RESUMEN
Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single-cell RNA sequencing, we uncovered both direct and indirect paths by which resident SG progenitors ordinarily differentiate into sebocytes, including transit through a Krt5+PPARγ+ transitional basal cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair-follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR2 signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.
Asunto(s)
Glándulas Sebáceas , Piel , Diferenciación Celular , Folículo Piloso , Células EpitelialesRESUMEN
Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single cell RNA-sequencing, we uncovered both direct and indirect paths by which these resident SG progenitors ordinarily differentiate into sebocytes, including transit through a PPARγ+Krt5+ transitional cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.
RESUMEN
Several methods for generating human-skin-equivalent (HSE) organoid cultures are in use to study skin biology; however, few studies thoroughly characterize these systems. To fill this gap, we use single-cell transcriptomics to compare in vitro HSEs, xenograft HSEs, and in vivo epidermis. By combining differential gene expression, pseudotime analyses, and spatial localization, we reconstruct HSE keratinocyte differentiation trajectories that recapitulate known in vivo epidermal differentiation pathways and show that HSEs contain major in vivo cellular states. However, HSEs also develop unique keratinocyte states, an expanded basal stem cell program, and disrupted terminal differentiation. Cell-cell communication modeling shows aberrant epithelial-to-mesenchymal transition (EMT)-associated signaling pathways that alter upon epidermal growth factor (EGF) supplementation. Last, xenograft HSEs at early time points post transplantation significantly rescue many in vitro deficits while undergoing a hypoxic response that drives an alternative differentiation lineage. This study highlights the strengths and limitations of organoid cultures and identifies areas for potential innovation.
Asunto(s)
Piel , Transcriptoma , Humanos , Transcriptoma/genética , Piel/metabolismo , Queratinocitos/metabolismo , Epidermis/metabolismo , Diferenciación Celular , OrganoidesRESUMEN
Spatial transcriptomic technologies and spatially annotated single-cell RNA sequencing datasets provide unprecedented opportunities to dissect cell-cell communication (CCC). However, incorporation of the spatial information and complex biochemical processes required in the reconstruction of CCC remains a major challenge. Here, we present COMMOT (COMMunication analysis by Optimal Transport) to infer CCC in spatial transcriptomics, which accounts for the competition between different ligand and receptor species as well as spatial distances between cells. A collective optimal transport method is developed to handle complex molecular interactions and spatial constraints. Furthermore, we introduce downstream analysis tools to infer spatial signaling directionality and genes regulated by signaling using machine learning models. We apply COMMOT to simulation data and eight spatial datasets acquired with five different technologies to show its effectiveness and robustness in identifying spatial CCC in data with varying spatial resolutions and gene coverages. Finally, COMMOT identifies new CCCs during skin morphogenesis in a case study of human epidermal development.
Asunto(s)
Comunicación Celular , Transcriptoma , Humanos , Comunicación Celular/genética , Perfilación de la Expresión Génica , Transducción de Señal , Simulación por Computador , Análisis de la Célula IndividualRESUMEN
Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.
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ARN , Espectrometría de Masas en Tándem , Animales , Ratones , Humanos , ARN/química , Cromatografía Liquida , Perfilación de la Expresión GénicaRESUMEN
Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.
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Proteína Quinasa 1 Activada por Mitógenos , Proteínas Represoras , Proteína con Dedos de Zinc GLI1 , Animales , Sitios de Unión , Células Cultivadas , Secuencia Conservada , Proteínas Hedgehog/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Unión Proteica , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. We use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity-PAGA, cellular entropy, and regulon analysis in stromal cells reveals a cancer-specific rewiring of fibroblasts, where STAT1, TGF-ß, and inflammatory signals induce a noncanonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, whose expression we validated in situ. Last, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro vismodegib-resistant BCC cell growth, Hedgehog signaling, and in vivo tumor growth in a BCC mouse model, validating HSP70's essential role in tumor growth and reinforcing the critical nature of tumor microenvironment cross-talk in BCC progression.
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Carcinoma Basocelular , Neoplasias Cutáneas , Animales , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ecosistema , Proteínas Hedgehog , Humanos , Ratones , Análisis de la Célula Individual , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Microambiente TumoralRESUMEN
Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.
