Detection of microrna expression in formalin-fixed paraffin-embedded materials with fluorescent in situ hybridization [abstract]

Non-coding regulatory RNAs designated microRNAs (miRNA) are small single-stranded RNAs that may suppress the protein outcome of target messenger RNAs and thereby regulate gene expression networks. To facilitate delineation of the role of microRNAs in cancer pathology, the goal of this study was to demonstrate the feasibility of detecting microRNA expression using commercially available formalin-fixed paraffin-embedded (FFPE) tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH) procedures with (a) different synthetic probes: regular custom DNA oligos vs. LNA incorporated DNA oligos complementary to mature microRNA sequence; (b) different tracers for the probes: biotin vs. digoxigenin; (c) different visualization: direct vs. TSA amplification; and (d) different blocking reagents for endogenous peroxidase. Finally, we performed mir-146a FISH on a commercially available oral cancer tissue microarray (TMA), which contains 40 cases of oral squamous cell carcinoma (OSCC) and 10 cases of normal epithelia from the human oral cavity. Spiny cells in most normal oral squamous epithelia were positive for mir-146a, while basal cells stained negative. In OSCC tissues, a correlation between a decrease in mir-146a and an increase in histological grade was observed. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissue. This detection is useful for studies on the participation of microRNA in oral cancer pathology, and may have potential prognostic or diagnostic value

Potential role of Kallikrein 5 expression in metastatic dissemination of oral squamous cell carcinoma (OSCC) [abstract]

OSCC is one of the top 10 causes of cancer deaths worldwide, with only a 50% 5-year survival rate, necessitating the discovery of novel biomarkers and therapeutic targets. Using cDNA microarray analyses, we identified expression of a panel of kallikreins (KLK 5, 7, 8, and 10) associated with formation of more aggressive OSCC tumors in a murine orthotopic OSCC model. The goal of the current study is to elucidate the function of KLKs in OSCC progression. In initial studies, KLK levels in malignant OSCC cells (SCC25) were compared to cells from normal oral mucosa (OKF/6) and pre-malignant oral keratinocytes (pp126) using qPCR. A marked elevation of all KLKs was observed in aggressive SCC25 cells relative to OKF/6 cells. In normal skin, KLKs 5, 7, and 8 are involved in desquamation during epidermal differentiation via proteolytic cleavage of the desmosomal cadherin component desmoglein 1 (Dsg1). As loss of cell-cell adhesion is prevalent in tumor metastasis, Dsg1 was evaluated by immunofluorescence and western blotting. SCC25 cells exhibit cleavage of Dsg1 which is blocked by treatment with a KLK inhibitor as well as by siRNA silencing of KLK5. Furthermore, cell-cell adhesion and barrier function assays demonstrate that silencing of KLK5 enforces cell-cell adhesion and improves epithelial barrier function. Current studies are focusing on overexpression KLK5 in normal oral mucosal cells (OKF/6) to further evaluate the role of KLK5 in desmosomal cadherin processing

Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

The noncoding RNA designated as microRNA (miRNA) is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH) procedures to detect miR-146a with (a) different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA) incorporated DNA oligonucleotides; (b) different reporters for the probes: biotin versus digoxigenin (DIG); (c) different visualization: traditional versus tyramide signal amplification (TSA) system; (d) different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC) and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value

Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration

Background Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. Methods We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. Results Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59 % were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30 % were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. Conclusions We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells

MicroRNA mir-146a expression in oral cancer tissues [abstract]

Recent advances in basic research have shown that the expression and function of microRNAs impact on gene expression regulation networks extensively. Ever increasing new knowledge from microRNA studies should be translated into medical practice if possible. Formalin-fixed paraffin-embedded (FFPE) human cancer tissues are a huge resource for delineating the role and potential application of microRNAs in cancer pathology. However, detecting microRNA in such cancer tissues with in situ hybridization is a challenge. The goal of this study was to demonstrate the feasibility of such approach in cancer research and practice

An Update on Immunohistochemistry in Translational Cancer Research

Immunohistochemistry (IHC) takes advantage of the specific binding between antigen and antibody to measure the presence and abundance of antigen while simultaneously providing morphologic context on a tissue section. Since the revolutionary application of heat-induced epitope retrieval methods on formalin-fixed paraffin-embedded tissues, which started in early 1990s, IHC has been routinely used in diagnostic pathology. This approach has also enabled mining of the rich archives of pathologic specimens for exploration in translational cancer research. Newer IHC biomarkers are being continuously found as aids in differential diagnosis, prediction of outcome or response to molecular-targeted therapies. These are prime examples for translational cancer research. The last decade has witnessed some significant improvements in the use of this technology. This review provides an overview on the current status of IHC as applied in translational cancer research, commenting on the underlying principles in specimen preparation, reagent choice, staining procedure, and results evaluation so that both beginners and seasoned users could appreciate the key factors and benefit from this update

Molecules of cell adhesion and extracellular matrix proteolysis in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity with a poor 5-year survival rate, due in large part to the presence of metastatic disease at initial diagnosis. In recent years, a number of studies have examined the oral tumor microenvironment to asses the potential dynamic balance between extracellular matrix deposition and proteolytic degradation as well as the cellular adhesion molecules that mediate adhesion to matrix and regulate tissue cohesion. The objective of this review is to provide a brief overview of the major matrix components, adhesion molecules and proteolytic enzymes in the oral tumor microenvironment and to summarize recent findings regarding the role of these complex molecular players in oral tumor progression

Potent, simplified derivatives of pateamine A

The present invention provides a compound of Formula I, all of its related stereoisomers, and their pharmaceutically acceptable salts, wherein A—B, K, Q, X, Y, Z, R and R1 are as defined in Claim 1. The present invention also provides processes for the preparation thereof, the use thereof in treating immune mediated disease and conditions, and pharmaceutical compositions for use in such therapyU

Potent, simplified derivatives of pateamine A

The present invention provides a compound of Formula I, all of its related stereoisomers, and their pharmaceutically acceptable salts, wherein A?B, K, Q, X, Y, Z, R and R1 are as defined in Claim 1. The present invention also provides processes for the preparation thereof, the use thereof in treating immune mediated disease and conditions, and pharmaceutical compositions for use in such therapyU

Potent, simplified derivatives of pateamine A

The present invention provides a compound of Formula I, all of its related stereoisomers, and their pharmaceutically acceptable salts, wherein A—B, K, Q, X, Y, Z, R and R1 are as defined in Claim 1. The present invention also provides processes for the preparation thereof, the use thereof in treating immune mediated disease and conditions, and pharmaceutical compositions for use in such therapyU