RESUMEN
How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.
Asunto(s)
Genómica , Mutación , Neoplasias Ováricas , Análisis de la Célula Individual , Neoplasias de la Mama Triple Negativas , Femenino , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Filogenia , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.
Asunto(s)
Variaciones en el Número de Copia de ADN , Resistencia a Antineoplásicos , Neoplasias de la Mama Triple Negativas/genética , Animales , Línea Celular Tumoral , Cisplatino/farmacología , Células Clonales/patología , Femenino , Aptitud Genética , Humanos , Ratones , Modelos Estadísticos , Trasplante de Neoplasias , Proteína p53 Supresora de Tumor/genética , Secuenciación Completa del GenomaRESUMEN
Single-cell RNA sequencing has enabled the decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types through unsupervised clustering followed by manual annotation or via 'mapping' to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data and both are prone to batch effects. To overcome these issues, we present CellAssign, a probabilistic model that leverages prior knowledge of cell-type marker genes to annotate single-cell RNA sequencing data into predefined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high-grade serous ovarian cancer and follicular lymphoma.
Asunto(s)
Perfilación de la Expresión Génica , Linfoma Folicular/patología , Probabilidad , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Microambiente Tumoral , Humanos , Linfoma Folicular/inmunologíaRESUMEN
Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) and PGC-1α-related coactivator (PRC). Suppression of PGC-1ß and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1ß, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Mitofagia , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia Celular/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Calorie restriction (CR) extends lifespan and has been shown to reduce age-related diseases including cancer, diabetes, and cardiovascular and neurodegenerative diseases in experimental models. Recent translational studies have tested the potential of CR or CR mimetics as adjuvant therapies to enhance the efficacy of chemotherapy, radiation therapy, and novel immunotherapies. Chronic CR is challenging to employ in cancer patients, and therefore intermittent fasting, CR mimetic drugs, or alternative diets (such as a ketogenic diet), may be more suitable. Intermittent fasting has been shown to enhance treatment with both chemotherapy and radiation therapy. CR and fasting elicit different responses in normal and cancer cells, and reduce certain side effects of cytotoxic therapy. Findings from preclinical studies of CR mimetic drugs and other dietary interventions, such as the ketogenic diet, are promising for improving the efficacy of anticancer therapies and reducing the side effects of cytotoxic treatments. Current and future clinical studies will inform on which cancers, and at which stage of the cancer process, CR, fasting, or CR mimetic regimens will prove most effective.
Asunto(s)
Restricción Calórica , Neoplasias/dietoterapia , Animales , HumanosRESUMEN
PTEN-induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up-regulated by overexpression of the major tumor suppressor, PTEN. Loss-of-function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinson's disease. Substantial work during the past decade has revealed that PINK1 regulates several primary cellular processes of significance in cancer cell biology, including cell survival, stress resistance, mitochondrial homeostasis and the cell cycle. Mechanistically, PINK1 has been shown to interact on a number of levels with the pivotal oncogenic PI3-kinase/Akt/mTOR signalling axis and to control critical mitochondrial and metabolic functions that regulate cancer survival, growth, stress resistance and the cell cycle. A cytoprotective and chemoresistant function for PINK1 has been highlighted by some studies, supporting PINK1 as a target in cancer therapeutics. This article reviews the function of PINK1 in cancer cell biology, with an emphasis on the mechanisms by which PINK1 interacts with PI3-kinase/Akt signalling, mitochondrial homeostasis, and the potential context-dependent pro- and anti-tumorigenic functions of PINK1.
Asunto(s)
Neoplasias/patología , Proteínas Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Neoplasias/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaRESUMEN
The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.
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Núcleo Celular , Variaciones en el Número de Copia de ADN , ADN Mitocondrial , Genoma Mitocondrial , Neoplasias , Análisis de la Célula Individual , Humanos , ADN Mitocondrial/genética , Análisis de la Célula Individual/métodos , Variaciones en el Número de Copia de ADN/genética , Núcleo Celular/genética , Neoplasias/genética , Neoplasias/patología , Línea Celular Tumoral , Animales , Mitocondrias/genética , Secuenciación Completa del Genoma/métodos , Ratones , Heteroplasmia/genéticaRESUMEN
Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of TP53 , LOH of BRCA1 or BRCA2 , and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.
RESUMEN
BACKGROUND: The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug-associated transcriptional cell states. RESULTS: We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. CONCLUSIONS: Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration, and re-treatment strategies.
