Marion Boyd Gillespie

Caspase-mediated cleavage of RNA-binding protein HuR regulates c-Myc protein expression after hypoxic stress.

Altered expression of RNA-binding proteins modulates gene expression in association with mRNAs encoding many proto-oncogenes, cytokines, chemokines, and proinflammatory factors. Hu antigen R (HuR), a ubiquitously expressed protein, controls a range of cellular functions such as tumor progression, apoptosis, invasion, and metastasis by stabilizing the AU-rich element located at the 3′-untranslated region (UTR) of target mRNAs. Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we report that during CoCl2-induced hypoxic stress, HuR is significantly overexpressed and undergoes caspase-dependent cleavage in head and neck squamous cell carcinoma cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3′-UTR of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3′-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression. These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells.

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA’s encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3′UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3′UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

Driving behaviors in patients with head and neck cancer during and after cancer treatment: a preliminary report

The purpose of this study was to explore the driving behaviors of head and neck cancer patients during and after cancer therapy.

Perioperative survival of elderly head and neck squamous cell carcinoma patients

To evaluate the survival benefit of surgery for elderly head and neck cancer (HNC) patients.

Diagnostic Value of RAS Mutations in Indeterminate Thyroid Nodules: Systematic Review and Meta-analysis

Objectives To determine the diagnostic value of HRAS, KRAS, and NRAS mutations in fine-needle aspiration biopsies of thyroid nodules that are nondiagnostic on cytology. Data Sources PubMed, Scopus, Embase, CINAHL. Review Methods Two authors independently searched the data sources. To be included, studies reported the RAS mutational status and postoperative histopathologic diagnosis of nodules that exhibited indeterminate cytology after fine-needle aspiration biopsy. Data were extracted to calculate sensitivity, specificity, and positive/negative predictive values of any HRAS, KRAS, or NRAS mutation. A meta-analysis was performed to generate pooled values for each parameter. Results A total of 7 studies with a combined 1025 patients met inclusion criteria. The pooled sensitivity of a RAS mutation for detecting cancer was 0.343 (95% confidence interval [95% CI], 0.198-0.506), while the pooled specificity was 0.935 (95% CI, 0.882-0.973). The weighted averages for positive predictive value and negative predictive value were 78.0% and 64.0%, respectively, with 68.0% accuracy. The positive likelihood ratio was 4.235 (95% CI, 1.506-11.910), and the negative likelihood ratio was 0.775 (95% CI, 0.630-0.953). Conclusion Our data suggest that testing for any RAS mutation is unlikely to change the clinical management of thyroid nodules that have indeterminate cytology. While a RAS mutation may rule in malignancy, the sensitivity of testing is low enough to merit further mutational analysis, repeat fine-needle aspiration, or surgical excision, even in the presence of a negative test.

Safety and Effectiveness of Upper Airway Stimulation via the Hypoglossal Nerve for Obstructive Sleep Apnea

Objectives: Determine safety and effectiveness of upper airway stimulation for treatment of obstructive sleep apnea (OSA) in a prospective, multi-center trial; with subsequent randomized, controlled therapy withdrawal evaluation. Methods: This study enrolled and screened 900 adult subjects. Inclusion criteria were moderate-to-severe OSA (apnea-hypopnea index [AHI] 20-50), prior continuous positive airway pressure (CPAP) failure or intolerance, and body mass index (BMI) < 32. All qualified subjects underwent further screening with drug-induced sleep endoscopy (DISE) to exclude those with concentric palatal collapse. 126 subjects meeting all inclusion/exclusion criteria then underwent surgical implantation of a right sided unilateral hypoglossal nerve stimulation electrode, with pulse generator and respiration sensor (Upper Airway Stimulation System, Inspire Medical Systems, Minnesota). Patients were evaluated at 2, 6, and 12 months. Primary outcome measures included AHI, oxygen desaturation index, Epworth Sleepiness Scale, and Functional Outcomes of Sleep Questionnaire at 12 months. Therapy withdrawal effect was evaluated by randomizing a subset of consecutive responders to one week of on or off therapy for controlled comparison. Results: All 126 subjects were successfully implanted. In general, subjects were middle aged (mean 54.5 ± 10.2 yrs), male (83%), with severe OSA (mean baseline AHI 32.0 ± 11.8). Transient dysarthria and dysphagia were noted in a minority of patients. Over 70% of subjects have completed the study, with the remaining scheduled to complete 12 month evaluation by April, 2013. Final safety and effectiveness outcomes will be presented. Conclusions: Implantation of an upper airway stimulation system involving unilateral hypoglossal nerve electrode placement appears safe and feasible, with transient dysarthria occurring infrequently. Effectiveness will be addressed after completion of 12 month follow-up for all subjects.

Abstract 4026: HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT.

