Squamous Cell Carcinoma Of Head And Neck Cells Research Articles

NOTCH1 signaling promotes protein stability of HER3 through the AKT pathway in squamous cell carcinoma of head and neck

Epidermal growth factor receptor (EGFR) remains the sole druggable molecular target other than the PD1/PD-L1 pathway with meaningful clinical benefit in squamous cell carcinoma of head and neck (SCCHN). Human epidermal growth factor receptor 3 (HER3) confers the resistance to EGFR-targeted treatment in SCCHN. Thus, it is essential to determine the distribution and regulatory mechanisms of HER3 in SCCHN. We explored the prevalence of HER3 expression and its distribution within SCCHN by immunohistochemical staining and clinicopathological correlations were analyzed. The regulatory mechanism of HER3 expression was then dissected in vitro, using RT-PCR, Western blotting, and immunoprecipitation in a set of SCCHN cell lines. Subsequent in vivo validation in the murine model was also performed. We found that concomitant high expression of HER3 and its ligand NRG1 in SCCHN is associated with the increased presence of regional lymphatic metastasis and the majority of HER3 is located on the differentiated tumor cells. Further investigation revealed that HER3 is under positive control of NOTCH1 through transcriptional activation and inhibition of protein degradation through the polyubiquitination machinery via AKT pathway and USP8 deubiquitinating enzyme. In addition, loss of function of NOTCH1 suppresses HER3 expression through increased phosphorylation of serine 473 of AKT in SCCHN cells, and promotes the aggressiveness of the tumor cells. These data indicated that the level of HER3 is regulated by NOTCH1 in SCCHN both transcriptionally and post-translationally, and NOTCH1 is in a higher hierarchy in the regulatory system of the AKT pathway. Since NOTCH1 is inactivated in approximately 10% of SCCHN cases and this aberration strongly impacts the AKT pathway and diminishes HER3, exclusion of patients with NOTCH1-inactivated SCCHN may be beneficial for future clinical trials of HER3-targeting antibodies.

Tumor-associated macrophages derived CCL18 promotes metastasis in squamous cell carcinoma of the head and neck

BackgroundAlternatively activated macrophages in tumor microenvironment is defined as M2 tumor-associated macrophages (M2 TAMs) that promote cancer progression. However, communicative mechanisms between M2 TAMs and cancer cells in squamous cell carcinoma of head and neck (SCCHN) remain largely unknown.MethodsQuantitative real-time PCR, western blotting, enzyme-linked immunosorbent assay and flow cytometry were applied to quantify mRNA and protein expression of genes related to M2 TAMs, epithelial–mesenchymal transition (EMT) and stemness. Wounding-healing and Transwell invasion assays were performed to detect the invasion and migration. Sphere formation assay was used to detect the stemness of SCCHN cells. RNA-sequencing and following bioinformatics analysis were used to determine the alterations of transcriptome.ResultsTHP-1 monocytes were successfully polarized into M2-like TAMs, which was manifested by increased mRNA and protein expression of CCL18, IL-10 and CD206. Conditioned medium from M2-like TAMs promoted the migration and invasion of SCCHN cells, which was accompanied by the occurrence of EMT and enhanced stemness. Importantly, CCL18 neutralizing antibody partially abrogated these effects that caused by conditional medium from M2-like TAMs. In addition, recombinant human CCL18 (rhCCL18) correspondingly promoted the malignant biological behaviors of SCCHN in vitro. Finally, RNA-sequencing analysis identified 331 up-regulated and 363 down-regulated genes stimulated by rhCCL18, which were statistically enriched in 10 cancer associated signaling pathways.ConclusionThese findings indicate that CCL18 derived from M2-like TAMs promotes metastasis via inducing EMT and cancer stemness in SCCHN in vitro.

