drug therapy , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Riluzole/pharmacology , Thiadiazoles/pharmacology , Animals , Apoptosis , Biological Availability , Cell Proliferation , Drug Therapy, Combination , Female , Humans , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Hairless , Neuroprotective Agents/pharmacology , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Frizzled 3 receptor (FZD3) plays an important role in the homeostasis of the neural crest and its derivatives, which give rise to pigment-synthesizing cells, melanocytes. While the role for FZD3 in specification of the melanocytic lineage from neural crest is well established, its significance in the formation of melanoma, its associated malignancy, is less understood. In this study we identified FZD3 as a critical regulator of human melanoma tumorigenesis. Down-regulation of FZD3 abrogated growth, colony-forming potential, and invasive capacity of patient-derived melanoma cells. Xenotransplantation of tumor cells with down-regulated FZD3 levels originating from melanomas carrying the BRAF(V600) mutation uniformly suppressed their capacity for tumor and metastasis formation. FZD3 knockdown leads to the down-regulation of the core cell cycle protein components (cyclins D1, E2, B1, and CDKs 1, 2, and 4) in melanomas with a hyperactive BRAF oncogene, indicating a dominant role of this receptor during melanoma pathogenesis. Enriched pathway analysis revealed that FZD3 inhibits transcriptional networks controlled by CREB5, FOXD1, and ATF3, which suppress the activity of MAPK-mediated signaling. Thus, FZD3 establishes a positive-feedback mechanism that activates MAPK signal transduction network, critical to melanoma carcinogenesis. Importantly, high levels of FZD3 mRNA were found to be correlated with melanoma advancement to metastatic stages and limited patient survival. Changes in gene-expression patterns mediated by FZD3 activity occur in the absence of nuclear ß-catenin function, thus representing an important therapeutic target for the melanoma patients whose disease progresses independent of canonical WNT signaling.
Subject(s)
Cell Proliferation/physiology , Down-Regulation , Frizzled Receptors/physiology , Melanoma/pathology , Neoplasm Metastasis , Wnt Signaling Pathway , Frizzled Receptors/genetics , Gene Expression Profiling , Humans , Melanoma/genetics , Melanoma/metabolism , Neoplasm InvasivenessABSTRACT
Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years.
Subject(s)
Melanoma/prevention & control , Radiation-Protective Agents/therapeutic use , Skin Neoplasms/prevention & control , Animals , Anticarcinogenic Agents/therapeutic use , Chemoprevention , Clinical Trials, Phase III as Topic , Drug Development , Drug Repositioning , Female , Humans , Male , Skin Neoplasms/drug therapyABSTRACT
In this perspective, we identify emerging frontiers in clinical and basic research of melanocyte biology and its associated biomedical disciplines. We describe challenges and opportunities in clinical and basic research of normal and diseased melanocytes that impact current approaches to research in melanoma and the dermatological sciences. We focus on four themes: (1) clinical melanoma research, (2) basic melanoma research, (3) clinical dermatology, and (4) basic pigment cell research, with the goal of outlining current highlights, challenges, and frontiers associated with pigmentation and melanocyte biology. Significantly, this document encapsulates important advances in melanocyte and melanoma research including emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, epidemiology, pigment biophysics and chemistry, and evolution.
