Head And Neck Cancer Cell Research Articles

SHP2 Is Overexpressed and Inhibits pSTAT1-Mediated APM Component Expression, T-cell Attracting Chemokine Secretion, and CTL Recognition in Head and Neck Cancer Cells

Human leukocyte antigen (HLA) class I antigen processing machinery (APM) component downregulation permits escape of malignant cells from recognition by cytotoxic T lymphocytes (CTL) and correlates with poor prognosis in patients with head and neck cancer (HNC). Activated STAT1 (pSTAT1) is necessary for APM component expression in HNC cells. We investigated whether an overexpressed phosphatase was responsible for basal suppression of pSTAT1 and subsequent APM component-mediated immune escape in HNC cells. Immunohistochemical staining and reverse transcription PCR of paired HNC tumors was performed for the phosphatases src homology domain-containing phosphatase (SHP)-1 and SHP2. Depletion of phosphatase activity in HNC and STAT1(-/-) tumor cells was achieved by siRNA knockdown. HLA class I-restricted, tumor antigen-specific CTL were used in IFN-γ ELISPOT assays against HNC cells. Chemokine secretion was measured after SHP2 depletion in HNC cells. SHP2, but not SHP1, was significantly upregulated in HNC tissues. In HNC cells, SHP2 depletion significantly upregulated expression of pSTAT1 and HLA class I APM components. Overexpression of SHP2 in nonmalignant keratinocytes inhibited IFN-γ-mediated STAT1 phosphorylation, and SHP2 depletion in STAT1(-/-) tumor cells did not significantly induce IFN-γ-mediated APM component expression, verifying STAT1 dependence of SHP2 activity. SHP2 depletion induced recognition of HNC cells by HLA class I-restricted CTL and secretion of inflammatory, T-cell attracting chemokines, RANTES and IP10. These findings suggest for the first time an important role for SHP2 in APM-mediated escape of HNC cells from CTL recognition. Targeting SHP2 could enhance T-cell-based cancer immunotherapy.

Abstract A41: Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells.

Abstract Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ~20% of HNC patients are clinically responsive the EGFR-specific therapeutic monoclonal antibody (mAb) cetuximab. In addition to blocking EGFR signaling that leads to tumor cell survival and proliferation, increasing evidence suggests that innate and adaptive immunity likely plays a significant role in patient response to cetuximab. Natural killer (NK) cells recognize the Fc-region of EGFR-bound cetuximab via their activating Fcγ receptor IIIa (FcγRIIIa, also known as CD16) and mediate tumor cell lysis by antibody dependent cellular cytotoxicity (ADCC). NK cells are increasingly recognized as key mediators of crosstalk bridging the innate and adaptive arms of the immune response to human malignancies. We have previously shown that in addition to being direct effectors of ADCC against cetuximab-coated HNC cells, NK cells are instrumental in indirectly promoting an anti-tumor adaptive immune response via secretion of immunomodulatory cytokines such as IFNγ, which is known to enhance dendritic cell (DC) antigen processing and presentation to CD8+ T cells. Multiple studies have consistently demonstrated a relative decrease in the CD56dimCD16bright NK cell subset, traditionally considered to be primarily cytotoxic effectors, and an increase in the immunoregulatory CD56brightCD16dim NK cell subset in the peripheral blood of patients with various malignancies. However, given that the immunoregulatory CD56brightCD16dim NK cells are generally considered to be the more prolific cytokine producing subset, it was unclear how this redistribution of NK cell subsets might affect the dynamics of cytokine-mediated NK:DC crosstalk in the presence of mAb-coated tumor cells. Using primary NK cells and monocyte-derived DC isolated from PBMC of healthy donors, we therefore sought to better elucidate the patterns and kinetics of IFNγ secretion by the two NK cell subsets in response to cetuximab-coated HNC cells in the presence of DC. In this context, we found that while CD56brightCD16dim NK cells express more of the activating NKG2D receptor, rapid (by 4hrs) IFNγ secretion by CD56dimCD16bright NK cells is a CD16-dependent process. CD56brightCD16dim NK cells only produced IFNγ following stimulation for at least 12 hours with a combination of IL-12 and IL-15. IL-12/IL-15 stimulation also induced IFNγ secretion by CD56dimCD16bright NK cells, though to a lesser extent as compared to the CD56brightCD16dim subset. Taken together, these data contribute significantly to a mechanistic understanding of how NK cells function to bridge innate and adaptive anti-tumor responses in the context of mAb cancer therapies. Understanding this link is of broad relevance to the optimization of current therapeutic mAbs and will help devise novel ways to improve clinical responses. Citation Format: Christopher A. Lord, Raghvendra M. Srivastava, Robert L. Ferris. Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A41.