Abstract 680: FLLL12 is a small molecule JAK2 inhibitor that inhibits JAK-STAT3 pathway in head and neck cancer

Abstract Purpose: We have recently reported that FLLL12, a synthetic curcumin analog, induces apoptosis of lung cancer cells through a death receptor (DR) 5-dependent pathway and in squamous cell carcinoma of head and neck (SCCHN) by modifying multiple Bcl-2 proteins. However, the direct molecular target (receptor) of FLLL12 is unknown. The purpose of the current study is to identify the receptor of FLLL12. Methods: Computer-aided molecular docking was used to predict molecular interaction. Isothermal titration calorimetry and thermal shift assays were used to confirm molecular interactions in a cell free system. SCCHN cell lines were used to study the effect of this interaction on JAK-STAT3 pathway. Protein expression levels were measured by Western blotting. Results: Computer-aided molecular docking modeling of FLLL12 and JAK2 (PDB code: 5CF5) predicted that FLLL12 fit well into the JAK2 catalytic pocket. Although curcumin also fit into the JAK2 catalytic pocket, the binding sites were different for FLLL12 and curcumin. Docking score for FLLL12, natural ligand and curcumin were -7.2, -12.1 and -6.9, respectively. FLLL12 also docked into the STAT3 catalytic pocket (PDB code: 5AX3). However, the docking score was worse than those for natural ligand and curcumin (-5.1, -7.3 and -7.4 respectively). We confirmed that FLLL12 interacted with JAK2 in a cell free system using recombinant JAK2. Isothermal titration calorimetry assay revealed one binding site for both FLLL12 and the FDA-approved JAK inhibitor ruxolitinib. The Ka values were 3.84E6±1.03E5 and 2.31E7±6.78E6 M-1, respectively. FLLL12 and JAK2 interactions were further confirmed by thermal shift assay. Kd values were 1.12±0.14 μM and 2.24 ±0.18 μM for ruxolitinib and FLLL12, respectively. Two hour pretreatment with FLLL12 also inhibited IL-6 induced phosphorylation of JAK2 and STAT3. Finally, FLLL12 strongly inhibited constitutively active STAT3 phosphorylation in SCCHN cell lines, a downstream effector of JAK. Conclusions: Our results strongly suggest that FLLL12 is a novel small molecule inhibitor for JAK2 and FLLL12-JAK2 interaction inhibits IL-6-induced as well as constitutively active STAT3 phosphorylation, thus has great promise for cancer prevention and therapy. Grant Support: Start-up fund from Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University and from Department of Pharmaceutical Sciences and Research, Marshall University. Citation Format: ARM Ruhul Amin, ASM Anisuzzaman, Abu B. Siddique, James R. Fuchs. FLLL12 is a small molecule JAK2 inhibitor that inhibits JAK-STAT3 pathway in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 680.

Abstract 876: Effective reduction of PD-L1 expression by simultaneous blockade of EGFR and HER3 (ErbB3) in head and neck cancer

Abstract Background: We previously reported that simultaneous blockade of EGFR and HER3 by antibodies cetuximab and MM-121, respectively, could more potently reduce tumor growth in both squamous cell carcinoma of head and neck (SCCHN) cell lines and patient derived xenograft (PDX) models (Jiang et al, MCT, 13:1826-36, 2014; Wang et al., CCR, 23:677-86, 2016). Recently, we tested a new anti-HER3 antibody CDX-3379 (provided by Celldex Therapeutics, Inc) in combination with cetuximab for treatment of SCCHN. The effects of this combination on expression of the immune check point regulator PD-L1 and regulatory pathways underlying these effects were examined. In fact, a Phase II clinical trial combining CDX-3379 with cetuximab in checkpoint and cetuximab refractory patients has already been initiated. Method: Antitumor effects of cetuximab and CDX-3379 combination were investigated in SCCHN PDX animal models. Both HPV positive and negative SCCHN cell lines, UM-SCC47 (HPV+), 93Vu147T (HPV+), FaDu (HPV-), and JHU-022 (HPV-), were used for western blot analysis to examine EGFR and HER3 downstream pathways and PD-L1 expression in the presence and absence of interferon γ (IFNγ) after treatment with cetuximab, CDX-3379, or both agents in combination. Results: The PDX study indicated that, similar to MM-121, combining CDX-3379 with cetuximab inhibited tumor growth more potently than cetuximab alone or the untreated control (control vs. combination: p < 0.0001; cetuximab vs. combination: p < 0.0001). Mechanistic in vitro studies demonstrated that in addition to co-blocking ERK and AKT pathways, and regardless of HPV status, the cetuximab and CDX-3379 combination reduced PD-L1 expression more effectively than any of the single agents in the presence or absence of IFNγ, which has been previously reported to be secreted by natural killer (NK) cells through cetuximab-mediated antibody-dependent cell mediated cytotoxicity, leading to induction of PD-L1 expression in vivo. Using an AKT inhibitor, we confirmed that the AKT pathway was one of the major regulatory pathways involved in regulation of PD-L1. Considering that HER3 exerts its inhibitory effect mainly through regulation of the AKT pathway, reducing AKT activity by addition of CDX-3379 to cetuximab contributed significantly to the reduction of PD-L1 expression. Conclusion: In addition to its anti-tumor effect, the combination of cetuximab and CDX-3379 significantly reduced PD-L1 expression. The role of this combination in the regulation of tumor immunology and the tumor microenvironment in SCCHN deserves further investigation. Citation Format: Zhuo Georgia Chen, Dongsheng Wang, Christopher C. Griffith, Sreenivas Nannapaneni, Conor Steuer, Mihir R. Patel, Mark W. El-Deiry, Xu Wang, Yuzi Zhang, Zhengjia Chen, Diego Alvarado, Dong M. Shin, Nabil F. Saba. Effective reduction of PD-L1 expression by simultaneous blockade of EGFR and HER3 (ErbB3) in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 876.

