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Intra-tumoral phenotypic heterogeneity promotes tumor relapse and therapeutic resistance and remains an unsolved clinical challenge. Decoding the interconnections among different biological axes of plasticity is crucial to understand the molecular origins of phenotypic heterogeneity. Here, we use multi-modal transcriptomic data-bulk, single-cell, and spatial transcriptomics-from breast cancer cell lines and primary tumor samples, to identify associations between epithelial-mesenchymal transition (EMT) and luminal-basal plasticity-two key processes that enable heterogeneity. We show that luminal breast cancer strongly associates with an epithelial cell state, but basal breast cancer is associated with hybrid epithelial/mesenchymal phenotype(s) and higher phenotypic heterogeneity. Mathematical modeling of core underlying gene regulatory networks representative of the crosstalk between the luminal-basal and epithelial-mesenchymal axes elucidate mechanistic underpinnings of the observed associations from transcriptomic data. Our systems-based approach integrating multi-modal data analysis with mechanism-based modeling offers a predictive framework to characterize intra-tumor heterogeneity and identify interventions to restrict it.
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SUMMARY: Understandably, conventional therapeutic strategies have focused on controlling primary tumors. We ask whether the cost of such strategies is actually an increased likelihood of metastatic relapse.
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Neoplasias , Humanos , Neoplasias/terapia , Microambiente TumoralRESUMEN
Intra-tumoral phenotypic heterogeneity promotes tumor relapse and therapeutic resistance and remains an unsolved clinical challenge. It manifests along multiple phenotypic axes and decoding the interconnections among these different axes is crucial to understand its molecular origins and to develop novel therapeutic strategies to control it. Here, we use multi-modal transcriptomic data analysis - bulk, single-cell and spatial transcriptomics - from breast cancer cell lines and primary tumor samples, to identify associations between epithelial-mesenchymal transition (EMT) and luminal-basal plasticity - two key processes that enable heterogeneity. We show that luminal breast cancer strongly associates with an epithelial cell state, but basal breast cancer is associated with hybrid epithelial/mesenchymal phenotype(s) and higher phenotypic heterogeneity. These patterns were inherent in methylation profiles, suggesting an epigenetic crosstalk between EMT and lineage plasticity in breast cancer. Mathematical modelling of core underlying gene regulatory networks representative of the crosstalk between the luminal-basal and epithelial-mesenchymal axes recapitulate and thus elucidate mechanistic underpinnings of the observed associations from transcriptomic data. Our systems-based approach integrating multi-modal data analysis with mechanism-based modeling offers a predictive framework to characterize intra-tumor heterogeneity and to identify possible interventions to restrict it.
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Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Recurrencia Local de Neoplasia/genética , Factores de Transcripción/genética , Epigénesis Genética/genéticaRESUMEN
Exciting advances in technologies to measure biological systems are currently at the forefront of research. The ability to gather data along an increasing number of omic dimensions has created a need for tools to analyze all of this information together, rather than siloing each technology into separate analysis pipelines. To advance this goal, we introduce a framework called the single-cell multi-modal generative adversarial network (scMMGAN) that integrates data from multiple modalities into a unified representation in the ambient data space for downstream analysis using a combination of adversarial learning and data geometry techniques. The framework's key improvement is an additional diffusion geometry loss with a new kernel that constrains the otherwise over-parameterized GAN. We demonstrate scMMGAN's ability to produce more meaningful alignments than alternative methods on a wide variety of data modalities and that its output can be used to draw conclusions from real-world biological experimental data.
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ABSTRACT: Phenotypic plasticity describes the ability of cancer cells to undergo dynamic, nongenetic cell state changes that amplify cancer heterogeneity to promote metastasis and therapy evasion. Thus, cancer cells occupy a continuous spectrum of phenotypic states connected by trajectories defining dynamic transitions upon a cancer cell state landscape. With technologies proliferating to systematically record molecular mechanisms at single-cell resolution, we illuminate manifold learning techniques as emerging computational tools to effectively model cell state dynamics in a way that mimics our understanding of the cell state landscape. We anticipate that "state-gating" therapies targeting phenotypic plasticity will limit cancer heterogeneity, metastasis, and therapy resistance. SIGNIFICANCE: Nongenetic mechanisms underlying phenotypic plasticity have emerged as significant drivers of tumor heterogeneity, metastasis, and therapy resistance. Herein, we discuss new experimental and computational techniques to define phenotypic plasticity as a scaffold to guide accelerated progress in uncovering new vulnerabilities for therapeutic exploitation.