Regulation of epidermal growth factor receptor signaling and erlotinib sensitivity in head and neck cancer cells by miR-7.

Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3′-untranslated region (3′-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.

The p53-reactivating small-molecule RITA enhances cisplatin-induced cytotoxicity and apoptosis in head and neck cancer

We evaluated whether the restoration of p53 function by the p53-reactivating small molecule RITA (reactivation of p53 and induction of tumor cell apoptosis enhances cisplatin-induced cytotoxicity and apoptosis in head-and-neck cancer (HNC). RITA induced prominent accumulation and reactivation of p53 in a wild-type TP53-bearing HNC cell line. RITA showed maximal growth suppression in tumor cells showing MDM2-dependent p53 degradation. RITA promoted apoptosis in association with upregulation of p21, BAX, and cleaved caspase-3; notably, the apoptotic response was blocked by pifithrin-α, demonstrating its p53 dependence. With increasing concentrations, RITA strongly induced apoptosis rather than G2-phase arrest. In combination therapy, RITA enhanced cisplatin-induced growth inhibition and apoptosis of HNC cells invitro and in vivo. Our data suggest that the restoration of p53 tumor-suppressive function by RITA enhances the cytotoxicity and apoptosis of cisplatin, an action that may offer an attractive strategy for treating HNC.

Abstract 1588: Synergistic apoptosis by combination of natural compound EGCG and resveratrol in head and neck cancer: Potential role for AKT-dependent signaling

Abstract Natural dietary agents have drawn great attention for cancer prevention because of their wide safety margin and ready availability. Green tea is one such agent that is widely used for chemoprevention (Amin et al. 2009, Kim et al., 2010). Recent clinical trials suggest that green tea alone might not be sufficient for chemoprevention of head and neck cancer (HNC) (Tsao et al., 2009). Therefore, identification of compounds which exhibit synergistic effects with green tea is warranted. In the current study, we investigated combination of green tea component EGCG and resveratrol (a major constituent of red wines and grapes), and found that their combination strongly induced apoptosis as measured by annexin-V staining and supported by cleavage of PARP and caspase 3. The combination of resveratrol (15 and 20 μM, which alone induce 10-15% apoptosis) with EGCG (30-80 μM, which also induce <10% apoptosis) strongly increased apoptosis (to 60-80%) in multiple HNC cell lines. Data analysis for the combination using CalcuSyn software suggests that the combination of EGCG and resveratrol has synergistic apoptotic effects with combination index between 0.4-0.7. Interestingly, a premalignant oral leukoplakia cell line was sensitive to relatively lower doses of the combination (5 and 10 μM resveratrol, 10 and 15 μM EGCG) than fully transformed cancer cell lines, suggesting that the combination of EGCG and resveratrol might be a suitable combination for prevention of HNC. To further elucidate the mechanism of synergistic apoptosis, we found that combination of EGCG and resveratrol strongly inhibited AKT and ERK phosphorylation along with its several downstream targets including p-mTOR, p-S6 and p-4EBP1. Retroviral transduction of constitutively active AKT significantly inhibited apoptosis induced by the combination (p=.003). We also found that the combination of EGCG and resveratrol strongly inhibited p-AMPK, a kinase that regulates autophagy, a protective mechanism during energy stress. The fact that inhibition of autophagy triggers apoptosis (Xu Y et al., 2011) suggests that inhibition of p-AMPK might also play a role in EGCG- and resveratrol-induced apoptosis. Furthermore, strong inhibition of anti-apoptotic Mcl-1 and survivin by the combination was observed as a consequence. Taken together, our studies identify a novel combination of two natural dietary agents, EGCG and resveratrol, which induces synergistic anti-tumor effects in both premalignant and fully transformed HNC cells by targeting multiple signal transduction pathways. The signaling pathways modulated by the combination of EGCG and resveratrol are critical for development of HNC, thus our study provides an important rationale for future preclinical and clinical development. (Supported by U01CA101244, R01CA112643, and P50CA128613 to DMS) 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 1588. doi:1538-7445.AM2012-1588

