miR-124 Regulates the Epithelial-Restricted with Serine Box/Epidermal Growth Factor Receptor Signaling Axis in Head and Neck Squamous Cell Carcinoma.

Epithelial-restricted with serine box (ESX), a member of the ETS transcription factor family, is elevated and regulates EGFR in head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms that contribute to ESX dysregulation remain to be elucidated. In this study, in silico analysis of the 3'-untranslated region (UTR) of ESX predicted two miR-124-binding sites. Delivery of miR-124 inhibited the 3'UTR ESX-driven reporter activity by 50% (P < 0.05) confirming ESX as a direct target of miR-124. Loss of miR-124 was found to be a frequent event in HNSCC. miR-124 expression was significantly depleted in the primary tumor compared with matched normal tissue in 100% (12/12) of HNSCC patients; relative mean miR-124 expression of 0.01197 and 0.00118 (P < 0.001, n = 12) in matched normal adjacent tissue and primary HNSCC tumor, respectively. Overexpression of miR-124 decreased ESX and EGFR levels in miR-124(low)/ESX(high)/EGFR(high) SCC15 HNSCC cells and reduced cell invasion, migration, proliferation, and colony formation. SCC15 cells with miR-124 restoration were less tumorigenic in vivo than miR-control SCC15 cells (70% inhibition, P < 0.01). Restoration of miR-124 in SCC15 cells enhanced the antiproliferative efficacy of the EGFR/Her2 tyrosine kinase inhibitors. Furthermore, recapitulation of EGFR in miR-124-overexpressing SCC15 cells was sufficient to completely block the antiproliferative effects of lapatinib and afatinib. Taken together, our work provides intriguing evidence that miR-124 is a novel therapeutic approach to reduce ESX/EGFR, and may be a tractable strategy to enhance the response rate of HNSCC patients to current anti-EGFR/Her2 therapies.

Suberoylanilide hydroxamic acid (SAHA) reverses chemoresistance in head and neck cancer cells by targeting cancer stem cells via the downregulation of nanog.

Acquisition of chemoresistance and metastatic phenotype are the major causes of treatment failure and mortality in head and neck squamous cell carcinoma (HNSCC) patients. Histone deacetylases (HDACs) have been shown to be overexpressed in many tumor types and directly linked to poor prognosis. In this study, we demonstrate that HDACs are markedly elevated in HNSCC. HDACs expression was further increase in cisplatin resistant cell lines (CisR). In addition, cisplatin-resistant cells showed enhanced stem cell properties and tumor metastasis. Depletion of HDAC1 and 2 in CisR cell lines significantly reversed cisplatin resistance and tumorsphere formation. Next, we tested the efficacy of Suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, by using both in vitro and in vivo models. SAHA significantly inhibited cell proliferation and synergistically enhanced the anti-proliferative effects of cisplatin. In addition, SAHA significantly decreased tumorsphere formation by markedly reducing nanog expression. In a SCID mouse xenograft model, SAHA significantly enhanced the anti-tumor effects of cisplatin treatment with no added systemic toxicity. Furthermore, SAHA and cisplatin combination treatment significantly decreased tumor metastasis and nanog expression, in vivo. Taken together, our results suggest that targeting HDACs with SAHA could be an effective treatment strategy for the treatment of HNSCC patients.

Down regulation of RhoC by microRNA-138 results in de-activation of FAK, Src and Erk1/2 signaling pathway in head and neck squamous cell carcinoma.

OBJECTIVE: RhoC a pro-metastatic oncogene is constitutively active in many head and neck squamous cell carcinomas. MicroRNA-138 which possesses a documented tumor suppressor function can bind to the 3'UTR of RhoC mRNA and inhibit its activity. We hypothesize that miR-138 can inhibit the function of RhoC and consequently the activation of downstream target molecules involve in the signaling cascade. For this reason we investigated the role of miR-138 in HNSCC. METHODS: In vitro studies were carried out to evaluate the role of miR-138 in HNSCC cell lines and in primary tumors obtained from HNSCC patients. Real time RT-PCR, Western blot, cell motility, invasion and colony formation assays were performed according to standard procedures. RESULTS: Data obtained by G-LISA and real time PCR shows an inverse correlation between RhoC expression and miR-138 in HNSCC cell lines. Additionally, we obtained a similar pattern of RhoC and miR-138 expression in primary tumors from HNSCC patients. Over expression of miR-138 in HNSCC lines showed down regulation of RhoC, as well as a decrease in cell motility, invasion colony and stress fiber formation. Furthermore, a significant down regulation was observed for FAK, Src and Erk(1/2) upon miR-138 overexpression. CONCLUSION: These findings strongly suggest that the inhibition of RhoC can be achieved by over expressing miR-138, which further attenuates the downstream signaling cascade leading to cancer progression and survival. Moreover, this study for the first time shows that down regulation of FAK, Src and Erk(1/2) by miR-138 overexpression is due to inhibition of RhoC in HNSCC.

