Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma.

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors.

Current Technologies and Recent Developments for Screening of HPV-Associated Cervical and Oropharyngeal Cancers.

Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, predominantly represented by cervical cancer and oropharyngeal squamous cell carcinoma. Because of the prevalence of the virus, persistence of infection, and long latency period, novel and low-cost methods are needed for effective population level screening and monitoring. We review established methods for screening of cervical and oral cancer as well as commercially-available techniques for detection of HPV DNA. We then describe the ongoing development of microfluidic nucleic acid-based biosensors to evaluate circulating host microRNAs that are produced in response to an oncogenic HPV infection. The goal is to develop an ideal screening platform that is low-cost, portable, and easy to use, with appropriate signal stability, sensitivity and specificity. Advances in technologies for sample lysis, pre-treatment and concentration, and multiplexed nucleic acid detection are provided. Continued development of these devices provides opportunities for cancer screening in low resource settings, for point-of-care diagnostics and self-screening, and for monitoring response to vaccination or surgical treatment.

Obesity Contributes to Ovarian Cancer Metastatic Success through Increased Lipogenesis, Enhanced Vascularity, and Decreased Infiltration of M1 Macrophages.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy, with high mortality attributable to widespread intraperitoneal metastases. Recent meta-analyses report an association between obesity, ovarian cancer incidence, and ovarian cancer survival, but the effect of obesity on metastasis has not been evaluated. The objective of this study was to use an integrative approach combining in vitro, ex vivo, and in vivo studies to test the hypothesis that obesity contributes to ovarian cancer metastatic success. Initial in vitro studies using three-dimensional mesomimetic cultures showed enhanced cell-cell adhesion to the lipid-loaded mesothelium. Furthermore, in an ex vivo colonization assay, ovarian cancer cells exhibited increased adhesion to mesothelial explants excised from mice modeling diet-induced obesity (DIO), in which they were fed a "Western" diet. Examination of mesothelial ultrastructure revealed a substantial increase in the density of microvilli in DIO mice. Moreover, enhanced intraperitoneal tumor burden was observed in overweight or obese animals in three distinct in vivo models. Further histologic analyses suggested that alterations in lipid regulatory factors, enhanced vascularity, and decreased M1/M2 macrophage ratios may account for the enhanced tumorigenicity. Together, these findings show that obesity potently affects ovarian cancer metastatic success, which likely contributes to the negative correlation between obesity and ovarian cancer survival.

Detecting microRNA in human cancer tissues with fluorescence in situ hybridization.

The technique of nucleic acid in situ hybridization is an effective method for identifying the existence and abundance of nucleic acids in tissue sections or cytological preparations. Such a method has the advantage of keeping morphological relationships intact while identifying changes at the molecular level. As a noncoding regulatory RNA, microRNA has been found to intricately control many physiological and pathological conditions. We provide here a representative fluorescence in situ hybridization protocol for microRNA detection, and note commonly used alternatives, and some troubleshooting points. The method described is based on formalin-fixed paraffin-embedded oral cancer tissues but should be broadly applicable to similarly processed tissues of other types of cancer.

Epidermal growth factor receptor-mediated membrane type 1 matrix metalloproteinase endocytosis regulates the transition between invasive versus expansive growth of ovarian carcinoma cells in three-dimensional collagen.

The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y(573)F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics.

Functional relevance of urinary-type plasminogen activator receptor-alpha3beta1 integrin association in proteinase regulatory pathways.

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.