MiR-1275 promotes cell migration, invasion and proliferation in squamous cell carcinoma of head and neck via up-regulating IGF-1R and CCR7

PurposemiRNAs can play vital role in migration, invasion and proliferation in Squamous cell carcinoma of head and neck (SCCHN). In our study, we attempted to validate the expression and function of miR-1275 in SCCHN, and we also identified the mechanism by which miR-1275 affects migration, invasion and proliferation of SCCHN. MethodsReal-time polymerase chain reaction (RT-PCR) was employed to evaluate the expression of miR-1275 in both SCCHN tissues and cell lines. The role of miR-1275 in SCCHN cells was verified by cell function experiments upon transfection with miR-1275 mimics and inhibitor. Western blot analysis was employed to test the target gene expression of miR-1275. Survival analysis was made with the information of SCCHN patients expressed miR-1275 from The Cancer Genome Atlas (TCGA) database. ResultsmiR-1275 expression was up-regulated in SCCHN tissues and advanced metastatic SCCHN cells. Increasing miR-1275 expression in SCCHN could promote cell migration, invasion and proliferation probably by upregulating Insulin-like growth factor 1 receptor (IGF-1R) and C-C chemokine receptor type 7(CCR7) protein levels, whereas inhibition of miR-1275 could lead the opposite effects, although others have already demonstrated that IGF-1R is a direct target of miR-1275. Survival analysis suggested that patients with lower miR-1275 expression may have a better outcome. ConclusionsHerein we report for the first time that miR-1275 could act as a tumor-promoter in SCCHN possibly by regulating its target gene via novel miRNA mechanisms. MiR-1275 plays an important role in promoting SCCHN progression. The miR-1275 may be a potential therapeutic target for SCCHN treatment in the future.

TRAF6 regulates tumour metastasis through EMT and CSC phenotypes in head and neck squamous cell carcinoma.

Epithelial–mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor‐associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N‐cadherin was down‐regulated and that of E‐cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF‐β1) and CAL27 similar to mesenchymal cells formed after TGF‐β1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor‐dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1‐positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.

Inhibition of CK2 by CX4945 to induce autophagy-mediated cell death through dephosphorylating acetyl-CoA carboxylase in squamous cell carcinoma of head and neck cancer.

e17503 Background: Accumulating evidence has shown that overexpression of casein kinase 2 (CK2) in patients with squamous cell carcinoma of head and neck (SCCHN) correlates with worsened survival. Strong expression of phosphorylated acetyl CoA carboxylase (pACC) was found to be an independent prognostic marker for patients with node-positive SCCHN. Computational analysis showed that ACC was a potential substrate for CK2, and CK2 inhibition can suppress ACC phosphorylation in non-cancer cells. CX-4945, also known as silmitasertib, is an orally administered, highly specific, ATP-competitive inhibitor of CK2 and is under clinical investigation for treating malignancies. Methods: We investigated the anticancer activity of CX-4945 in 3 aggressive SCCHN cell lines. Autophagy induction and interference with cellular metabolism were characterized. Results: CX-4945 induced autophagic cell death in SCCHN cells. CX-4945 treatment caused time- and dose-dependent lipid droplet accumulation, inhibition of ACC phosphorylation, depressed cellular levels of acetyl Co-A, and severely suppressed mitochondrial respiration, without altering the expression of stress-related proteins in the endoplasmic reticulum. Conclusions: The findings demonstrated that inhibition of CK2 by CX-4945 contributed to deranged cellular energy metabolism in SCCHN cells, leading to autophagy-mediated programmed cell death. CX-4945 targeting to CK2 may be a promising therapeutic strategy against SCCHN cancer.