Subject(s)
Biomedical Research , Melanocytes/pathology , Melanoma/pathology , Animals , Disease Models, Animal , Drug Resistance, Neoplasm , Humans , Melanoma/epidemiology , Melanoma/prevention & control , Melanoma/therapy , PigmentationABSTRACT
The International Federation of Pigment Cell Societies (IFPCS) held its XXIII triennial International Pigment Cell Conference (IPCC) in Denver, Colorado in August 2017. The goal of the summit was to provide a venue promoting a vibrant interchange among leading basic and clinical researchers working on leading-edge aspects of melanocyte biology and disease. The philosophy of the meeting, entitled Breakthroughs in Pigment Cell and Melanoma Research, was to deliver a comprehensive program in an inclusive environment fostering scientific exchange and building new academic bridges. This document provides an outlook on the history, accomplishments, and sustainability of the pigment cell and melanoma research community. Shared progress in the understanding of cellular homeostasis of pigment cells but also clinical successes and hurdles in the treatment of melanoma and dermatological disorders continue to drive future research activities. A sustainable direction of the societies creates an international forum identifying key areas of imminent needs in laboratory research and clinical care and ensures the future of this vibrant, diverse and unique research community at the same time. Important advances showcase wealth and breadth of the field in melanocyte and melanoma research and include emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, precision bench-to-bedside approaches, epidemiology, pigment biophysics and chemistry, and evolution. This report recapitulates highlights of the federate meeting agenda designed to advance clinical and basic research frontiers from melanoma and dermatological sciences followed by a historical perspective of the associated societies and conferences.
Subject(s)
Internationality , Melanocytes/pathology , Awards and Prizes , HumansABSTRACT
BACKGROUND: Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. METHODS: The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. RESULTS: Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. CONCLUSIONS: The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.
Subject(s)
Melanoma/enzymology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Systems Biology , Cell Line, Tumor , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Transcription Factors/metabolism , Transcriptome/drug effectsABSTRACT
BACKGROUND: Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1+ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities. METHODS: Using 1D 1H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1+ VPCs positively correlated with a shift in metabolism and cellular growth. RESULTS: Specifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm2 exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development. CONCLUSIONS: These results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Animals , Cell Count , Cell Line , Endothelial Cells/cytology , Mice , Signal TransductionABSTRACT
INTRODUCTION: Prolonged fasting in northern elephant seals (NES) is characterized by a reliance on lipid metabolism, conservation of protein, and reduced plasma insulin. During early fasting, glucose infusion previously reduced plasma free fatty acids (FFA); however, during late-fasting, it induced an atypical elevation in FFA despite comparable increases in insulin during both periods suggestive of a dynamic shift in tissue responsiveness to glucose-stimulated insulin secretion. OBJECTIVE: To better assess the contribution of insulin to this fasting-associated shift in substrate metabolism. METHODS: We compared the responses of plasma metabolites (amino acids (AA), FFA, endocannabinoids (EC), and primary carbon metabolites (PCM)) to an insulin infusion (65 mU/kg) in early- and late-fasted NES pups (n = 5/group). Plasma samples were collected prior to infusion (T0) and at 10, 30, 60, and 120 min post-infusion, and underwent untargeted and targeted metabolomics analyses utilizing a variety of GC-MS and LC-MS technologies. RESULTS: In early fasting, the majority (72%) of metabolite trajectories return to baseline levels within 2 h, but not in late fasting indicative of an increase in tissue sensitivity to insulin. In late-fasting, increases in FFA and ketone pools, coupled with decreases in AA and PCM, indicate a shift toward lipolysis, beta-oxidation, ketone metabolism, and decreased protein catabolism. Conversely, insulin increased PCM AUC in late fasting suggesting that gluconeogenic pathways are activated. Insulin also decreased FFA AUC between early and late fasting suggesting that insulin suppresses triglyceride hydrolysis. CONCLUSION: Naturally adapted tolerance to prolonged fasting in these mammals is likely accomplished by suppressing insulin levels and activity, providing novel insight on the evolution of insulin during a condition of temporary, reversible insulin resistance.
ABSTRACT
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.