Abstract 669: Disparities in head and neck cancer: A case for chemoprevention with vitamin D

Abstract Worldwide head and neck cancer (HNC) is the sixth most frequently diagnosed cancer in men and is associated with significant morbidity, mortality and economic loss. African Americans (AA) experience significantly more HNC compared to Caucasian Americans (CA) and they have markedly reduced 5-year survival rates, 40% compared to 61% among CA. AA bear a disproportionate burden of HNC for reasons that are incompletely understood, but likely due to the complex interplay of environmental, genetic and health care access issues. An inverse association for vitamin (vit) D status and HNC cancer has been reported and hypovitaminosis D in AA is well documented; yet, vit D status has not been assessed in HNC patients stratified by race. Thus, objective of this prospective pilot study is to access vit D status in newly diagnosed AA and CA HNC patients by measuring the circulating metabolite of vit D, vit-D 25-OH and by assessing dietary levels utilizing the Block vit D/Calcium screener. Circulating calcium, parathyroid, and 1,25-Dihydroxy vit D are also being measured. In addition, to improve our understanding of mechanisms associated with the potential cancer protective effects of vit D in the context of HNC two HNC cell lines were treated with 2 µM cholecalciferol for 6 hours and protein harvested for proteome profiling. To date, 18 HNC patients have been evaluated, 8 AA and 10 CA. Mean circulating vit D 25-OH levels were 27.3 (+ 9.9) and 20.0 (+ 5.9) ng/mL in CA and AA, respectively supporting that vit D levels are suppressed to a greater extent in AA compared to CA HNC patients, even in sunny Florida. Dietary vit D estimates were non-significantly higher in AA HNC patients compared to CA who had higher total vit D levels, supporting differences in metabolism or potentially supplement use. None of the AA patients had vit D 25-OH levels > 30 ng/mL, a level suggested for optimum regulation of PTH, calcium absorption and bone density; 30% of CA patients expressed levels above 30 ng/mL. Proteome profiling of CAL27 and SSC25 HNC cell lines following vit D treatment revealed inverse modulation of proteins linked to HNC. Proteins down-regulated by vit D treatment included nucleophosmin (NPM), lactoylglutathione, heat shock protein beta-1 (HSPB1) Peroxiredoxin-1 (PRDX1) and Ras-related protein Rap-2b. Recent reports have linked NPM with the regulation of the oncogene FOXM1, also a potential biomarker for early detection. Disease-free survival rate is improved in HNC patients with low NPM expression. Similarly, HSPB1 has been shown to be over expressed in HNC and silencing of HSPB1 has been shown to decrease metastatic behavior in HSNCC. PRDX1 displays upregulation in oral premalignancy and is an indicator for local recurrence. Histone H2A type was up-regulated by Vitamin D. Study results support that newly diagnosed AA HNC patients have depressed vit D levels compared to CA cases and that vit D treatment inversely targets select proteins dysregulated in HNC. 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 669. doi:1538-7445.AM2012-669

Abstract 4919: Pathway profiling of head and neck cancer cell lines

Abstract Introduction: Cell lines retain key genetic aberrations of the originating tumors, which frequently correlate with sensitivity to specific inhibitors. We performed genetic and pathway based profiling of 40 head and neck cancer (HNC) cell lines using three publicly available methods for pathway profiling based on expression and copy number data. Methods: 40 HNC cell lines were analyzed for expression using expression arrays (Agilent), and copy number using array comparative hybridization (aCGH) and/or Nanostring methodologies. Integrative genomic analysis was performed to determine pathway activity using pathway analysis techniques previously described by Gatza et al. (BFRM), Pe'er et al (CONEXIC), Vaske et al. (PARADIGM). Results: Frequent pathway activation was identified in >10 well established signalling pathways for HNC including TP53, PI3K-AKT, TP63/SOX2, Cyclin D1, MYC etc, as well as several - for HNC previously unknown - signalling pathways. Cell lines could be associated with expression-based biologic subtypes in HNC tissues suggesting that specific cell lines should be used preferentially for modelling and therapeutic evaluation for HNC subtypes (including HPV(+) cell lines with characteristics of HPV(+) tumors). The three pathway analysis platforms identified a large number of pathways with some overlap in particular between BFRM and CONEXIC, whereas PARADIGM profiled a much wider set of pathways. Both CONEXIC and PARADIGM employed copy number data, whereas BFRM relied solely on expression data. Conclusion: Pathway alterations characteristic of head and neck cancers were identified in virtually all HNC cell lines. Pathway based classification of cell lines was achieved and correlated with pathway activity in HNC tumor tissues. Three pathway-profiling approaches provided a large set of pathway activity with some significant overlap that may have translational relevance. Validation studies are currently on-going. 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 4919. doi:1538-7445.AM2012-4919