Genetic and chemical targeting of epithelial-restricted with serine box reduces EGF receptor and potentiates the efficacy of afatinib.

EGF receptor (EGFR) is elevated in more than 90% of head and neck squamous cell carcinoma (HNSCC). However, a majority of patients with HNSCC do not respond to anti-EGFR therapeutics. Insensitivity to EGFR inhibitors may be due to kinase-independent actions of EGFR and/or activation of Her2. Strategies to reduce EGFR and Her2 protein levels in concert may be an optimal approach to enhance the efficacy of current anti-EGFR molecules. In this study, knockdown of epithelial-restricted with serine box (ESX) decreased EGFR and Her2 promoter activity, expression, and levels. ESX was elevated in primary HNSCC tumors and associated with increased EGFR and Her2. Genetic ablation of ESX decreased EGFR and Her2 levels and enhanced the antiproliferative effects of EGFR/Her2 tyrosine kinase inhibitors (TKI), lapatinib and afatinib. Biphenyl isoxazolidine, a novel small-molecule ESX inhibitor, reduced EGFR and Her2 levels and potentiated the antiproliferative efficacy of afatinib. Single-agent biphenyl isoxazolidine retarded the in vivo tumorigenicity of CAL27 cells. Importantly, the combination of biphenyl isoxazolidine and afatinib was significantly superior in vivo and resulted in a 100% response rate with a 94% reduction in tumor volume. Targeting EGFR/Her2 levels with an ESX inhibitor and EGFR/Her2 kinase activity with a TKI simultaneously is a highly active therapeutic approach to manage HNSCC. Our work provides evidence to support the further development of ESX inhibitors as an adjuvant to enhance the response rate of patients with HNSCC to current anti-EGFR/Her2 therapeutics.

Multiparameter analysis, including EMT markers, on negatively enriched blood samples from patients with squamous cell carcinoma of the head and neck.

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

YM155 reverses cisplatin resistance in head and neck cancer by decreasing cytoplasmic survivin levels.

Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus-negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC.

Confocal images of circulating tumor cells obtained using a methodology and technology that removes normal cells.

A completely negative enrichment technology was used to detect circulating tumor cells, CTCs, in the peripheral blood of head and neck cancer patients. Of 32 blood samples, 63% contained CTCs and the number of CTCs identified per mL of blood collected ranged from 0 to 214. The final purity ranged from 1 CTC in 9 total cells to 1 CTC in 20,000 total cells, the final purity being both a function of the number of CTCs and the performance of the specific enrichment. Consistent with previous reports, CTC were positively identified if: (1) they contained a nucleus based on DAPI stain, (2) stained positive for cytokeratins, and (3) have a high nuclei to cytoplasmic ratio. In addition, for a blood sample to be considered positive for CTCs, the enriched sample must be positive for epithelial growth factor receptor, EGFR, as measured by RT-PCR. While most of the blood samples were obtained during surgery, a number were taken prior to and during surgery. In all of the pre- and postsurgery paired samples, significant numbers of CTCs were detected. A number of these enriched samples were observed under confocal microscope in addition to the microscopic observations under traditional wide-field fluorescent microscope. As expected, the FITC stained cytokeratins appeared in the cytoplasm and the average size of these positively stained cells, on the cytospin, was in the range of 8-12 mum. Future studies will involve the investigation if cancer stem cell and mesenchymal markers are present on these CTCs and correlations of patient outcome to the number and type of CTC present.

Genome-wide hypomethylation in head and neck cancer is more pronounced in HPV-negative tumors and is associated with genomic instability.

Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells.

