RESUMEN
The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , Queratinocitos/patología , Mutagénesis Insercional/métodos , Análisis de Secuencia de ADN/métodos , Neoplasias Cutáneas/genética , Proteína de Unión a CREB/genética , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Coactivador 2 del Receptor Nuclear/genética , Neoplasias Cutáneas/patologíaRESUMEN
Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.
Asunto(s)
Adenocarcinoma/genética , Elementos Transponibles de ADN , Neoplasias Mamarias Experimentales/genética , Fosfohidrolasa PTEN/genética , Adenocarcinoma/secundario , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Genes Supresores de Tumor , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares , Neoplasias Mamarias Experimentales/patología , Ratones Transgénicos , Mutagénesis , Mutación Missense , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.
Asunto(s)
ADN de Neoplasias/genética , Genes Relacionados con las Neoplasias/genética , Hibridación in Situ/métodos , Leucemia Mieloide/genética , Mutagénesis Insercional/genética , Análisis de Secuencia de ADN/métodos , Algoritmos , Animales , Biomarcadores de Tumor/genética , Elementos Transponibles de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Mieloide/patología , Masculino , Ratones , Proteínas de Neoplasias/genética , Programas Informáticos , Transposasas/genéticaRESUMEN
There is an impetus on development of implantable biomaterials with the characteristics of improved biodegradability, bio-absorbability and wound healing activities. The milk proteins have valuable nutritional and biological properties, which lead to the promotion of quality health. In this study, whey protein isolate or WPI (highly aggregated) and its component lactalbumin (less aggregated) were melt blended with polycaprolactone (PCL) and then compression moulded into thin sheets ( approximately 1mm thickness). The effects of structural morphologies of the proteins on the mechanical, morphological, in vitro enzymatic degradation, and cytotoxicity and cell proliferation characteristics of the biomaterials were examined. In general, the tensile strength and modulus of the biomaterials decreased with increasing protein content. Compared to WPI, lactalbumin showed a better compatibility with the PCL matrix as observed in the mechanical properties and scanning electron microscopic morphology. The biomaterials exhibited a good retention of the mechanical characteristics after digestion in a physiologically simulated fluid containing trypsin enzyme. However, lactalbumin containing biomaterials showed a better retention of the tensile properties compared to WPI containing biomaterials. The cell culture studies indicated that the biomaterials have no cytotoxic effects, moreover they enhanced the proliferation of L929 cells compared to the pure PCL. Finally, this study indicated that the PCL based biomaterials with a protein content of 20wt% may be applied in fabrication of implantable devices for soft tissue engineering, where it requires a reasonably low to moderate mechanical strength (e.g., approximately 10MPa tensile strength), and improved biodegradability, biocompatibility and tissue healing activities.