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Transición Epitelial-Mesenquimal , Neoplasias , Adaptación Fisiológica , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
Therapies that prevent metastatic dissemination and tumor growth in secondary organs are severely lacking. A better understanding of the mechanisms that drive metastasis will lead to improved therapies that increase patient survival. Within a tumor, cancer cells are equipped with different phenotypic and functional capacities that can impact their ability to complete the metastatic cascade. That phenotypic heterogeneity can be derived from a combination of factors, in which the genetic make-up, interaction with the environment, and ability of cells to adapt to evolving microenvironments and mechanical forces play a major role. In this review, we discuss the specific properties of those cancer cell subgroups and the mechanisms that confer or restrict their capacity to metastasize.
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Inhibition of metastatic cancer cell colonization and outgrowth is arguably one of the greatest therapeutic challenges. Reporting in Cancer Discovery, Liu et al. (2018) describe how homophilic interactions of CD44, a classical breast cancer stem cell marker, drive tumor cell aggregation outside the primary tumor to augment their metastatic potential.
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Neoplasias de la Mama , Agregación Celular , Humanos , Receptores de Hialuranos , Células Madre NeoplásicasRESUMEN
Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1ß (IL-1ß)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1ß maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1ß expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.
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Neoplasias de la Mama/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/metabolismo , Células Mieloides/metabolismo , Microambiente Tumoral , Animales , Antiinflamatorios/farmacología , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Comunicación Celular , Diferenciación Celular , Línea Celular Tumoral , Plasticidad de la Célula , Proliferación Celular , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Inflamación/prevención & control , Interleucina-1beta/genética , Interleucina-1beta/farmacología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Metástasis Linfática , Ratones Desnudos , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/patología , Transducción de Señal , Factores de Tiempo , Ensayos Antitumor por Modelo de Xenoinjerto , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.
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Empalme Alternativo/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Filaminas/genética , Células Madre Mesenquimatosas/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Exones/genética , Femenino , Filaminas/metabolismo , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Receptores de Hialuranos/metabolismo , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Sistemas de Lectura Abierta/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed "dropout," which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations.
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Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Línea Celular , Epistasis Genética/genética , Redes Reguladoras de Genes/genética , Humanos , Cadenas de Markov , MicroARNs/genética , ARN Mensajero/genética , Programas InformáticosRESUMEN
Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-ß4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.
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Carcinoma/metabolismo , Integrina beta4/metabolismo , Mesodermo/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Carcinoma/genética , Carcinoma/mortalidad , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Humanos , Integrina beta4/genética , Mesodermo/citología , Pronóstico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Carcinoma cells that are induced to suppress their epithelial features and upregulate mesenchymal gene expression programs acquire traits that promote an invasive and metastatic phenotype. This is achieved through the expression of a program termed the epithelial-to-mesenchymal transition (EMT)-a fundamental cell-biological process that plays key roles in embryogenesis and wound healing. Re-activation of the EMT during cancer promotes disease progression and enhances the metastatic phenotype by bestowing upon previously benign carcinoma cell traits such as migration, invasion, resistance to anoikis, chemoresistance and tumour-initiating potential. Herein, we discuss recent insights into the function of the EMT and cancer cell plasticity during cancer progression, with a focus on their role in promoting successful completion of the later stages of the metastatic cascade.
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Neoplasias de la Mama/patología , Plasticidad de la Célula/fisiología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Plasticidad de la Célula/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Humanos , Metástasis de la NeoplasiaRESUMEN
By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.
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División Celular/fisiología , Senescencia Celular/fisiología , Mitocondrias/fisiología , Células Madre/fisiología , Células Madre/ultraestructura , División Celular/genética , Línea Celular , Senescencia Celular/genética , Humanos , Mitocondrias/ultraestructuraRESUMEN
Identifying the cancer cells-of-origin is of great interest, as it holds the potential to elucidate biologic mechanisms inherent in the normal cell state that have been co-opted to drive the oncogenic cell state. An emerging concept, proposed here, states that cancer stem cells, key players in cancer initiation and metastasis, arise when transit-amplifying cells with mutant genomes dedifferentiate and enter the stem cell state. This model contrasts with the notion that cancer stem cells are the direct products of neoplastically transformed normal tissue stem cells.
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Carcinogénesis , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre/metabolismoRESUMEN
The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFß. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.
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Neoplasias de la Mama/patología , Cromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/genética , Células Epiteliales/patología , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
BACKGROUND: Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models. CONTENT: The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity. SUMMARY: Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies.
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Neoplasias/patología , Humanos , Modelos BiológicosRESUMEN
The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-ß and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.
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Comunicación Autocrina , Neoplasias de la Mama/metabolismo , Mama/citología , Células Madre Neoplásicas/metabolismo , Comunicación Paracrina , Células Madre/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/patología , Movimiento Celular , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.