Asunto(s)
Resistencia a Antineoplásicos , Análisis de la Célula Individual , Transcriptoma , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Humanos , Animales , Resistencia a Antineoplásicos/genética , Femenino , Ratones , Variaciones en el Número de Copia de ADN , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transición Epitelial-Mesenquimal/genéticaRESUMEN
Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to existing ExM protocols is the equipping of different kinds of molecules, with different kinds of anchoring moieties, so they can all be pulled apart from each other by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables proteins and RNAs to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and reduces cost (by 2-10-fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this united ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructures, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues and may support the visualization of other kinds of biomolecular species as well. uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.
Asunto(s)
Compuestos Epoxi , Microscopía , Humanos , Animales , Modelos Animales de Enfermedad , Polímeros , ARNRESUMEN
Functional characterization of the cancer clones can shed light on the evolutionary mechanisms driving cancer's proliferation and relapse mechanisms. Single-cell RNA sequencing data provide grounds for understanding the functional state of cancer as a whole; however, much research remains to identify and reconstruct clonal relationships toward characterizing the changes in functions of individual clones. We present PhylEx that integrates bulk genomics data with co-occurrences of mutations from single-cell RNA sequencing data to reconstruct high-fidelity clonal trees. We evaluate PhylEx on synthetic and well-characterized high-grade serous ovarian cancer cell line datasets. PhylEx outperforms the state-of-the-art methods both when comparing capacity for clonal tree reconstruction and for identifying clones. We analyze high-grade serous ovarian cancer and breast cancer data to show that PhylEx exploits clonal expression profiles beyond what is possible with expression-based clustering methods and clear the way for accurate inference of clonal trees and robust phylo-phenotypic analysis of cancer.
Asunto(s)
Neoplasias Ováricas , Árboles , Femenino , Humanos , Árboles/genética , Transcriptoma , Evolución Clonal , Recurrencia Local de Neoplasia , Neoplasias Ováricas/genética , Células Clonales , Análisis de la Célula Individual/métodosRESUMEN
Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.
Asunto(s)
Neoplasias de la Mama , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Animales , Teorema de Bayes , Neoplasias de la Mama/genética , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Triple-negative breast cancers (TNBCs), accounting for approximately 15% of breast cancers, lack targeted therapy. A hallmark of cancer is metabolic reprogramming, with one-carbon metabolism essential to many processes altered in tumor cells, including nucleotide biosynthesis and antioxidant defenses. We reported that folate deficiency via folic acid (FA) withdrawal in several TNBC cell lines results in heterogenous effects on cell growth, metabolic reprogramming, and mitochondrial impairment. To elucidate underlying drivers of TNBC sensitivity to folate stress, we characterized in vivo and in vitro responses to FA restriction in two TNBC models differing in metastatic potential and innate mitochondrial dysfunction. METHODS: Metastatic MDA-MB-231 cells (high mitochondrial dysfunction) and nonmetastatic M-Wnt cells (low mitochondrial dysfunction) were orthotopically injected into mice fed diets with either 2 ppm FA (control), 0 ppm FA, or 12 ppm FA (supplementation; in MDA-MB-231 only). Tumor growth, metabolomics, and metabolic gene expression were assessed. MDA-MB-231 and M-Wnt cells were also grown in media with 0 or 2.2 µM FA; metabolic alterations were assessed by extracellular flux analysis, flow cytometry, and qPCR. RESULTS: Relative to control, dietary FA restriction decreased MDA-MB-231 tumor weight and volume, while FA supplementation minimally increased MDA-MB-231 tumor weight. Metabolic studies in vivo and in vitro using MDA-MB-231 cells showed FA restriction remodeled one-carbon metabolism, nucleotide biosynthesis, and glucose metabolism. In contrast to findings in the MDA-MB-231 model, FA restriction in the M-Wnt model, relative to control, led to accelerated tumor growth, minimal metabolic changes, and modest mitochondrial dysfunction. Increased mitochondrial dysfunction in M-Wnt cells, induced via chloramphenicol, significantly enhanced responsiveness to the cytotoxic effects of FA restriction. CONCLUSIONS: Given the lack of targeted treatment options for TNBC, uncovering metabolic vulnerabilities that can be exploited as therapeutic targets is an important goal. Our findings suggest that a major driver of TNBC sensitivity to folate restriction is a high innate level of mitochondrial dysfunction, which can increase dependence on one-carbon metabolism. Thus, folate deprivation or antifolate therapy for TNBCs with metabolic inflexibility due to their elevated levels of mitochondrial dysfunction may represent a novel precision-medicine strategy.
