RESUMEN
ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.
RESUMEN
We characterized a prospective endometrial carcinoma (EC) cohort containing 138 tumors and 20 enriched normal tissues using 10 different omics platforms. Targeted quantitation of two peptides can predict antigen processing and presentation machinery activity, and may inform patient selection for immunotherapy. Association analysis between MYC activity and metformin treatment in both patients and cell lines suggests a potential role for metformin treatment in non-diabetic patients with elevated MYC activity. PIK3R1 in-frame indels are associated with elevated AKT phosphorylation and increased sensitivity to AKT inhibitors. CTNNB1 hotspot mutations are concentrated near phosphorylation sites mediating pS45-induced degradation of ß-catenin, which may render Wnt-FZD antagonists ineffective. Deep learning accurately predicts EC subtypes and mutations from histopathology images, which may be useful for rapid diagnosis. Overall, this study identified molecular and imaging markers that can be further investigated to guide patient stratification for more precise treatment of EC.
Asunto(s)
Neoplasias Endometriales , Metformina , Proteogenómica , Femenino , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Estudios Prospectivos , Neoplasias Endometriales/tratamiento farmacológico , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Metformina/farmacologíaRESUMEN
CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.
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Edición Génica , Neoplasias , Animales , Ratones , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Neoplasias/genética , Neoplasias/terapia , Reparación del ADN por Recombinación , Modelos Animales de EnfermedadRESUMEN
Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer. PREVENTION RELEVANCE: PI3K protein is abnormally high in breast precancerous lesions. This preclinical study demonstrates that the FDA-approved anti-PI3K inhibitor alpelisib can prevent breast cancer and thus warrant future clinical trials in high-risk women.
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Lesiones Precancerosas , Tiazoles , Animales , Ratones , Femenino , Hiperplasia/tratamiento farmacológico , Tiazoles/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Lesiones Precancerosas/tratamiento farmacológico , Moduladores de los Receptores de Estrógeno , Fosfatidilinositol 3-Quinasa Clase IRESUMEN
Signal transducer and activator of transcription 5 (STAT5) promotes cell survival and instigates breast tumor formation, and in the normal breast it also drives alveolar differentiation and lactogenesis. However, whether STAT5 drives a differentiated phenotype in breast tumorigenesis and therefore impacts cancer spread and metastasis is unclear. We found in two genetically engineered mouse models of breast cancer that constitutively activated Stat5a (Stat5aca) caused precancerous mammary epithelial cells to become lactogenic and evolve into tumors with diminished potential to metastasize. We also showed that STAT5aca reduced the migratory and invasive ability of human breast cancer cell lines in vitro. Furthermore, we demonstrated that STAT5aca overexpression in human breast cancer cells lowered their metastatic burden in xenografted mice. Moreover, RPPA, Western blotting, and studies of ChIPseq data identified several EMT drivers regulated by STAT5. In addition, bioinformatic studies detected a correlation between STAT5 activity and better prognosis of breast cancer patients. Together, we conclude that STAT5 activation during mammary tumorigenesis specifies a tumor phenotype of lactogenic differentiation, suppresses EMT, and diminishes potential for subsequent metastasis.
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Neoplasias de la Mama , Factor de Transcripción STAT5 , Animales , Femenino , Humanos , Ratones , Mama/patología , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/patología , Factor de Transcripción STAT5/metabolismoRESUMEN
Leucine-rich repeat-containing G protein-coupled receptors 4, 5, and 6 (LGR4/5/6) play critical roles in development and cancer. The widely accepted mechanism is that these proteins, together with their R-spondin ligands, stabilize Wnt receptors, thus potentiating Wnt signaling. Here we show that LGR4 enhanced breast cancer cell metastasis even when Wnt signaling was deactivated pharmacologically or genetically. Furthermore, LGR4 mutants that cannot potentiate Wnt signaling nevertheless promoted breast cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Multiomic screening identified EGFR as a crucial mediator of LGR4 activity in cancer progression. Mechanistically, LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a Wnt-independent LGR4-EGFR signaling axis with broad implications for cancer progression and targeted therapy. SIGNIFICANCE: This work demonstrates a Wnt-independent mechanism by which LGR4 promotes cancer metastasis.See related commentary by Stevens and Williams, p. 4397.
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Receptores ErbB/metabolismo , Metástasis de la Neoplasia , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Femenino , Células HEK293 , Humanos , Técnicas In Vitro , Estimación de Kaplan-Meier , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Proteoma/metabolismo , Análisis de Matrices Tisulares , Ubiquitina/metabolismo , Vía de Señalización WntRESUMEN
Chemotherapy employs anti-cancer drugs to stop the growth of cancerous cells, but one common obstacle to the success is the development of chemoresistance, which leads to failure of the previously effective anti-cancer drugs. Resistance arises from different mechanistic pathways, and in this critical review, we focus on the Fanconi Anemia (FA) pathway in chemoresistance. This pathway has yet to be intensively researched by mainstream cancer researchers. This review aims to inspire a new thrust toward the contribution of the FA pathway to drug resistance in cancer. We believe an indepth understanding of this pathway will open new frontiers to effectively treat drug-resistant cancer.