Exome sequencing reveals recurrent REV3L mutations in cisplatin-resistant squamous cell carcinoma of head and neck.

Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in squamous cell carcinoma of head and neck (SCCHN) patients. Therefore, validated predictive biomarkers are required to identify patients most likely to benefit from this therapeutic option. To characterize the genetic landscape of cisplatin-treated SCCHN genomes and identify potential predictive biomarkers for dacomitinib sensitivity, we performed whole exome sequencing on 18 cisplatin-resistant metastatic SCCHN tumors and their matched germline DNA. Platinum-based chemotherapy elevated the mutation rates of SCCHN compared to chemotherapy-naïve SCCHNs. Cisplatin-treated SCCHN genomes uniquely exhibited a novel mutational signature characterized by C:G to A:T transversions at CCR sequence contexts that may have arisen due to error-prone translesional synthesis. Somatic mutations in REV3L, the gene encoding the catalytic subunit of DNA polymerase ζ involved in translesional synthesis, are significantly enriched in a subset of patients who derived extended clinical benefit to dacomitinib (P = 0.04). Functional assays showed that loss-of-function of REV3L dramatically enhanced the sensitivity of SCCHN cells to dacomitinib by the loss of both translesion synthesis and homologous recombination pathways. Our data suggest that the ‘platinum’ mutational signature and inactivation of REV3L may inform treatment options in patients of recurrent SCCHN.

CCR7 regulates cell migration and invasion through MAPKs in metastatic squamous cell carcinoma of head and neck

Migration and invasion of tumor cells are essential prerequisites for the formation of metastasis in malignant diseases. Previously, we have reported that CC chemokine receptor 7 (CCR7) regulates the mobility of squamous cell carcinoma of head and neck (SCCHN) cells through several pathways, such as integrin and cdc42. In this study, we investigated the connection between CCR7 and mitogen-activated protein kinase (MAPK) family members, and their influence on cell invasion and migration in metastatic SCCHN cells. Western blotting, immunostaining and fluorescence microcopy were used to detect the protein expression and distribution of MAPKs, and the Migration assay, Matrigel invasion assay and wound-healing assay to detect the role of MAPKs in CCR7 regulating cell mobility. To analyze the correlation between CCR7 and MAPK activity and clinicopathological factors immunohistochemical staining was emplyed. The results showed stimulation of CCL19 and the activation of CCR7 could induce ERK1/2 and JNK phosphorylation, while it had no efect on p38. After activation, ERK1/2 and JNK promoted E-cadherin low expression and Vimentin high expression. The MAPK pathway not only mediated CCR7 induced cell migration, but also mediated invasion speed. The immunohistochemistry results showed that CCR7 was correlated with the phosphorylation of ERK1/2 and JNK in SCCHN, and these molecules were all associated with lymph node metastasis. Therefore, our study demonstrates that MAPK members (ERK1/2 and JNK) play a key role in CCR7 regulating SCCHN metastasis.

Chemokine receptor 7 enhances cell chemotaxis and migration of metastatic squamous cell carcinoma of head and neck through activation of matrix metalloproteinase-9.

The mechanisms leading to squamous cell carcinoma of head and neck (SCCHN) metastasis are not fully understood. Although evidence shows that the chemokine receptor 7 (CCR7) and its ligand CCL19 may regulate tumor dissemination, their role is not clearly defined in SCCHN. Matrix metalloproteinases break consisting of tissue barrier to the surrounding tissue invasion and metastasis by destroying the balance of matrix degradation of the basement membrane of tumor cells and extracellular matrix (ECM). We used chemotaxis and migration assays, western blotting, gelatin zymography, actin polymerization assay, immunofluorescence staining and immunohistochemical analysis to explore whether MMP-9 can be activated by CCL19 (CCR7's ligand) and its role in SCCHN. The experiments were performed in the metastatic SCCHN cell line PCI-37B after pre-incubation of the cells with CCL19 and SB-3CT (inhibitor of MMP-9). Our results demonstrated that CCR7 favors PCI-37B cell chemotaxis and migration, upregulation of MMP-9 protein and motivates the activity of MMP-9 protein, induces reorganization of the actin cytoskeleton and upregulation of MMP-9 protein. SB-3CT can block all these effects. Collectively, our data indicated that CCR7 regulates cell chemotaxis and migration via MMP-9 in metastatic SCCHN, and these results provide a basis for new strategies in preventing metastases of SCCHN.

Chemokine Receptor 7 (CCR7) Gene Expression Is Regulated by NF-κB and Activator Protein 1 (AP1) in Metastatic Squamous Cell Carcinoma of Head and Neck (SCCHN)

The chemokine receptor CCR7 is a seven-transmembrane domain G-protein-coupled receptor that facilitates leukocyte migration to regional lymph nodes. Aberrant CCR7 expression in a number of human malignancies has been linked to pro-survival, -invasive, and -metastatic pathways. We demonstrate here that up-regulation of CCR7 in squamous cell carcinoma of the head and neck (SCCHN) patient tumors correlates with lower survival because of metastatic disease. Because of this important oncogenic phenotype, we investigated the mechanisms that regulate CCR7 expression in these tumors. Interestingly, the inflammatory transcription factor NF-κB has been associated with a more aggressive SCCHN phenotype. Immunohistochemical staining of a SCCHN tumor cohort (n = 47) strongly linked NF-κB staining and CCR7 expression in SCCHN. Thus, we investigated whether NF-κB contributes to metastatic disease by promoting CCR7 expression in SCCHN tumor cells. We characterized four novel, potential NF-κB binding sites in the 1000-bp promoter region upstream of the CCR7 gene, using luciferase, ChIP, and EMSA. However, NF-κB inhibition only resulted in partial reduction in CCR7 expression, prompting consideration of other co-regulators of CCR7. Indeed, cooperation between NF-κB and AP1 transcription factors, which are often co-activated, is crucial to the regulation of CCR7 mRNA expression in metastatic SCCHN cells. Thus, our findings support an important biological role for inflammatory NF-κB and AP1 in the regulation of CCR7 expression in metastatic SCCHN. As such, CCR7, NF-κB, and AP1 could be potentially useful therapeutic targets in controlling the progression and metastasis of SCCHN tumors.

PKCα take part in CCR7/NF-κB autocrine signaling loop in CCR7-positive squamous cell carcinoma of head and neck

Metastatic squamous cell carcinoma of head and neck (SCCHN) has been shown to express chemokine receptor 7 (CCR7). The role of nuclear factor-κB (NF-κB) in propagating an autocrine signaling loop in CCR7-positive SCCHN cells may provide a clinically useful biomarker of disease status and response to therapy. In this article, we hypothesized that PKCα might be involved in the CCR7/NF-κB autocrine signaling loop. Results showed that CCL19 induced the activation of PKCα, and the increased activity of PKCα was abolished by CCR7 mAb. PKCα inhibition with Gö6976 led to significant reduction in the activation and nuclear translocation of NF-κB induced by CCL19. Immunohistochemical assay also showed that CCR7 and PKCα were highly expressed in SCCHN and correlated with each other, which was significantly related to lymph node metastasis and clinical stage. Taken together, PKCα is involved in the CCR7/NF-κB autocrine signaling loop.

HDAC inhibitor vorinostat enhances the antitumor effect of gefitinib in squamous cell carcinoma of head and neck by modulating ErbB receptor expression and reverting EMT