Asunto(s)
Dietoterapia/métodos , Ácido Fólico/administración & dosificación , Neoplasias Mamarias Experimentales/dietoterapia , Neoplasias de la Mama Triple Negativas/dietoterapia , Animales , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Metabolómica , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
We have previously shown that withdrawal of folic acid led to metabolic reprogramming and a less aggressive phenotype in a mouse cell model of triple-negative breast cancer (TNBC). Herein, we evaluate the effects of folic acid withdrawal on transcriptomic profiles in these cells. Murine cell lines were originally derived from a pool of spontaneous mammary tumors grown in MMTV-Wnt1 transgenic mice. Based on their differential molecular characteristics and metastatic potential, these cell lines were previously characterized as non-metastatic epithelial (E-Wnt), non-metastatic mesenchymal (M-Wnt) and metastatic mesenchymal (metM-Wntliver) cells. Using custom two-color 180K Agilent microarrays, we have determined gene expression profiles for three biological replicates of each subtype kept on standard medium (2.2 µM folic acid) or folic acid-free medium for 72 h. The analyses revealed that more genes were differentially expressed upon folic acid withdrawal in M-Wnt cells (1884 genes; Benjamini-Hochberg-adjusted P-value <0.05) compared to E-Wnt and metM-Wntliver cells (108 and 222 genes, respectively). Pathway analysis has identified that type I interferon signaling was strongly affected by folic acid withdrawal, with interferon-responsive genes consistently being upregulated upon folic acid withdrawal in M-Wnt cells. Of note, repressed interferon signaling has been established as one of the characteristics of aggressive human TNBC, and hence reactivation of this pathway may be a promising therapeutic approach. Overall, while our study indicates that the response to folic acid withdrawal varies by molecular subtype and cellular phenotype, it also underscores the necessity to further investigate one-carbon metabolism as a potential therapeutic means in the treatment of advanced TNBC.
Asunto(s)
Ácido Fólico/administración & dosificación , Transcriptoma , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Medios de Cultivo , Transición Epitelial-Mesenquimal , Femenino , Ratones , Ratones Transgénicos , Vía de Señalización WntRESUMEN
Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone.
Asunto(s)
Biomarcadores de Tumor/genética , Cistadenocarcinoma Seroso/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Modelos Estadísticos , Neoplasias Ováricas/genética , Análisis de la Célula Individual/métodos , Programas Informáticos , Neoplasias de la Mama Triple Negativas/genética , Animales , Células Clonales , Cistadenocarcinoma Seroso/patología , Femenino , Humanos , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/patología , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and ß-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFß. Cytoplasmic PDLIM2 was associated with active ß-catenin and ectopic expression of PDLIM2 was sufficient to increase ß-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated ß-catenin activity and which may be amenable to therapies that target these pathways. SIGNIFICANCE: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the ß-catenin and adhesion signaling pathways. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2619/F1.large.jpg.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Adhesión Celular , Proteínas con Dominio LIM/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , beta Catenina/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Femenino , Células HEK293 , HumanosRESUMEN
BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. RESULTS: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. CONCLUSIONS: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.
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Genómica/métodos , Neoplasias/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Animales , Frío , Colagenasas , Humanos , Ratones , Péptido Hidrolasas , Estrés Fisiológico , TranscriptomaRESUMEN
The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.
Asunto(s)
Reprogramación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Perilipina-4/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Reprogramación Celular/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Gotas Lipídicas/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Prevalence of obesity, an established risk factor for many cancers, has increased dramatically over the past 50 years in the United States and across the globe. Relative to normoweight cancer patients, obese cancer patients often have poorer prognoses, resistance to chemotherapies, and are more likely to develop distant metastases. Recent progress on elucidating the mechanisms underlying the obesity-cancer connection suggests that obesity exerts pleomorphic effects on pathways related to tumor development and progression and, thus, there are multiple opportunities for primary prevention and treatment of obesity-related cancers. Obesity-associated alterations, including systemic metabolism, adipose inflammation, growth factor signaling, and angiogenesis, are emerging as primary drivers of obesity-associated cancer development and progression. These obesity-associated host factors interact with the intrinsic molecular characteristics of cancer cells, facilitating several of the hallmarks of cancer. Each is considered in the context of potential preventive and therapeutic strategies to reduce the burden of obesity-related cancers. In addition, this review focuses on emerging mechanisms behind the obesity-cancer link, as well as relevant dietary interventions, including calorie restriction, intermittent fasting, low-fat diet, and ketogenic diet, that are being implemented in preclinical and clinical trials, with the ultimate goal of reducing incidence and progression of obesity-related cancers.
Asunto(s)
Dieta/métodos , Neoplasias/prevención & control , Obesidad/dietoterapia , Carcinogénesis/metabolismo , Progresión de la Enfermedad , Humanos , Incidencia , Neoplasias/epidemiología , Neoplasias/etiología , Obesidad/complicaciones , Obesidad/metabolismo , Factores de RiesgoRESUMEN
IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, ß1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.