Abstract LB-431: Augmented induction of tumor antigen-specific CTL by cetuximab in head and neck cancer patients does not correlate with Fcγ receptor IIIa genotype, but relies on NK:DC cross talk mediated by interferon-γ and NKG2D

Abstract Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ∼20% of HNC patients are clinically responsive to treatment with the EGFR-specific monoclonal antibody (mAb) cetuximab. To date, no reliable biomarker of clinical efficacy has been identified to predict clinical response to cetuximab therapy in HNC. Modest, yet statistically significant correlations of Fcγ receptor (FcγR) IIIa polymorphisms with clinical outcome have been noted in B cell lymphoma, breast, and colorectal cancer patients treated with rituximab, trastuzumab, and cetuximab, respectively. Therefore, we have investigated whether a known FcγRIIIa polymorphism in natural killer (NK) cells at amino acid position 158 (valine [V] vs. phenylalanine [F]) correlates with the anti-tumor activity of cetuximab in immunodeficient mice engrafted with human HNC tumors as well as with the induction of EGFR-specific cytotoxic T lymphocytes (CTL) and clinical course in HNC patients treated with cetuximab. When injected into immunodeficient mice, human NK cells expressing the V allele at this position demonstrated significantly greater ability to control the growth of xenografted HNC tumors. However, we did not find a significant correlation between FcγRIIIa genotype and disease free survival (p=0.683) in a cohort of 107 consecutive HNC patients treated with regimens incorporating cetuximab. Furthermore, we demonstrate for the first time that in the presence of cetuximab-opsonized HNC cells, NK cells trigger maturation of dendritic cells (DC), leading to augmented TA-specific cross-priming of EGFR and MAGE-specific CTL in vitro, and in vivo in cetuximab-treated HNC patients. The significantly higher frequencies of EGFR-specific CTL we found in HLA-A2+ PBMC from cetuximab-treated HNC patients relative to cetuximab-naive HNC patients (p<0.001) also did not correlate with FcγRIIIa genotype, but their induction was dependent on NK:DC cross talk mediated by interferon-γ and NKG2D. Taken together, our data indicate that cetuximab treatment augments the priming of both EGFR and non-EGFR TA-specific cellular immunity in vivo, thereby providing a novel immune mechanism for enhanced anti-tumor activity of TA-specific mAb relevant to clinical response, as well as a potential biomarker for monitoring this response in mAb-treated cancer patients. 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 LB-431. doi:1538-7445.AM2012-LB-431

Abstract 5136: Identification of sensitizers of platinum-based therapy in head and neck cancer using a functional genomics approach

Abstract Objective The prevalence and mortality for head and neck cancer (HNC) has not improved significantly in the last several decades. It is the sixth most common cancer with an estimated 600,000 new cases diagnosed worldwide every year and has ∼50% 5-year survival rate. Of these, 47000 new cases are diagnosed in the US annually. The most common risk factors for HNC are tobacco and alcohol use but a growing subset is also attributed to human papillomavirus (HPV) infection. Late stage HNCs are treated with a combination of surgery, radiation, and chemotherapy (often platinum-based), but the cancers treated with platinum-based chemotherapy often develop drug resistance. Identification of target genes using high-throughput RNAi (HT-RNAi) gene-silencing technology has become an increasingly popular method in the field of cancer research. The objective of our study is to use this functional genomics based approach to identify genes that, when silenced, increase the sensitivity of the platinum drugs. This would possibly help us in identifying potentiators of platinum-based drugs to more effectively treat HNC patients. Methods We performed drug dose response (DDR) experiments on nine HNC cell lines; including two are HPV-positive lines, to identify cisplatin-resistant and -sensitive cancer cell lines. To identify sensitizers of cisplatin response, we used a siRNA library targeting 572 kinases. Each kinase is represented by two different siRNA sequences in the library. Based on the data mined from published literature on head and neck cancer as well as genes thought to confer a functional relationship between HPV infection and HNC, a more focused siRNA library was also designed and included in the screening. Cell lines were reverse transfected with the siRNAs in a 384 well format, dosed with cisplatin after 24 hours and the plates were read 72 hours later. Results We calculated the inhibitory concentrations using DDR experiments for all the nine HNC cell lines and identified cisplatin-resistant (UMSCC47 and SCC9) and -sensitive (Cal27, UMSCC14C, and SCC090) HNC cell lines. HT-RNAi experiments have been performed on the cisplatin-resistant cell lines, one of which is HPV-positive. After data analysis, confirmation of silencing and validation of drug modulation will be done on a subset of candidate genes. Conclusions HNC patients with surgically non-resectable tumors could have improved treatment with chemotherapeutic drugs; however, acquired drug resistance often inhibits a successful treatment. Identification of sensitizing targets may provide hope for a better outcome, but may also allow for use of smaller doses of more toxic drugs, such as cisplatin. In addition, this study may also identify alternate genes that can be targeted in the subset of HPV-positive HNC. 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 5136. doi:1538-7445.AM2012-5136