Potentiation of epidermal growth factor receptor (EGFR) inhibitors is required in squamous cell carcinoma of head and neck (SCCHN) to improve their therapeutic index. We demonstrated that the histone deacetylase inhibitor vorinostat in combination with the EGFR tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in SCCHN cells, including cells resistant to gefitinib. We provided evidence suggesting that differential modulation of ErbB receptors together with reversion of epithelial-to-mesenchymal transition (EMT) by vorinostat represent mechanistic bases for the observed synergism. We demonstrated in epithelial CAL27 cells expressing EGFR, ErbB2, and ErbB3 that vorinostat downregulated the expression and signaling of all three receptors. In gefitinib-resistant KB and Hep-2 cells, both of which had undergone EMT and expressed very low levels of ErbB3, vorinostat reverted the mesenchymal phenotype by inducing both E-cadherin and ErbB3 and downregulating vimentin as well as EGFR and ErbB2. Both transcriptional and post-translational mechanisms were involved in the modulation of ErbB receptors by vorinostat. Attenuation of all ErbB transcripts in CAL27 cells as well as induction of ErbB3 transcript in Hep-2 and KB cells was seen upon vorinostat treatment. We showed that vorinostat induced ubiquitination of EGFR and ErbB2 and targeted them predominantly to lysosome-degradation in all cell lines, while the induction of ErbB3-ubiquitination in CAL27 cells led to proteasomes-degradation. Overall, this study suggests that the vorinostat/gefitinib combination represents a promising therapeutic strategy that warrants further clinical evaluation in SCCHN, including tumors intrinsically resistant to EGFR-inhibitors.

Abstract 3958: IL-6 stimulates cell motility in squamous cell carcinoma of head and neck (SCCHN) cells through CK2 phosphorylation and stabilization of TWIST

Abstract Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://hits.isb-sib.ch/cgi-bin/PFSCAN), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addtion, mutation of the putative CK2 phosphorylation site (S18/S20) in TWIST decreased the amount of phospho- ATP incorporated by TWIST in an in vitro kinase assay and altered TWIST stability. In soft agar colony forming assays, the CK2 inhibitor, DMAT, was found to decrease the number of IL-6 stimulated colonies formed by SCCHN cells. To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN and supports the previous findings from other researchers. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway. 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 3958.

Effect of chemokine receptor 7 small interfering RNA on proliferation and invasion of squamous cell carcinoma of head and neck

To study the effect of chemokine receptor 7 (CCR-7) small interfering RNA (siRNA) on proliferation and invasion of squamous cell carcinoma of head and neck (SCCHN). CCR-7 siRNA was co-transfected into SCCHN cell line PCI-4B using Lipofectamine 2000. CCR-7 protein level was detected by western blotting. SCCHN cell proliferation was detected by MTT, and the change of actin cytoskeleton observed by confocal laser scanning microscope. Transwell assays were used to determine chemotaxis and invasion of SCCHN cells. The activity and nuclear translocation of nuclear factor-kappa B (NF-kappa B) were detected by TransAM NF-kappa B p65 kit and fluorescence microscope respectively. After CCR-7 siRNA transfection, the protein level of CCR-7 was significantly decreased. The changes induced by CCL-19, including increased proliferation rate, polarized actin polymerization, increased chemotaxis rate and invasion rate, were all abolished by CCR-7 siRNA transfection. CCR-7 siRNA also diminished CCL-19-induced NF-kappaB activation and nuclear translocation. CCR-7 siRNA could inhibit expression of CCR-7 and diminish the increased proliferation and invasion of SCCHN induced by CCL-19 in vitro. CCR-7 siRNA may provide a potential treatment strategy for SCCHN.

ΔNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck

The human p63 gene codes for multiple protein isoforms and is commonly over-expressed in squamous cell carcinoma of head and neck (SCCHN). This expression is predominantly of the DeltaN- and beta-isoforms, the former lacking the p53-related transactivation domain. p63 can activate or repress transcription of p53 and p73 target genes, but also has unique transcriptional targets and, unlike other p53 family members, is required for normal development and differentiation of squamous epithelia. We have identified novel targets of p63, using microarray analysis of SCCHN cells that stably over-express individual DeltaNp63 isoforms. All three isoforms induced expression of the cancer stem cell marker, CD44, with the DeltaNp63beta isoform showing strongest induction. Using chromatin immunoprecipitation, we were unable to show direct binding of p63 to the CD44 promoter, but found that p63 specifically increased expression of CD44 lacking variant exon 2. Each of the DeltaNp63 isoforms up-regulated expression of keratins 6A and 14 and down-regulated expression of keratins 4 and 19, in keeping with their expression patterns in SCCHN. The data strengthen the idea that p63 has key roles in regulating normal and abnormal differentiation processes through both induction and repression of genes with opposite functions. The identification of up-regulation and differential splicing of CD44 following p63 over-expression indicates roles in the regulation of adhesion, metastasis and the cancer stem cell phenotype.

