Enoxacin induces oxidative metabolism and mitigates obesity by regulating adipose tissue miRNA expression.

MicroRNAs (miRNAs) have been implicated in oxidative metabolism and brown/beige adipocyte identity. Here, we tested whether widespread changes in miRNA expression promoted by treatment with the small-molecule enoxacin cause browning and prevent obesity. Enoxacin mitigated diet-induced obesity in mice, and this was associated with increased energy expenditure. Consistently, subcutaneous white and brown adipose tissues and skeletal muscle of enoxacin-treated mice had higher levels of markers associated with thermogenesis and oxidative metabolism. These effects were cell autonomous since they were recapitulated in vitro in murine and human cell models. In preadipocytes, enoxacin led to a reduction of miR-34a-5p expression and up-regulation of its target genes (e.g., Fgfr1, Klb, and Sirt1), thus increasing FGF21 signaling and promoting beige adipogenesis. Our data demonstrate that enoxacin counteracts obesity by promoting thermogenic signaling and inducing oxidative metabolism in adipose tissue and skeletal muscle in a mechanism that involves, at least in part, miRNA-mediated regulation.

Overexpression of miR-21-5p as a predictive marker for complete tumor regression to neoadjuvant chemoradiotherapy in rectal cancer patients.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the preferred treatment strategy for locally advanced rectal cancer. However, complete tumor regression is observed in a significant proportion of patients after nCRT, making them ideal candidates for alternative treatment strategies to this considerably morbid procedure. Identification of such patients based on clinical findings (complete clinical response - cCR) is difficult mainly because it relies on subjective clinical and imaging studies. Our goal was to identify biomarkers capable of predicting complete response to nCRT. METHODS: We analyzed miRNA expression profile using deep sequencing in rectal tumor biopsies prior to nCRT. Differential expression was investigated by EdgeR for a training (n = 27) and a validation (n = 16) set of patients to identify miRNAs associated with treatment response (complete vs. incomplete). In vitro experiments with two cancer cell lines were also performed in order to evaluate the possible role of miRNAs on response to nCRT. RESULTS: We found 4 miRNAs differentially expressed between complete and incomplete responders to nCRT. In addition, validation was performed using an independent group of patients and miR-21-5p was confirmed as being overexpressed in complete responders. Overall sensitivity and specificity of miR-21-5p expression in predicting complete response to nCRT was 78% and 86% respectively. Interestingly, in a subset of patients with cCR followed by early local recurrence, the expression level of miR-21-5p was considerably low, similarly to incomplete responders. We also found SATB1, a miR-21-5p target gene and known multidrug resistance gene, whose expression was inversely correlated with miR-21-5p expression. Finally, we performed functional experiments and showed that miR-21-5p and SATB1 may be directly involved with poor response to nCRT in rectal cancer patients. CONCLUSIONS: This study suggests miR-21-5p as a promising predictive biomarker, which should aid in the selection of patients with cCR to nCRT that potentially could be spared from radical surgery.

Identification of human exons overexpressed in tumors through the use of genome and expressed sequence data.

Alternative splicing is one of the major sources of the large transcriptional diversity found in human cells. Splicing variants have been shown to be associated with features like spreading and progression in several human tumors. Therefore, such variants may be of great importance as both diagnostic and therapeutic tools. Here, by using a set of criteria regarding the expression pattern of splicing variants and statistical analyses, we were able to screen the genome for exons overexpressed in tumors of specific tissues. However, as in other analyses attempting to identify tumor-associated variants, our list of candidates was seriously inflated with cases of genes differentially expressed in tumors. To exclude these cases and increase the probability of finding bona fide regulated splicing variants, we performed a serial analysis of gene expression (SAGE), excluding those genes that were shown to be upregulated in tumors. This allowed us to predict the overexpression of single exons in specific tumors. Our final group of candidates includes 1,386 exons belonging to 638 genes. Experimental validation of a few candidates in normal tissue, tumor cell lines, and patient samples suggests that most of these candidates are indeed tumor-associated exons. Further functional classification of our candidate genes shows that our final list is slightly inflated with cancer-related genes.