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 x 10(9) to 8.04 x 10(3) cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log(10)) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominantly epithelial or mesenchymal characteristics?).

Molecular signatures of metastasis in head and neck cancer.

BACKGROUND: Metastases are the primary cause of cancer treatment failure and death, yet metastatic mechanisms remain incompletely understood. METHODS: We studied the molecular basis of head and neck cancer metastasis by transcriptionally profiling 70 samples from 27 patients-matching normal adjacent tissue, primary tumor, and cervical lymph node metastases. RESULTS: We identified tumor-associated expression signatures common to both primary tumors and metastases. Use of matching metastases revealed an additional 46 dysregulated genes associated solely with head and neck cancer metastasis. However, despite being metastasis-specific in our sample set, these 46 genes are concordant with genes previously discovered in primary tumors that metastasized. CONCLUSIONS: Although our data and related studies show that most of the metastatic potential appears to be inherent to the primary tumor, they are also consistent with the notion that a limited number of additional clonal changes are necessary to yield the final metastatic cell(s), albeit in a variable temporal order.

Tumor suppressor activity of CCAAT/enhancer binding protein alpha is epigenetically down-regulated in head and neck squamous cell carcinoma.

Tumor suppressor CCAAT enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in cell cycle control and cellular differentiation. In a recent study, microarray expression profiling on head and neck squamous cell carcinoma (HNSCC) samples identified significant C/EBPalpha down-regulation, correlating with poor prognosis. However, the mechanisms of C/EBPalpha down-regulation remained elusive. C/EBPalpha has been previously found to provide an antiproliferative role in lung cancer, and our laboratory showed that its down-regulation involves epigenetic mechanisms. This prompted us to investigate the involvement of epigenetics in down-regulating C/EBPalpha in HNSCC. Here, we show that C/EBPalpha is down-regulated in HNSCC by loss of heterozygosity and DNA methylation, but not by gene mutation. We found a consistently methylated upstream regulatory region (-1,399 bp to -1,253 bp in relation to the transcription start site) in 68% of the HNSCC tumor samples, and DNA demethylation using 5-aza-2'-deoxycytidine treatment was able to significantly restore C/EBPalpha mRNA expression in the HNSCC cell lines we tested. In addition, C/EBPalpha overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In conclusion, we showed for the first time not only that C/EBPalpha has tumor suppressor activity in HNSCC, but also that it is down-regulated by DNA promoter methylation.

The effects of reactive species on the tumorigenic phenotype of human head and neck squamous cell carcinoma (HNSCC) cells.

Sustained inflammation up-regulates the reactive species (RS) generating enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). While clinical data show that levels of iNOS and COX-2 are increased in epithelium during the transformation of dysplasia to overt head and neck squamous cell carcinoma (HNSCC), the mechanisms by which their overexpression contributes to HNSCC development have not been completely delineated. This study assessed the effects of RS on parameters associated with the HNSCC tumorigenic phenotype inclusive of activation of NF-kappaB (in situ immunostaining and reporter assay) and production of proinflammatory and proangiogenic proteins (ELISA analyses). Our data, which show both reactive oxygen and nitrogen species activated NF-kappaB, and that all RS donors evaluated increased HNSCC cellular production of vascular endothelial growth factor, IL-8 and epidermal growth factor receptor proteins, imply inflammation associated RS promote HNSCC by their abilities to modulate intracellular signaling and affect gene expression.

p16 gene alterations in locally advanced squamous cell carcinoma of the head and neck.

We previously documented the presence of mutations/deletions in the tumor suppressor gene p16 in squamous cell carcinoma of the head and neck (SCCHN). However, the association of these p16 alterations with clinical outcome is unknown. In this study, RNA was isolated from 19 frozen SCCHN from 19 patients who were previously enrolled in the OSU intensification regimen 2. Quantitative real-time RT-PCR and direct sequencing analysis was then performed on the specimens to detect p16 gene alterations. Clinical outcome for each patient was updated and correlated with the p16 alterations found. Five tumor specimens were found to have no or very low expression of p16 when compared with normal tissue. The remaining 14 tumor samples demonstrated overexpression of p16 relative to the level of expression in normal tissue. Sequence analysis of the p16 RT-PCR product from these specimens allowed identification of mutational changes in the coding sequence of p16 in four of the SCCHN specimens. Subsequent analysis of clinical outcome associated with locoregional/distant failure demonstrated no correlation with either altered expression of p16 or mutational status of p16. Results from this study indicate that p16 alterations are frequently found in this cohort of SCCHN. However, p16 alterations alone do not appear to be associated with clinical outcome.

Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer.

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

Expression of the serine protease DESC1 correlates directly with normal keratinocyte differentiation and inversely with head and neck squamous cell carcinoma progression.

BACKGROUND: As part of ongoing studies aimed at identifying the molecular events involved in head and neck squamous cell carcinoma progression, we recently isolated a novel serine protease, DESC1. This study was conducted to further characterize DESC1. METHODS: Specimens of normal, dysplastic, and carcinomatous oropharyngeal mucosa (n = 31) were evaluated for DESC1 immunoreactivity using standard streptavidin-biotin immunoperoxidase techniques. Between-lesion stain intensity values were analyzed using multiple Wilcoxon tests. DESC1 expression was also evaluated in cultured human keratinocytes after induction of differentiation by calcium challenge, with subsequent real-time reverse transcriptase-polymerase chain reaction quantification. RESULTS: DESC1 immunoreactivity decreased as lesions approached a malignant phenotype. Post hoc testing comparing the different lesion types and DESC1 staining values showed significance between "normal" and "carcinoma" (p = .0017) groups. Induction of normal keratinocyte differentiation by calcium challenge was accompanied by an increase in DESC1 expression (p = .002). CONCLUSIONS: These results suggest downregulation of DESC1 during squamous cell carcinoma progression and upregulation during normal epithelial differentiation.

Oral graft-versus-host disease and programmed cell death: pathogenetic and clinical correlates.

Graft-versus-host disease (GVHD) is an untoward complication of bone marrow transplantation. It is characterized by an immune-mediated attack by donor immune cells against various host cells and tissues, a process which may be associated with significant morbidity in affected patients. Oral lesions are a common sequelae and can serve as a highly predictive index to the presence of systemic GVHD. The oral lesions of GVHD are clinically and histologically lichenoid in nature and can be a challenge in terms of management. Ulcerated and painful mucosal lesions may represent a significant impediment to normal eating habits and nutritional intake, necessitating appropriate diagnosis and treatment. Importantly, recent evidence has indicated that programmed cell death, or apoptosis, is the major constituent in the pathogenesis of GVHD. Apoptosis not only plays a major role in normal growth and ontogeny, but has been shown to contribute to a wide spectrum of both inflammatory and neoplastic disorders. Since knowledge of apoptotic molecular pathways is requisite for understanding GVHD, the purpose of this paper is to provide a fundamental overview of the predominant apoptotic mechanisms implicated in the pathogenesis of GVHD and to relate these findings to the oral complications of the disease. Finally, we will discuss management strategies for diagnosing and treating the oral lesions of GVHD. By explicating the molecular events in the apoptotic pathway, unique therapeutic and pharmacologic strategies for regulating apoptosis may be developed in the future, reducing the morbidity associated with conditions like GVHD.

Expression and biological effect of urodele fibroblast growth factor 1: relationship to limb regeneration.

Fibroblast growth factors (FGFs) have been previously implicated in urodele limb regeneration. Here, we examined expression of FGF-1 by blastema cells and neurons and investigated its involvement in wound epithelial formation and function and in the trophic effect of nerves. Neurons innervating the limb and blastema cells in vivo and in vitro expressed the FGF-1 gene. The peptide was present in blastemas in vivo. Wound epithelium thickened when recombinant newt FGF-1 was provided on heparin-coated beads, demonstrating that the FGF-1 was biologically active and that the wound epithelium is a possible target tissue of FGF. FGF-1 did not stimulate accessory limb formation. FGF-1 was as effective as 10% fetal bovine serum in maintaining proliferative activity of blastema cells in vitro but was unable to maintain growth of denervated, nerve-dependent stage blastemas when provided on beads or by injection. FGF-1 had a strong stimulating effect on blastema cell accumulation and proliferation of limbs inserted into the body cavity that were devoid of an apical epithelial cap (AEC). These results show that FGF-1 can signal wound epithelium cap formation and/or function and can stimulate mesenchyme accumulation/proliferation in the absence of the AEC but that FGF-1 is not directly involved in the neural effect on blastema growth.