Thiazolidinedione (TZD) exposure and risk of squamous cell carcinoma of head and neck (SCCHN)

1511 Background: Peroxisome proliferator-activated receptor γ (PPARγ), a nucleic acid receptor, heterodimerizes with retinoic acid X receptor (RXR). Ligand activation of RXR leads to decreased proliferation of SCCHN cell lines. TZDs (syn: glitazones), used to treat diabetes mellitus (DM), are ligands for the RXR:PPARγ heterodimer. A retrospective study of diabetics attending the Veterans Affairs (VA) health care system was undertaken to assess the association between TZD exposure and the risk of developing SCCHN. Methods: Data were obtained from the electronic patient database VISN 16 (covering 10 VA institutions) for males aged ≥40 presenting with DM and no prior SCCHN between 10/01/96 and 12/31/03. DM and SCCHN diagnoses were identified using ICD9 codes. Subjects were followed until 12/31/05, and analyzed for SCCHN risk via Cox regression, using time-dependent covariates to model exposure to TZDs, insulin and other anti-DM agents. Results: 130,406 subjects met the study criteria. Median (quartiles) age at DM presentation was 64 (55–72).18.5% were African-American, 61.3% were Caucasian, and 20.2% were other/unknown. The table shows TZD exposure, SCCHN incidence and hazard ratios for SCCHN with exposure to TZD alone or in combination with insulin and other anti-DM agents after adjusting for age, race, BMI, and HbA1C. Conclusions: A 41%-55% reduction in the risk of SCCHN was observed in diabetic subjects treated with TZD alone, with insulin, and with other anti-DM agents. The decrease was statistically significant (P<0.05) for TZD alone and TZD with other agents, but not for TZD with insulin. These findings suggest a protective effect of TZD on the development of SCCHN. The mechanism is not clearly known but possibly mediated through RXR:PPARγ pathway. [Table: see text] No significant financial relationships to disclose.

Effects of celecoxib on vascular endothelial growth factor (VEGF) gene expression in squamous cell carcinoma of head and neck (SCCHN) patients receiving 5-fluorouracil and cisplatin

5603 Background: Induction of cyclooxygenase-2 (COX-2) gene expression in SCCHN cells results in an increase in VEGF expression. Several phase II studies using celecoxib, a selective COX-2 inhibitor, in combination with chemotherapy have shown to increase chemotherapy efficacy in patients with non-small cell lung and colorectal cancers. The purpose of this pilot study is to examine VEGF gene expression in SCCHN patients receiving celecoxib and chemotherapy with an intention to develop a novel therapeutic regimen. Methods: This is an open-label one-arm phase II study with ongoing accrual. Recurrent or metastatic SCCHN patients who meet the eligibility criteria are given celecoxib 400mg po bid starting 3 days before first course of chemotherapy and continuing through out study period. Chemotherapy is repeated every 28 days with cisplatin 100 mg/m2 IV infusion 2 hours on day 1, then 5-fluorouracil 1000 mg/m2 IV infusion 24 hours daily for 5 days. Evaluation of treatment response is conducted after 3 cycles of chemotherapy. Twenty ml blood sample and 24-hour urine are collected on 4 days prior to 1st cycle of chemotherapy (before taking celecoxib), day 1 and day 5 of each chemotherapy cycle, and are subject to VEGF and PGE2 measurement by enzyme immunoassay kits from Amersham. Additionally, peripheral blood mononuclear cells are used for VEGF RT-PCR. Results: We have analyzed the first 3 patients’ blood samples for VEGF gene expression. One patient’s VEGF gene expression remained unchanged during the first and second cycles of treatment. Two patients’ VEGF gene expression increased 1.5- to 2-fold by the end of first cycle, and increased to four-fold by the end of 2nd cycle. All three patients were found to have progression of disease. Analysis of serum prostaglandin E2 is ongoing. Further results will be presented. Conclusions: Celecoxib can not effectively suppress induction of VEGF gene expression in SCCHN patients receiving chemotherapy with cisplatin and 5-fluorouracil, and may not enhance chemotherapy efficacy in this setting. Future study using celecoxib and VEGF inhibitor in combination with chemotherapy in SCCHN is planned. No significant financial relationships to disclose.