Caracterização de um novo antígeno tumoral: CTSP-1 / Characterization of a new tumor antigen: CTSP-1

A necessidade de identificar novos antígenos tumorais que possam ser utilizados no tratamento e diagnóstico do câncer, tem levado ao desenvolvimento de técnicas cada vez mais eficientes na detecção dos mesmos. Antígenos de diferentes categorias foram identificados e caracterizados, sendo os antígenos cancer-testis (CT) e os antígenos de diferenciação (CD) os de maior importância clínica, dado seu restrito padrão de expressão. Utilizando uma estratégia de alinhamento de seqüências expressas no genoma humano, identificamos um novo transcrito localizado no cromossomo 21, denominado CTSP-1, que apresenta alta similaridade com o antígeno tumoral NY-BR-1... Além disso, avaliamos seu padrão de expressão em diferentes tecidos normais, linhagens celulares tumorais e amostras de tumores de pacientes... Através de immunoblotting foi possível a identificação de uma banda específica de 22kDa, correspondente ao peso esperado da proteína CTSP-1 em extrato total de testículo normal. Em seguida, através da imunohistoquímica, verificamos uma marcação preferencial nas espermatogônias e células de Leydig de testículo normal. Nos tecidos com amostras pareadas normal/tumor (mama e próstata), apenas as amostras tumorais foram fortemente marcadas. Posteriormente, a proteína CTSP-1 recombinante foi utilizada na investigação de anticorpos específicos em plasma de pacientes com câncer. Aproximadamente 150 amostras foram analisadas, das quais 20% apresentaram resposta imune humoral contra a proteína CTSP-1. Estes resultados revelam que o CTSP-1 é um novo antígeno tumoral da categoria dos CTs, com expressão restrita a tumor e testículo e com alta imunogenicidade em pacientes com câncer...(AU)

The need to identify new tumor antigens to be used in cancer treatment and diagnosis has lead to the development of efficient techniques for this purpose. Antigens from different categories have been identified and characterized and, among those, the cancer-testis (CT) and the cancer differentiation (CD) antigens are of the greatest clinicai interest dueto their restricted expression partem. Using alignments between expressed sequences and the Human Genome Sequence, we identified a new gene located on chromosome 21, named CTSP-1. This gene has a high similarity to the tumor antigen NY-BR-1, which encodes for a tissue specific transcription factor and is a potential target for cancer immunotherapy. In order to verify ifthe CTSP-1 gene is really a new tumor antigen, we performed its complete characterization. Using different techniques, we were able to obtain the complete sequence of the CTSP-1 gene and to identify different alternative polyadenilation and splicing forms. Moreover, we analyzed the CTSP-1 expression pattern in normal tissues, tumor cell lines and tumor samples. CTSP-1 showed a restricted expression pattern, being expressed only in testis among normal tissues, in different tumor celllines (9/22) and in different tumor types (7 4/178), which matches with the expression pattern of Cancer-Testis antigens. Afterwards, the recombinant CTSP-1 protein was expressed in a heterologous system and used for the generation of polyclonal antibody in mice. This antiboby was used in immunoblotting and immunohistochemistry experiments for the detection of CTSP-1 protein in normal testis and paired normal and tumor samples from breast and prostate. Using immunoblotting, a 22kDa specific band was identified in testis total protein extract, corresponding to the expected molecular weight of the CTSP-1 protein. Using immunohistochemistry, we verified the preferential staining of germ cells and Leydig cells in normal testis. Among tissues with paired normal/tumor samples, only tumor samples were strongly stained. The CTSP-1 recombinant protein was also used in the search for specific antibodies in plasma from cancer patients. Approximately 150 samples were analyzed, of which 20% showed a humoral immune response against the CTSP-1 protein. Taken together, these results confirm that the CTSP-1 gene is a new tumor antigen from the Cancer-Testis category, with restricted expression in testis and tumors and with high immunogenicity in cancer patients (AU)