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Reparación del ADN , Resistencia a Antineoplásicos , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Antineoplásicos/uso terapéutico , Biomarcadores/metabolismo , Reparación del ADN/efectos de los fármacos , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Tissue injury induces changes in cellular identity, but the underlying molecular mechanisms remain obscure. Here, we show that upon damage in a mouse model, epidermal cells at the wound edge convert to an embryonic-like state, altering particularly the cytoskeletal/extracellular matrix (ECM) components and differentiation program. We show that SOX11 and its closest relative SOX4 dictate embryonic epidermal state, regulating genes involved in epidermal development as well as cytoskeletal/ECM organization. Correspondingly, postnatal induction of SOX11 represses epidermal terminal differentiation while deficiency of Sox11 and Sox4 accelerates differentiation and dramatically impairs cell motility and re-epithelialization. Amongst the embryonic genes reactivated at the wound edge, we identify fascin actin-bundling protein 1 (FSCN1) as a critical direct target of SOX11 and SOX4 regulating cell migration. Our study identifies the reactivated embryonic gene program during wound repair and demonstrates that SOX11 and SOX4 play a central role in this process.
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Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción SOXC/genética , Cicatrización de Heridas/genética , Heridas y Lesiones/genética , Animales , Diferenciación Celular/genética , Movimiento Celular/genética , Citoesqueleto/metabolismo , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Matriz Extracelular , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Factores de Transcripción SOXC/metabolismo , Heridas y Lesiones/embriologíaRESUMEN
The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with ß-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity.
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Carcinogénesis , Carcinoma de Células Escamosas/fisiopatología , Lipocalina 2/metabolismo , Neoplasias Cutáneas/fisiopatología , Proteína 1 Similar al Factor de Transcripción 7/metabolismo , beta Catenina/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , RatonesRESUMEN
Wnt signaling through ß-catenin plays a crucial role in skin development and homeostasis. Disruption or hyperactivation of this pathway results in skin defects and diseases (Lim and Nusse, Cold Spring Harb Perspect Biol 5(2), 2013). Monitoring Wnt signaling in skin under normal and abnormal conditions is therefore critical to understand the role of this pathway in development and homeostasis.In this chapter, we provide methods to detect Wnt/ß-catenin (canonical) signaling in the skin. We present a comprehensive list of Wnt reporter mice and detail the processing of skin tissue to detect reporter genes. From this list, we focus on the three most recent lines that, according to reports, are the most sensitive in skin. Additionally, we describe a protocol to detect nuclear ß-catenin, a hallmark of active Wnt signaling, although this technique should be used with caution due to its limited sensitivity. The techniques outlined below will be useful for detecting active Wnt signaling in skin.
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Biología Molecular/métodos , Piel/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo , Animales , Homeostasis , Ratones , Piel/crecimiento & desarrolloRESUMEN
Cell migration is an integral part of re-epithelialization during skin wound healing, a complex process involving molecular controls that are still largely unknown. Here we identify a novel role for Tcf3, an essential transcription factor regulating embryonic and adult skin stem cell functions, as a key effector of epidermal wound repair. We show that Tcf3 is upregulated in skin wounds and that Tcf3 overexpression accelerates keratinocyte migration and skin wound healing. We also identify Stat3 as an upstream regulator of Tcf3. We show that the promigration effects of Tcf3 are non-cell autonomous and occur independently of its ability to interact with ß-catenin. Finally, we identify lipocalin-2 as the key secreted factor downstream of Tcf3 that promotes cell migration in vitro and wound healing in vivo. Our findings provide new insights into the molecular controls of wound-associated cell migration and identify potential therapeutic targets for the treatment of defective wound repair.
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Proteínas de Fase Aguda/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Movimiento Celular/genética , Queratinocitos , Lipocalinas/metabolismo , Proteínas Oncogénicas/metabolismo , Repitelización/genética , Factor de Transcripción STAT3/metabolismo , Piel/metabolismo , Cicatrización de Heridas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Movimiento Celular/fisiología , Lipocalina 2 , Ratones , Ratones Noqueados , Repitelización/fisiología , Piel/citología , Cicatrización de Heridas/fisiología , beta Catenina/metabolismoRESUMEN
PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.
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Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Elementos Transponibles de ADN/genética , Mamíferos/metabolismo , Transgenes/genética , Transposasas/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Codón/genética , Genoma/genética , Células HEK293 , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Humanos , Ratones , Mutagénesis Insercional/métodos , Dedos de Zinc/genéticaRESUMEN
Hair follicles cyclically degenerate and regenerate throughout adult life and require regular stem cell activation to drive the cycle. In the resting phase of the hair cycle, hair follicle stem cells are maintained in a quiescent state until they receive signals to proliferate. We found that the forkhead transcription factor Foxp1 is crucial for maintaining the quiescence of hair follicle stem cells. Loss of Foxp1 in skin epithelial cells leads to precocious stem cell activation, resulting in drastic shortening of the quiescent phase of the hair cycle. Conversely, overexpression of Foxp1 in keratinocytes prevents cell proliferation by promoting cell cycle arrest. Finally, through both gain- and loss-of-function studies, we identify fibroblast growth factor 18 (Fgf18) as the key downstream target of Foxp1. We show that exogenously supplied FGF18 can prevent the hair follicle stem cells of Foxp1 null mice from being prematurely activated. As Fgf18 controls the length of the quiescent phase and is a key downstream target of Foxp1, our data strongly suggest that Foxp1 regulates the quiescent stem cell state in the hair follicle stem cell niche by controlling Fgf18 expression.
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Ciclo Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Folículo Piloso/citología , Proteínas Represoras/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Recuento de Células , Puntos de Control del Ciclo Celular , Proliferación Celular , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión de Mamíferos/citología , Factores de Crecimiento de Fibroblastos/genética , Células HEK293 , Humanos , RatonesRESUMEN
Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies.