Ana L. Nocito

Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus–associated lesions as a marker for progression to malignancy

High‐risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h‐Dlg for ubiquitin‐mediated proteolysis. The h‐Dlg oncosuppressor is associated with cell–cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h‐Dlg expression in HPV‐associated lesions. We analyzed h‐Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR‐colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPV‐associated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV‐positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late‐stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability.

Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity.

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

Alteration of the microRNA-122 regulatory network in rat models of hepatotoxicity

MicroRNAs are small RNA molecules that post-transcriptionally regulate gene expression. MicroRNA-122 is the most abundant and specific liver microRNA. Hepatotoxicity involves a significant alteration of liver gene expression. The aim of this work was to evaluate the microRNA-122 regulatory network in models of hepatotoxicity induced by thioacetamide or carbon tetrachloride. We report that the toxins decreased the expression of microRNA-122, which corresponded with an increase in two target genes: Cyclin G1 and the cationic amino acid transporter CAT-1. We found a decreased expression of its precursor, pri-microRNA-122, and of the transcription factors that specifically bind its promoter: CCAAT/enhancer-binding protein alpha, and members of the hepatocyte nuclear factor family. Therefore, microRNA-122 expression levels are under transcriptional control during hepatotoxicity. We propose that the changes observed are associated with the liver response to cope with the injury caused by the hepatotoxins, likely through a cell proliferation process to repair the damaged tissue.

DLG1 polarity protein expression associates with the disease progress of low-grade cervical intraepithelial lesions

Human Discs large tumour suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes. We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n=30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n=23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n=4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n=3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p<0.00001). This work contributes to the knowledge of DLG1 biological functions, suggesting that its expression may have an important role in the progression of early dysplastic cervical lesions, giving prognostic information.

Abstract 701: TUMOR lymphocyte infiltration, expression of CD4, CD8, FOXP3, CD34 molecules and their relationship with tumor evolution after primary treatment in breast cancer patients

Considering that, approximately, only half of breast cancer patients respond to present therapies, a better knowledge of breast cancer biology, one of the most common neoplasms in women, will enable to design better therapeutic approaches. It has been proposed that different tumor characteristics like inflammatory infiltrate intensity, number of T regulatory (Tregs) cells and density of tumor vasculature could be useful to anticipate the response to therapy. Our aim was: a) to analyze the lymphocytic infiltrate and the expression of CD4, CD8, CD34 (endothelial marker) and Foxp3 (Treg marker) in primary tumors of breast cancer patients, and b) to relate them with clinical evolution (disease free: DF or relapsed: R) after 5 years from the primary treatment (surgery). There were analyzed archive samples of ductal mammary tumors (n=27), mostly in stages I and II. The expression of CD4 and CD8 in intra- and peri-tumoral lymphocytes was determined by immunohistochemistry (IHC) and confirmed by confocal microcopy; also the expression of Foxp3 in lymphocytes and tumor cells and CD34 in endothelial cells was evaluated by IHC. The lymphocytic infiltrate was assessed in histological sections stained with hematoxylin-eosin. The quantification was done in 20 high magnification fields and a score ranging from 0 (null) to 6 (high) was utilized. In DF patients (n=22) the intratumoral lymphocytic infiltrate (median [range]: 3 [0-6]) was more intense than that of R patients (n=5), (2 [0-2]), (P=0.05). For CD4, CD8, CD34 and Foxp3 molecules no differences between groups of patients were observed, being their expression highly variable: the score for CD34 varied from 1 to 6 and for the other three molecules from 0 to 6, in either group. In conclusion, the presence of an intense intratumoral lymphocytic infiltrate could be a prognostic marker of good response to treatment, at least in the first 5 years after surgery. These results warrant the study of higher number of tumor samples, since its confirmation could give support for its use in the clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 701. doi:1538-7445.AM2012-701

Abstract 3146: The involvement of Foxp3 or CD34 expression and tumor lymphocyte infiltration in the prognosis of breast carcinoma patients

Breast cancer is a heterogeneous disease with different known biological subclasses some of which are associated with immune cell infiltration. Studies in mice have shown that thymus-derived CD4+CD25+ regulatory T cells (Treg; FOXP3+ lymphocytes) inhibit the antitumor immune response. The presence of inflammatory cells may represent an immune response to the tumor, but could also potentially exhibit pro-tumor effects including stimulation of tumor invasion or angiogenesis. Angiogenesis has an important role in the progression of breast cancer. Microvessel density has been found to be strongly associated with features of tumor aggression as larger size and poor differentiation. It was observed that different characteristics of tumors like immune cells infiltration, number of Tregs and blood vessels density would be useful tools to anticipate the response to the therapy. The aim of this study was to analyze different cellular characteristics of primary tumors of patients with breast cancer and their relationship to the response to therapy. Samples of breast carcinomas (Stage I and II) (n=14) were analyzed. Foxp3 and CD34 expression was determined immunohistochemically on formalin-fixed, parafin-embedded tumor sections. Also, peritumor and intratumor lymphocytes infiltration was evaluated by hematoxylin-eosin staining. For quantification in 20 high magnification fields, a 0 (nule expression) to 6 (high expression) score was utilized. Confocal microcopy for Foxp3 was also used. Lymphocytes were mainly found in the intratumor (IT) area. Twelve over 14 patients (DF) that had a disease free survival of approximately 5 years showed an IT score for lymphocyte infiltration [median (range): 4 (2-6)] higher than that of 2/14 patients that relapsed (R) [2 (2-2)], p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3146. doi:10.1158/1538-7445.AM2011-3146