Role of c-MET in lung cancer, malignant mesothelioma, and head and neck cancer

9539 Background: c-MET is a potential therapeutic target with its aberrant signaling mediating invasive cellular program in cancers. Novel therapies are needed for lung cancer, mesothelioma and squamous cell carcinoma of head and neck (SCCHN) as their outcomes are poor despite standard therapy. Methods: We examined c-MET expression in cell lines and paraffin-embedded tumor tissues of lung cancer, mesothelioma and SCCHN using immunoblotting and immunohistochemistry (IHC) techniques. Activated-MET (pMET) was examined using phospho-MET antibodies in IHC in these tumor tissues, and using immunofluorescence (IF) in non-small cell lung cancer (NSCLC) A549 cells. Potential for targeted inhibition of c-MET was tested using small interfering RNA (siRNA) technique. siRNA-inhibition of c-MET expression was examined using immunoblotting, and the resultant inhibition of cell viability was studied using standard MTT assay. Results: There was varying c-MET expression in small cell lung cancer (SCLC) cell lines (N=10); and majority of the 9 NSCLC cell lines and 2 SCCHN cell lines had overexpression. In tumor tissues, c-MET expression was seen in 100% of thoracic malignancies (N=32). Strong expression was evident in 6/9 adenocarcinoma, 3/7 large cell, 3/7 squamous cell, and 3/5 carcinoid tumor). c-MET expression was also seen in tumor tissues of mesothelioma: 46% (N=13), and SCCHN: 54.5% (N=11). Activated pY1003-MET and pY1230/1234/1235-MET can also be demonstrated among all the tumor types. IF study showed that HGF dramatically increased pMET in A549 cells. c-MET expression was successfully inhibited using siRNA technique by >50% in the NSCLC A549 and mesothelioma H2373 cell lines. siRNA down-regulation of c-MET in A549 cells resulted in inhibition of viability (16.3±7.4% at 48hr; 57.1±7.2% at 72hr). Conclusions: c-MET is widely expressed in thoracic malignancies, mesothelioma and SCCHN with a potential role as novel therapeutic target. c-MET specific siRNA can successfully inhibit c-MET expression leading to significant inhibition of cell viability, and further inhibition strategies targeting the receptor will be sought for clinical applications. No significant financial relationships to disclose.

Role of c-MET in lung cancer, malignant mesothelioma, and head and neck cancer

9539 Background: c-MET is a potential therapeutic target with its aberrant signaling mediating invasive cellular program in cancers. Novel therapies are needed for lung cancer, mesothelioma and squamous cell carcinoma of head and neck (SCCHN) as their outcomes are poor despite standard therapy. Methods: We examined c-MET expression in cell lines and paraffin-embedded tumor tissues of lung cancer, mesothelioma and SCCHN using immunoblotting and immunohistochemistry (IHC) techniques. Activated-MET (pMET) was examined using phospho-MET antibodies in IHC in these tumor tissues, and using immunofluorescence (IF) in non-small cell lung cancer (NSCLC) A549 cells. Potential for targeted inhibition of c-MET was tested using small interfering RNA (siRNA) technique. siRNA-inhibition of c-MET expression was examined using immunoblotting, and the resultant inhibition of cell viability was studied using standard MTT assay. Results: There was varying c-MET expression in small cell lung cancer (SCLC) cell lines (N=10); and majority of the 9 NSCLC cell lines and 2 SCCHN cell lines had overexpression. In tumor tissues, c-MET expression was seen in 100% of thoracic malignancies (N=32). Strong expression was evident in 6/9 adenocarcinoma, 3/7 large cell, 3/7 squamous cell, and 3/5 carcinoid tumor). c-MET expression was also seen in tumor tissues of mesothelioma: 46% (N=13), and SCCHN: 54.5% (N=11). Activated pY1003-MET and pY1230/1234/1235-MET can also be demonstrated among all the tumor types. IF study showed that HGF dramatically increased pMET in A549 cells. c-MET expression was successfully inhibited using siRNA technique by >50% in the NSCLC A549 and mesothelioma H2373 cell lines. siRNA down-regulation of c-MET in A549 cells resulted in inhibition of viability (16.3±7.4% at 48hr; 57.1±7.2% at 72hr). Conclusions: c-MET is widely expressed in thoracic malignancies, mesothelioma and SCCHN with a potential role as novel therapeutic target. c-MET specific siRNA can successfully inhibit c-MET expression leading to significant inhibition of cell viability, and further inhibition strategies targeting the receptor will be sought for clinical applications. No significant financial relationships to disclose.