Abstract 4051: A mouse model of head and neck cancer with infrared-based molecular imaging

Abstract Introduction Head and neck cancer (HNC) is notoriously difficult to treat and the five-year survival rate has improved very little over the past thirty years. Head and neck squamous cell carcinomas (HNSCC) mouse models are invaluable tools for furthering mechanistic HNC research. Molecular imaging (MI) is a promising new modality that may improve current HNC mouse models. Visible florescent proteins (VFPs) have been widely used with animal models for tumor detection using MI. However, the low emission wavelength of VFP results in its overlapping with the auto-fluorescence of many tissues and causes insufficient penetration into deeper tissues, preventing the application of VFP to track local and metastatic tumor progressions. The recent development of infrared-fluorescent protein (IFP) with Biliverdin as a chromophore has greatly advanced in vivo molecular imaging of deep tissues in animals. Hypothesis The ultimate goal of this study is to develop a novel orthotopic mouse model of HNC with regional spreading and distant metastasis tumors that closely mimics human HNC and can be accurately detected by a noninvasive IFP-based molecular imaging (MI) system. Methods A stable human HNC cell line expressing IFP was established. This stable cell line was used for generating a mouse model with HNC. The tumor formation and tumor progression with this model were accurately monitored by a MI system. Results The IFP signal was clearly detected in our mouse model of HNC. Scanned imaging demonstrated a progression pattern of tumor growth in this model. The tumor can be detected earlier with molecular imaging than by conventional external caliper measurement. Unlike surgical measurement, the tumor can be quantified without disturbing the tumor environment. More important, our data suggest that local-regional metastasis tumors can be detected with this model. Conclusion This novel animal model represents an orthotopic human HNC model. The IFP signals penetrate tissues well and can be detected in the deep tissues with this model. Thus, our model has the potential to be used for monitoring tumor spreading and metastasis of advanced stage HNC. 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 4051. doi:1538-7445.AM2012-4051

Abstract 1768: Enhanced cytotoxicity and apoptosis of cisplatin by p53-reactivating small molecule RITA in head and neck cancer

Abstract Overexpression of MDM2 inactivates the tumor suppressive function of p53 and a small-molecule reactivation of p53 and induction of tumor cell apoptosis (RITA) disrupts the MDM2-p53 interaction. We evaluated whether the restoration of p53 function by RITA enhances cytotoxicity and apoptosis of cisplatin in head-and-neck cancer (HNC). RITA was tested in four human HNC cell lines differing in TP53 status. In vitro and in vivo growth suppression, cell cycle arrest, apoptosis after RITA treatment individually or in combination with cisplatin were assessed. RITA induced prominent accumulation and reactivation of p53 in a wild-type TP53-bearing tumor cell line. RITA showed maximal growth suppression in tumor cells showing MDM2-dependent p53 degradation. RITA upregulated p21, BAX, and cleavage of caspase-3 but the p53-dependent apoptosis was blocked by pifithrin-μ. Strong induction of apoptosis rather than G2-phase arrest was observed along with increased dose of RITA. In combination therapy, RITA enhanced the growth inhibition and apoptosis of HNC by cisplatin in vitro and in vivo. Our data suggest that the restoration of p53 tumor suppressive function by RITA enhances cytotoxicity and apoptosis of cisplatin, which may offer an attractive strategy for treating HNC. 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 1768. doi:1538-7445.AM2012-1768

