RESUMO
We have performed whole transcriptome sequencing of 5-FU resistant and 5-FU sensitive tumors generated in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. Tumors were generated using the DMBA/TPA model of chemically induced carcinogenesis [1], tumor-bearing mice were subsequently treated with 5-FU, and tumor growth as well as response to treatment was monitored by measuring tumor volume twice a week. Based on these measurements, we selected two 5-FU resistant and two 5-FU sensitive tumors and performed whole transcriptome sequencing and in order to identify differentially expressed transcripts between the two sets. Data obtained is deposited and available through NCBI SRA (reference number SRP155180 - https://www.ncbi.nlm.nih.gov/sra/?term=SRP155180).
RESUMO
The insufficient ability of specialized cells such as neurons, cardiac myocytes, and epidermal cells to regenerate after tissue damage poses a great challenge to treat devastating injuries and ailments. Recent studies demonstrated that a diverse array of cell types can be directly derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or somatic cells by combinations of specific factors. The use of iPSCs and direct somatic cell fate conversion, or transdifferentiation, holds great promise for regenerative medicine as these techniques may circumvent obstacles related to immunological rejection and ethical considerations. However, producing iPSC-derived keratinocytes requires a lengthy two-step process of initially generating iPSCs and subsequently differentiating into skin cells, thereby elevating the risk of cellular damage accumulation and tumor formation. In this study, we describe the reprogramming of mouse embryonic fibroblasts into functional keratinocytes via the transient expression of pluripotency factors coupled with directed differentiation. The isolation of an iPSC intermediate is dispensable when using this method. Cells derived with this approach, termed induced keratinocytes (iKCs), morphologically resemble primary keratinocytes. Furthermore they express keratinocyte-specific markers, downregulate mesenchymal markers as well as the pluripotency factors Oct4, Sox2, and Klf4, and they show important functional characteristics of primary keratinocytes. iKCs can be further differentiated by high calcium administration in vitro and are capable of regenerating a fully stratified epidermis in vivo. Efficient conversion of somatic cells into keratinocytes could have important implications for studying genetic skin diseases and designing regenerative therapies to ameliorate devastating skin conditions.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Regulação da Expressão Gênica , Queratinócitos/citologia , Queratinócitos/transplante , Regeneração/genética , Animais , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Embrião de Mamíferos , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transgenes , Tretinoína/farmacologiaRESUMO
Tumorigenesis is a complex, multistep process that depends on numerous alterations within the cell and contribution from the surrounding stroma. The ability to model macroscopic tumor evolution with high fidelity may contribute to better predictive tools for designing tumor therapy in the clinic. However, attempts to model tumor growth have mainly been developed and validated using data from xenograft mouse models, which fail to capture important aspects of tumorigenesis including tumor-initiating events and interactions with the immune system. In the present study, we investigate tumor growth and therapy dynamics in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. We show that the rate of tumor growth and the effects of therapy are highly variable and mouse specific using a Gompertz model to describe tumor growth and a two-compartment pharmacokinetic/ pharmacodynamic model to describe the effects of therapy in mice treated with 5-FU. We show that inter-mouse growth variability is considerably larger than intra-mouse variability and that there is a correlation between tumor growth and drug kill rates. Our results show that in vivo tumor growth and regression in a double transgenic mouse model are highly variable both within and between subjects and that mathematical models can be used to capture the overall characteristics of this variability. In order for these models to become useful tools in the design of optimal therapy strategies and ultimately in clinical practice, a subject-specific modelling strategy is necessary, rather than approaches that are based on the average behavior of a given subject population which could provide erroneous results.
Assuntos
Carcinogênese , Transformação Celular Neoplásica , Modelos Animais de Doenças , Camundongos Transgênicos , Animais , Carcinógenos , Humanos , Camundongos , NeoplasiasRESUMO
Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro(TM) 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.
Assuntos
Neoplasias da Mama/enzimologia , Imunoensaio/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Eletroforese Capilar/métodos , Feminino , Humanos , Isoenzimas/análise , Isoenzimas/metabolismo , Lapatinib , Redes e Vias Metabólicas , Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/métodos , Oxidiazóis/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/análise , Quinazolinas/farmacologiaRESUMO
A rise in technologies for epigenetic reprogramming of cells to pluripotency, highlights the potential of understanding and manipulating cellular plasticity in unprecedented ways. Increasing evidence points to shared mechanisms between cellular reprogramming and the carcinogenic process, with the emerging possibility to harness these parallels in future therapeutics. In this review, we present a synopsis of recent work from oncogenic viruses which contributes to this body of knowledge, establishing a nexus between infection, cancer, and stemness.
Assuntos
Carcinogênese , Epigênese Genética , Regulação da Expressão Gênica , Vírus Oncogênicos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , HumanosRESUMO
We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, â¼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cromatografia de Afinidade/métodos , Glicoproteínas/análise , Lectinas/química , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Glicoproteínas/química , Glicoproteínas/classificação , Glicoproteínas/metabolismo , Humanos , Lectinas/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteoma/análise , Proteoma/químicaRESUMO
During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.