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Neoplasias , ARN , Nucleósidos/metabolismo , ARN/metabolismoRESUMEN
Basal cell carcinoma (BCC) is a locally invasive epithelial cancer that is primarily driven by the Hedgehog (HH) pathway. Advanced BCCs are a critical subset of BCCs that frequently acquire resistance to Smoothened (SMO) inhibitors and identifying pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify a PI3K pathway expression signature in BCC. Pharmacological inhibition of PI3K activity in BCC cells significantly reduces cell proliferation and HH signaling. However, treatment of Ptch1fl/fl ; Gli1-CreERT2 mouse BCCs with the PI3K inhibitor BKM120 results in a reduction of tumor cell growth with no significant effect on HH signaling. Downstream PI3K components aPKC and Akt1 showed a reduction in active protein, whereas their substrate, cyclin-dependent kinase inhibitor p21, showed a concomitant increase in protein stability. Our results suggest that PI3K promotes BCC tumor growth by kinase-induced p21 degradation without altering HH signaling.
RESUMEN
Advanced basal cell carcinomas (BCCs) are driven by the Hedgehog (HH) pathway and often possess inherent resistance to SMO inhibitors. Identifying and targeting pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify an MTOR expression signature in BCC. Pharmacological inhibition of MTOR activity in BCC cells significantly reduces cell proliferation without affecting HH signalling. Similarly, treatment of the Ptch1 fl/fl ; Gli1-CreERT2 mouse BCC tumor model with everolimus reduces tumor growth. aPKC, a downstream target of MTOR, shows reduced activity, suggesting that MTOR promotes tumor growth by activating aPKC and demonstrating that suppressing MTOR could be a promising target for BCC patients.
Asunto(s)
Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Serina-Treonina Quinasas TOR , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Basocelular/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Everolimus/farmacología , Everolimus/uso terapéutico , Proteínas Hedgehog/metabolismo , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Ratones , Receptor Patched-1/genética , Proteína Quinasa C/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/farmacología , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
The advent of immune checkpoint therapy for metastatic skin cancer has greatly improved patient survival. However, most skin cancer patients are refractory to checkpoint therapy, and furthermore, the intra-immune cell signaling driving response to checkpoint therapy remains uncharacterized. When comparing the immune transcriptome in the tumor microenvironment of melanoma and basal cell carcinoma (BCC), we found that the presence of memory B cells and macrophages negatively correlate in both cancers when stratifying patients by their response, with memory B cells more present in responders. Moreover, inhibitory immune signaling mostly decreases in melanoma responders and increases in BCC responders. We further explored the relationships between macrophages, B cells and response to checkpoint therapy by developing a stochastic differential equation model which qualitatively agrees with the data analysis. Our model predicts BCC to be more refractory to checkpoint therapy than melanoma and predicts the best qualitative ratio of memory B cells and macrophages for successful treatment.
RESUMEN
Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.
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Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Matriz Extracelular/metabolismo , Ontología de Genes , Factores de Intercambio de Guanina Nucleótido/metabolismo , Folículo Piloso/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteína smad3/metabolismo , Transactivadores/metabolismo , Regulación hacia ArribaRESUMEN
How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.
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Diferenciación Celular , Células Epidérmicas/citología , Homeostasis , Células Madre/citología , Comunicación Celular/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Prepucio/citología , Prepucio/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Recién Nacido , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Modelos Biológicos , Transducción de Señal , Células Madre/metabolismoRESUMEN
Basosquamous carcinoma (BSC) is a rare form of skin cancer with both basaloid and squamous morphology. Chiang et al. (2019) genetically define BSCs and demonstrate that BSCs likely originate as basal cell carcinomas that partially squamatize through accumulation of ARID1A mutations and RAS/MAPK pathway activation.
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Carcinoma Basocelular , Carcinoma Basoescamoso , Neoplasias Cutáneas , Adaptación Fisiológica , Humanos , MutaciónRESUMEN
Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.
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Síndrome del Nevo Basocelular/metabolismo , Carcinoma Basocelular/metabolismo , Cilios/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas ras/metabolismo , Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/patología , Carcinogénesis , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Mutación/genética , Piridinas/uso terapéutico , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología , Animales , Ataxia/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Neoplasias/genética , Proteínas/metabolismo , Células de Purkinje/fisiología , Ataxias Espinocerebelosas/metabolismo , Degeneraciones Espinocerebelosas/metabolismo , Degeneraciones Espinocerebelosas/fisiopatología , Familia-src Quinasas/metabolismoRESUMEN
Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.
Asunto(s)
Actinas/metabolismo , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Células 3T3 , Proteína 3 Relacionada con la Actina/genética , Animales , Axonema/fisiología , Cuerpos Basales/metabolismo , Línea Celular , Activación Enzimática/fisiología , Regulación de la Expresión Génica/fisiología , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Polimerizacion , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Sporadic and basal cell nevus syndrome basal cell carcinomas show differential response rates to Smoothened inhibitors. Chiang et al. demonstrate notable decreases in UV-induced mutagenesis, total mutation load, genomic instability, and drug-resistant mutations among basal cell nevus syndrome basal cell carcinomas using whole exome sequencing, which may explain the differences in drug response rates.