Gene set enrichment analysis in closed pyometra detected by IPA analysis.

<p>Gene set enrichment analysis in closed pyometra detected by IPA analysis.</p

Immunohistochemical panel to characterize canine prostate carcinomas according to aberrant p63 expression

<div><p>An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases.</p></div

Data validation.

<p>(A) Boxplot illustrating MM (normal) and ULs (tumour) normalised to obtain relative expression values for all samples evaluated by RT-qPCR. <i>P</i> = paired <i>t</i> test significance. **<i>P</i> = 0.006; ***<i>P</i> = 0.0002; Immunostaining frequency for the (B) FGFR1 and (C) IGFBP5 proteins. The <i>P</i> values (Fisheŕs test) were obtained based on the comparison of the MM and ULs immunostaining results.</p

Molecular Expression Profile Reveals Potential Biomarkers and Therapeutic Targets in Canine Endometrial Lesions

<div><p>Cystic endometrial hyperplasia (CEH), mucometra, and pyometra are common uterine diseases in intact dogs, with pyometra being a life threatening disease. This study aimed to determine the gene expression profile of these lesions and potential biomarkers for closed-cervix pyometra, the most severe condition. Total RNA was extracted from 69 fresh endometrium samples collected from 21 healthy female dogs during diestrus, 16 CEH, 15 mucometra and 17 pyometra (eight open and nine closed-cervixes). Global gene expression was detected using the Affymetrix Canine Gene 1.0 ST Array. Unsupervised analysis revealed two clusters, one mainly composed of diestrus and CEH samples and the other by 12/15 mucometra and all pyometra samples. When comparing pyometra with other groups, 189 differentially expressed genes were detected. <i>SLPI</i>, <i>PTGS2/COX2</i>, <i>MMP1</i>, <i>S100A8</i>, <i>S100A9</i> and <i>IL8</i> were among the top up-regulated genes detected in pyometra, further confirmed by external expression data. Notably, a particular molecular profile in pyometra from animals previously treated with exogenous progesterone compounds was observed in comparison with pyometra from untreated dogs as well as with other groups irrespective of exogenous hormone treatment status. In addition to <i>S100A8</i> and <i>S100A9</i> genes, overexpression of the inflammatory cytokines <i>IL1B</i>, <i>TNF</i> and <i>IL6</i> as well as <i>LTF</i> were detected in the pyometra from treated animals. Interestingly, closed pyometra was more frequently detected in treated dogs (64% <i>versus</i> 33%), with <i>IL1B</i>, <i>TNF</i>, <i>LBP</i> and <i>CXCL10</i> among the most relevant overexpressed genes. This molecular signature associated with potential biomarkers and therapeutic targets, such as <i>CXCL10</i> and <i>COX2</i>, should guide future clinical studies. Based on the gene expression profile we suggested that pyometra from progesterone treated dogs is a distinct molecular entity.</p></div

Protein-protein interaction (PPI) networks in closed and open pyometra.

<p>PPI networks based on altered genes exclusively detected in closed pyometra and open pyometra and their interactive partners built and visualized with Navigator v.2.3. Potential candidates for biomarkers (B blue circles) and targets for therapy (Drugs, D orange circles) in closed pyometra and open pyometra are highlighted. Triangles represent the genes with altered expression in each group and are color-coded according to Gene Ontology (GO). Upright and inverted triangles indicate overexpressed and underexpressed genes, respectively.</p

Gene set enrichment analysis in closed pyometra detected by IPA analysis.

<p>Gene set enrichment analysis in closed pyometra detected by IPA analysis.</p

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

<div><p>Background</p><p>Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40–50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs.</p> <p>Methodology</p><p>We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data.</p> <p>Principal Findings</p><p>The integrated analysis identified the top 30 significant genes (<i>P</i><0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional <i>in silico</i> analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (<i>P</i> = 0.006 and <i>P</i><0.01, respectively) and IGFBP5 (<i>P</i> = 0.0002 and <i>P</i> = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium.</p> <p>Conclusions</p><p>The integrative genomic and transcriptomic approach indicated that <i>FGFR1</i> and <i>IGFBP5</i> amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.</p> </div

Characterization of basal cell markers in normal prostate samples, canine p63+ PC and canine p63- PC.

<p>(A) p63 immunohistochemical expression in a normal prostate gland. Positive basal cells were observed in a discontinuous basal cell layer. (B) p63+ tumor showing diffuse nuclear p63 expression by neoplastic cells. (C) p63- tumor presenting no p63 expression (blue staining). Few non-neoplastic basal cells with nuclear p63 expression (internal control–black arrows) were observed. (D) Immunohistochemical staining of high molecular weight cytokeratin (HMWC) in a normal prostate gland. Positive staining was observed in few basal cells (brown staining—white arrows) establishing a discontinuous basal cell layer. (E and F) Diffuse HMWC expression in p63+ and p63- tumors, respectively. (G) CK5 immunohistochemical expression in canine normal samples. There was a discontinuous basal cell layer with few CK5 positive cells (brown staining). (H) p63+ tumor showing diffuse CK5 expression (brown staining) in all neoplastic cells. (I) p63 tumor showing no CK5 expression by neoplastic cells (blue staining). An atrophic prostatic gland (black arrow) was observed presenting diffuse CK5 expression (internal control). (J) CK8/18 expression in normal prostate. Positive cytoplasmic expression was observed in the epithelial luminal cells (brown staining). p63+ (K) and p63 (L) tumors presented diffuse cytoplasmic staining in all neoplastic cells (brown staining). The negative staining can be observed as a blue signal.</p

Seventy-five modulators obtained from integrative analysis.

<p><b>In bold</b>, the top 30 modulators based on the highest scores on CONEXIC; Hg18: Human genome version 18 (Mar 2006 NCBI36); ^*miRNA target prediction; positive (+) and negative (−) signs indicate the gene status with regard to the genomic gains and losses and up- or down-regulated gene expression, respectively. ^£Regions which usually not are involved in chromosomal breakpoints.</p

Protein-protein interaction network of 75 modulators.

<p>The 75 modulators were used to query the I2D database to obtain their interacting partners (and also interactions among the modulators). I2D v. 2.0 contained data on 62 modulators, which resulted in a large network of 1,456 proteins and 29,530 interactions. The upward triangles represent up-regulated genes, and the downward triangles represent down-regulated genes. The red and green triangle lines represent genes in amplified and deleted regions, respectively. The biological processes that the modulators are involved are represented by different colours, per the legend, and the triangle size corresponds with the score, i.e., larger triangles depict the highest scores. Direct physical interactions between modulators are represented by blue lines. The remainder of the network is partially transparent to reduce the clutter. The network visualisation and analysis was performed in NAViGaTOR 2.3.</p