Up to 60% of oropharyngeal cancers and 25% of head and neck squamous cell carcinomas (HNSCC) are high-risk human papillomavirus (HPV) positive, primarily HPV16. Differences in risk factors, age of presentation and clinical behavior of these tumors indicate that HPV+ and HPV- tumors develop with different molecular mechanisms and are biologically distinct. Based upon a gene expression profile comparison of HPV- and HPV+ HNSCC specimens, obtained from the Medical University of South Carolina ENT/Head and Neck Surgery clinic, we identified 384 candidate genes differentially expressed (125 genes up-regulated and 223 down-regulated) in HPV+ in comparison with HPV- tumors. GO analysis of the microarray data indicated that gene expression changes in HPV+ tumors affected primarily proliferation and cell cycle control, while HPV- tumors showed changes in genes involved in EMT, cell motility, and angiogenesis. We selected five genes (c-MET, TP53, TGF-beta2, BRCA1 and SIX1) for further analysis in a set of 44 tumor and four normal (tonsil and uvula) samples. Seventeen tumor samples were from African American (AA) patients, and 27 from European Americans (EA). The prevalence of HPV infection was 30% in samples from AA patients, and 65% in those from EA patients. Most HPV+ tumor samples (20 of 24) were positive for HPV16 and a subset of the HPV16+ tumor samples (55%) expressed HPV16 E7, as documented by real-time RT/PCR using tumor-derived RNA. The prevalence of active HPV infections (where E7 was expressed) was 17% in AA and 50% in EA patients. These tumor samples were utilized to explore the expression of the selected genes by real-time RT/PCR. Data were normalized to normal human keratinocytes based on three reference genes derived from a panel of seven candidate reference genes, using Normfinder, BestKeeper and GeNorm. Normalized relative quantities for each gene of interest were then calculated using qbasePLUS (Biogazelle.) c-MET was overexpressed, when compared to control tissue, in all tumor samples but levels of c-MET were the highest in HPV- EA samples, and comparatively lower in HPV+ samples. TP53 was overexpressed in HPV+ samples; TGF-beta2 and Six1 were clearly overexpressed in all tumor samples, compared to control tissue samples; and BRCA1 levels were higher in HPV+ samples, compared to HPV- samples and controls. These data are consistent with our microarray results that indicate that HPV- tumors exhibit gene expression profiles indicative of activation of EMT. We are investigating the molecular interactions that lead to overexpression of SIX1, c-MET and BRCA1 in these tumors, and the functional consequences of this overexpression in terms of cell growth and EMT. Citation Format: Swati Tomar, Sangeeta Kowli, Diego Altomare, Christian Graves, Susannah Kassler, Natalie A. Sutkowski, Marion Boyd Gillespie, Saundra H. Glover, Kim E. Creek, Lucia A. Pirisi-Creek. HPV16-positive and HPV16-negative head and neck squamous cell carcinomas display different patterns of expression of genes involved in the control of growth and EMT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4026. doi:10.1158/1538-7445.AM2013-4026

Abstract 5110: Divergent gene expression profiles in HPV-positive and HPV-negative head and neck squamous cell carcinomas

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Up to 60% of oropharyngeal cancers and 25% of head and neck squamous cell carcinomas (HNSCC) are due to high-risk Human Papillomavirus (HPV), primarily HPV16. Differences in risk factors, age of presentation and clinical behavior of these tumors indicate that HPV+ and HPV- tumors develop with different molecular mechanisms and are biologically distinct. To further investigate the molecular characteristics of these tumors, we compared the gene expression profiles of three HPV- and four HPV+ HNSCC specimens, obtained from the Medical University of South Carolina ENT/Head and Neck Surgery clinic. All HPV+ tumors were positive for HPV16 and expressed E7, as documented by real-time RT/PCR using tumor-derived RNA. Gene expression profiles were determined on Agilent 4x44K human oligonucleotide microarrays. Identification of differentially expressed genes (>2-fold change, p<0.05) and pathway analysis were performed using GeneSifter Software (Geospiza). Overall, 125 genes were up-regulated and 223 were down-regulated in HPV+ in comparison with HPV- tumors. Gene Ontology analysis of the dataset revealed that pathways related to cell cycle control, mitotic checkpoint, regulation of cell proliferation, programmed cell death and DNA metabolism were heavily affected in HPV+ tumors, while pathways affected in HPV- tumors included regulation of cell motility and migration, angiogenesis and integrin-mediated signaling. We confirmed by Real-Time RT/PCR the differential expression of a panel of genes of interest on these samples and several additional HNSCC specimens, and then we extended our follow-up analysis to specimens from over 30 new cases of HNSCC. Among the genes we targeted for follow-up is the MET oncogene, which encodes the hepatocyte growth factor/scatter factor receptor. MET is overexpressed about 5-fold in HPV- tumors, in comparison with HPV+ tumors actively expressing E7. The MET product, which controls growth, invasion and metastasis in cancer cells, has been recently proposed as a new therapeutic target for HNSCC. Supported by grant #P20MD001770 from the NIH/NIMHD. 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 5110. doi:1538-7445.AM2012-5110

Trends and Outcomes in the Management of Upper Esophageal Disorders

Objective: 1) Compare the outcomes of 2 surgical modalities used to treat hypopharyngeal diverticulum and esophageal stenosis at the Medical University of South Carolina (MUSC) and analyze the economic efficiency of each. 2) Compare the MUSC experience with statewide treatment in South Carolina (SC) Medicare patients. Method: Retrospective analysis was conducted of 50 patients treated at MUSC. The 2 approaches utilized were endoscopic laser diverticulotomy and transcervical open diverticulectomy with cricopharyngeal myotomy. SC Medicare data was collected for patients undergoing these procedures. Primary outcomes investigated were intraoperative complications, operative charge, hospital stay, and postoperative complications. Results: Statistically significant differences between the 2 operative groups at MUSC were observed in length of stay (1.08 days endoscopic group and 4.75 days transcervical open group; P > .0001) and operative charge (US

Immunomodulation of Vitamin D in Head and Neck Cancer

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