Epigenetic and genetic alterations-based molecular classification of head and neck cancer

The long-term survival rates for patients diagnosed with advanced head and neck cancer (HNC) remain poor. Many perplexing factors, including etiology and comorbidity, lead to different molecular malfunctions of HNC cells and determine the prognosis of the disease. Traditional diagnostic methods are limited in that they fail to provide an effective classification diagnosis, such as a more precise prediction of prognosis and decisions for personalized treatment regimens. Recently, molecular biology techniques, especially epigenetic and genetic techniques, have been developed that have enabled us to gain a greater insight into the molecular pathways underlying the cancers. Translating the research into a format that will facilitate effective molecular classification, support personalized treatment and determine prognosis remains a challenge. In this review, the authors provide an overview of cancer epigenetic and genetic alterations, tissue banks, and several promising biomarkers or candidates that may ultimately prove to be beneficial in a clinical setting for patients with HNC.

S-Nitrosylation of mitogen activated protein kinase phosphatase-1 suppresses radiation-induced apoptosis

Radiotherapy is a key modality for head and neck cancer (HNC) treatment. Mitogen activated protein kinase phosphatase-1 (MKP-1) protein levels are elevated in various tumors and are negatively correlated with efficacy of chemo- or radio-therapy. However, the mechanisms underlying the moderate radiosensitivity of HNC and the increased MKP-1 protein levels are still dismal. Here we show that S-nitrosylation of MKP-1 on Cysteine 258 enhances MKP-1 protein stability, phosphatase activity, and MKP-1-mediated anti-apoptotic effect on HNC radiotherapy. Co-culturing MKP-1 transfected HNC cell lines with activated macrophages for mimicking the microenvironment of the irradiated cancer cells further confirms that S-nitrosylation-mediated increase of MKP-1 activity correlates with decrease of HNC radiosensitivity. Therefore, S-nitrosylation of MKP-1 presents a novel mechanism underlying the enhanced MKP-1 expression levels and MKP-1-mediated radio-resistance in head and neck cancer.

Targeting GPCR-Mediated p70S6K Activity May Improve Head and Neck Cancer Response to Cetuximab

Epidermal growth factor receptor (EGFR) overexpression is correlated with decreased survival in head and neck cancer (HNC) where the addition of EGFR inhibition to standard chemoradiation approaches has improved treatment responses. However, the basis for the limited efficacy of EGFR inhibitors in HNC is incompletely understood. G-protein-coupled receptors (GPCR) have been shown to be overexpressed in HNC where GPCR activation induces HNC growth via both EGFR-dependent and -independent pathways. We hypothesized that targeting GPCR-induced EGFR-independent signaling would improve the efficacy of EGFR inhibition. Using a high-throughput phosphoproteome array, we identified proteins that were phosphorylated in HNC cells where EGFR expression was downmodulated by RNA interference (RNAi) in the presence or absence of a GPCR ligand. We confirmed the findings from the array by Western blotting followed by in vitro and in vivo phenotypic assays. p70S6K phosphorylation was elevated approximately sixfold in EGFR siRNA-transfected cells treated with a GPCR ligand. In addition to RNAi-mediated EGFR downmodulation, GPCR-mediated phosphorylation of p70S6K was modestly increased by EGFR inhibitor cetuximab approved by the Food and Drug Administration. Biopsies from cetuximab-treated patients also displayed increased phospho-p70S6K staining compared with pretreatment biopsies. HNC cells were growth inhibited by both genetic and pharmacologic p70S6K targeting strategies. Furthermore, p70S6K targeting in combination with cetuximab resulted in enhanced antitumor effects in both in vitro and in vivo HNC models. These results indicate that increased phosphorylation of p70S6K in cetuximab-treated patients may be due to increased GPCR signaling. Therefore, the addition of p70S6K targeting strategies may improve treatment responses to EGFR inhibition.