ABSTRACT
The lysine demethylase 3A (KDM3A, JMJD1A or JHDM2A) controls transcriptional networks in a variety of biological processes such as spermatogenesis, metabolism, stem cell activity, and tumor progression. We matched transcriptomic and ChIP-Seq profiles to decipher a genome-wide regulatory network of epigenetic control by KDM3A in prostate cancer cells. ChIP-Seq experiments monitoring histone 3 lysine 9 (H3K9) methylation marks show global histone demethylation effects of KDM3A. Combined assessment of histone demethylation events and gene expression changes presented major transcriptional activation suggesting that distinct oncogenic regulators may synergize with the epigenetic patterns by KDM3A. Pathway enrichment analysis of cells with KDM3A knockdown prioritized androgen signaling indicating that KDM3A plays a key role in regulating androgen receptor activity. Matched ChIP-Seq and knockdown experiments of KDM3A in combination with ChIP-Seq of the androgen receptor resulted in a gain of H3K9 methylation marks around androgen receptor binding sites of selected transcriptional targets in androgen signaling including positive regulation of KRT19, NKX3-1, KLK3, NDRG1, MAF, CREB3L4, MYC, INPP4B, PTK2B, MAPK1, MAP2K1, IGF1, E2F1, HSP90AA1, HIF1A, and ACSL3. The cancer systems biology analysis of KDM3A-dependent genes identifies an epigenetic and transcriptional network in androgen response, hypoxia, glycolysis, and lipid metabolism. Genome-wide ChIP-Seq data highlights specific gene targets and the ability of epigenetic master regulators to control oncogenic pathways and cancer progression.
Subject(s)
Gene Expression Regulation, Neoplastic , Jumonji Domain-Containing Histone Demethylases/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Transcription, Genetic , Cell Line, Tumor , Chromatin Immunoprecipitation , Computational Biology/methods , Enzyme Activation , Epigenesis, Genetic , Gene Expression Profiling , Gene Knockdown Techniques , Gene Regulatory Networks , Genomics/methods , High-Throughput Nucleotide Sequencing , Histones/metabolism , Humans , Male , Methylation , Molecular Sequence Annotation , Protein Binding , Signal TransductionABSTRACT
Histone methylation is an epigenetic modification of chromatin undergoing dynamic changes and balancing tissue-specific demands of proliferation and differentiation. In cancer, aberrant histone methylation can facilitate oncogenic and tumor suppression programs by modulating gene expression. Histone remodelers such as lysine methyltransferases and lysine demethylases are seemingly opposite or contrary forces but may be part of an interconnected network complementing each other. We identify several layers of molecular communication where epigenetic master regulators engage in crosstalk between tumor metabolism and histone remodeling. Epigenetic master regulators have the ability to cooperate with members of the transcriptional machinery, DNA methyltransferases, as well as other histone modifiers. High-throughput sequencing and omics data in combination with cancer systems biology analysis have the power to prioritize regulatory events epigenome-wide.
Subject(s)
Epigenesis, Genetic , Histone Demethylases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Neoplasms/enzymology , High-Throughput Nucleotide Sequencing , Histone Demethylases/genetics , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Humans , Neoplasms/geneticsABSTRACT
Molecular insights from genome and systems biology are influencing how cancer is diagnosed and treated. We critically evaluate big data challenges in precision medicine. The melanoma research community has identified distinct subtypes involving chronic sun-induced damage and the mitogen-activated protein kinase driver pathway. In addition, despite low mutation burden, non-genomic mitogen-activated protein kinase melanoma drivers are found in membrane receptors, metabolism, or epigenetic signaling with the ability to bypass central mitogen-activated protein kinase molecules and activating a similar program of mitogenic effectors. Mutation hotspots, structural modeling, UV signature, and genomic as well as non-genomic mechanisms of disease initiation and progression are taken into consideration to identify resistance mutations and novel drug targets. A comprehensive precision medicine profile of a malignant melanoma patient illustrates future rational drug targeting strategies. Network analysis emphasizes an important role of epigenetic and metabolic master regulators in oncogenesis. Co-occurrence of driver mutations in signaling, metabolic, and epigenetic factors highlights how cumulative alterations of our genomes and epigenomes progressively lead to uncontrolled cell proliferation. Precision insights have the ability to identify independent molecular pathways suitable for drug targeting. Synergistic treatment combinations of orthogonal modalities including immunotherapy, mitogen-activated protein kinase inhibitors, epigenetic inhibitors, and metabolic inhibitors have the potential to overcome immune evasion, side effects, and drug resistance.