Molecular Chaperones as a Common Set of Proteins That Regulate the Invasion Phenotype of Head and Neck Cancer

The goal of this study was to establish a common set of molecules that regulate cell invasion in head and neck cancer (HNC). Five invasive sublines derived from HNC cell lines were established using the Matrigel selection method. Proteomic technology, MetaCore algorithm, and reverse transcriptase-PCR methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional analyses and clinical association studies were applied to examine the significance of the molecules. Fifty-two proteins were identified in more than two of the four independent proteomic experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differentially expressed in five sublines, Hsp90α, Hsp90β, Hsp90-B1/Gp96, Hsp70-A5/Grp78, and HYOU1, that upregulate, whereas Hsp60 and glucosidase-α neutral AB (GANAB) downregulate. Four molecules were further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78 considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly associated with advanced cancer (P < 0.001 to P = 0.047, respectively, for the four molecules) and poor survival (P < 0.001 to P = 0.025, respectively, for the four molecules). Our study defined molecular chaperones as a common set of proteins that regulate the invasion phenotype of HNC. Loss of the tumor suppression function of Hsp60 or GANAB and acquisition of the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as prognostic markers and targets for cancer drug development.

Abstract 2358: Molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer

Abstract Purpose: The goal of this study was to establish a common set of molecules that regulate cell invasion in head and neck cancer (HNC). Experimental design: Five invasive sublines derived from HNC cell lines were established using the Matrigel selection method. Proteomic technology, MetaCore™ algorithm, and RT-PCR methods were used to search for molecules that contribute to the invasion phenotype. Cellular functional analyses and clinical association studies were applied to examine the significance of the molecules. Results: Fifty-two proteins were identified in more than two of the four independent proteomic experiments, including 10 (19%) molecular chaperones. Seven chaperones were confirmed to be differentially expressed in five sublines: Hsp90α, Hsp90β, Hsp90-B1/Gp96, Hsp70-A5/Grp78, HYOU1 up-regulation, while Hsp60 and GANAB down-regulation. Four molecules with the highest differential expressions (&amp;gt; 4-fold) were further investigated. In all cell lines, knockdown of Hsp60 or GANAB and silencing of Gp96 or Grp78 considerably enhanced or reduced cell migration and invasion, respectively. Clinical association studies consistently revealed that low levels of Hsp60 or GANAB and high levels of Gp96 or Grp78 are significantly associated with advanced cancer (P&amp;lt;0.001 to P = 0.047, respectively, for the four molecules) and poor survival (P&amp;lt;0.001 to P=0.025, respectively, for the four molecules). Conclusion: In conclusion, our study defined molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer. Loss of the tumor suppression function of Hsp60 or GANAB and acquisition of the oncogenic function of Gp96 or Grp78 contribute to aggressive cancers. These molecules may serve as prognostic markers and targets for cancer drug development. 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 2358. doi:10.1158/1538-7445.AM2011-2358

Abstract 3967: Identification of metadherin as a novel target of miR-375 in head and neck cancer

Abstract Purpose: The objective of this study is to investigate the significance of miR-375 and its target metadherin in head and neck cancer (HNC) patients. Methods and Results: Expression of miR-375 was measured by quantitative RT-PCR and observed to be significantly under expressed in both head and neck squamous cell (HNSCC) and nasopharyngeal carcinoma (NPC) samples by factors of 5.0 and 4.4, respectively. One mechanism of miR-375 silencing appears to be promoter methylation. Using a combination of experimentally-observed transcript changes after miR-375 re-expression in HNC cell lines, with in silico prediction algorithms, metadherin was identified as a novel target of miR-375. Luciferase reporter assays confirmed an inhibitory interaction between miR-375 and the 3’UTR of metadherin. Functional characterizations of miR-375 and metadherin were then conducted using FaDu (hypopharyngeal squamous cell) and C666-1 (nasopharyngeal) cancer cell lines. In vitro re-expression of miR-375 as well as knock-down of metadherin using siRNA markedly decreased cell viability and clonogenic survival (30% – 45%), cell migration (50% – 80%), and in vivo tumour forming ability in both cancer models. In vitro cell invasion was also significantly decreased by miR-375 over-expression and metadherin knock-down in FaDu cells (&amp;gt;90%). Western blot analyses demonstrated that metadherin knock-down resulted in reduced phosphorylation of PKB, indicating that the PI(3)K pathway may be involved in mediating these effects. Using qRT-PCR on 100 NPC and 22 HNSCC archival samples, there was an inverse relationship between metadherin over-expression with miR-375 under-expression, corroborating the relevance of this axis in primary human HNC tissues. Furthermore, NPC patients whose tumours expressed high levels of metadherin (defined as &amp;gt; median) experienced significantly lower overall and disease-free survival (5-year OS of 82%, DFS of 73%) compared to patients with low (&amp;lt; median) expression levels (5-year OS of 66%, DFS of 56%). Specifically, this reduced survival was most significantly related to distant metastases, wherein high metadherin expression was significantly associated with distant relapse (5-year distant relapse rates: 26% vs. 5% for high vs. low expressors, respectively). Conclusions: We report that the miR-375:metadherin control mechanism may represent a novel oncogenic pathway driving human HNC progression, potentially mediated in part by the PI(3)K cascade. Most importantly, metadherin over-expression was a significant predictor of distant metastases, a major clinical challenge in HNC given the improvements in loco-regional control as a consequence of technical advances in radiation therapy delivery. Hence, targeting this pathway could be a potentially novel therapeutic strategy to improve outcome for HNC patients. 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 3967. doi:10.1158/1538-7445.AM2011-3967

Tumor‐derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor-derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)-dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C-C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor-derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor-derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

Hyaluronan-grafted particle clusters loaded with Mitomycin C as selective nanovectors for primary head and neck cancers

CD44, a well-documented cell surface receptor, is involved in cell proliferation, migration, signaling, adhesion, differentiation and angiogenesis, which are important properties for normal and cancerous cell function. We recently developed particle clusters coated with hyaluronan (termed gagomers; GAG), and showed that they can deliver the insoluble drug paclitaxel directly into CD44-over-expressing tumors in a mouse tumor model. Here, we tested primary head and neck cancers (HNC) and normal cells taken from the same patient, and found that although CD44 expression in both types of cells was high, GAGs bind only to the cancerous cells in a selective manner. We next formulated the anti cancer agent mitomycin C (MMC) in the GAGs. MMC-based chemoradiation is a potential treatment for HNC, however, due to patient’s toxicity, MMC is not part of the standard treatment of HNC. MMC encapsulation efficiency was about 70% with a half-life drug efflux of 1.2 ± 0.3 days. The Ex vivo study of the targeted MMC-GAG showed significant increase in the therapeutic effect on HNC cells (compared to free MMC), while it had no effect on normal cells taken from the same patient. These results demonstrate the specificity of the nanovectors towards head and neck cancers, which might be applicable as future therapy to many CD44-expressing tumors.

Abstract 4026: Suppression of carcinogenic phenotypes by DSG3 silencing occurs via a plakoglobin-mediated signaling pathway

Abstract DSG3 has been reported to act as an oncogene in head and neck cancers (HNCs), but the underlying mechanism of its oncogenic activity is unclear. In this study, we aimed to validate the proposed oncogenic function of DSG3 and investigate its downstream regulatory mechanisms. When we stably transfected HNC cells with a short hairpin DSG3-RNAi (shDSG3) to suppress DSG3 expression, reductions in cell growth (70%) and colony formation (&amp;gt;90%) were observed, in addition to cell cycle arrest at the G0/G1 phase. Both cell migration and invasion abilities were also decreased, to 10% and 16%, respectively, after one day. Because DSG3 is associated with plakoglobin in desmosomes, we examined whether or not the phenotypic alterations that occur following DSG3 knockdown were mediated through the plakoglobin signaling pathway. Immunoprecipitation and immunofluorescent staining analyses revealed that DSG3 knockdown disrupted the interaction of DSG3 with plakoglobin and induced the translocation of plakoglobin from the cytoplasm to the nucleus. Using a luciferase reporter assay and western blot analysis, we found that DSG3 knockdown significantly reduced TCF/LEF transcriptional activity and subsequently suppressed the expression of the downstream target genes of the TCF/LEF transcription factors, including c-myc, cyclin D1, and MMP7, which are involved in the regulation of cell proliferation and invasion. An in vivo xenograft study showed that knockdown DSG3 expression significantly inhibited tumor growth for 2 months in BALB/C nude mice and also reduces c-myc, cyclin D1 and MMP7 expression. In conclusion, we found that DSG3 has an oncogenic function in HNC. The suppression of DSG3 expression can interfere with the plakoglobin-mediated signaling pathway, subsequently leading to a reduction of pathogenicity in HNC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4026.