Search

Supplementary Figure Legends - PDF file 151K, Legend for Supplementary Figures S1-S6

Supplementary Figure S2 PDF - PDF file 414K, Activation of autophagy in AMC-HN-9 cells

Supplementary Methods - PDF file 168K, Additional experimental procedures, including western blot, RT-PCR, cell cycle analysis, etc. Also includes supplementary references

CD47 is expressed in all human cancer cells, including head and neck cancer, and initiates a signaling cascade to inhibit macrophage phagocytosis. However, the mechanism underlying CD47 overexpression has not been elucidated in radioresistant head and neck cancer. The present study demonstrated that decreased Tristetraprolin (TTP) expression induced a sustained overexpression of CD47 using reverse transcription-quantitative PCR and western blotting, and that CD47 overexpression prevented phagocytosis using a phagocytosis assay in a radioresistant HN31R cell line. Subsequently, using TTP transfection, RNA interference, duel-luciferase assay and EMSA, it was revealed that TTP transfection enhanced phagocytosis through degradation of CD47 mRNA by directly binding to CD47 AREs within the CD47 3'UTR. Based on our previous study, methylation-specific PCR and western blotting revealed that DNMT1 was overexpressed in radioresistant HN31R cell line and TTP expression was decreased epigenetically by DMNT1 associated DNA methylation. Overall, these findings provided novel insight into the role of TTP as a biomarker of CD47-positive head and neck cancer patients.

Sirt6 is involved in multiple biological processes, including aging, metabolism, and tumor suppression. Sirt1, another member of the sirtuin family, functionally overlaps with Sirt6, but its role in tumorigenesis is controversial. In this study, we focused on cell death in association with Sirt6/Sirt1 and reactive oxygen species (ROS) in head and neck squamous cell carcinomas (HNSCCs). Sirt6 induced cell death, as widely reported, but Sirt1 contributed to cell death only when it was suppressed by Sirt6 via regulation of MDM2. Sirt6 and Sirt6-mediated suppression of Sirt1 upregulated ROS, which further led to HNSCC cell death. These results provide insight into the molecular roles of Sirt6 and Sirt1 in tumorigenesis and could therefore contribute to the development of novel strategies to treat HNSCC., Cancer: A trio of proteins to tackle tumors New understanding of the interactions between three proteins sheds light on their role in either promoting or restricting the development of tumors called squamous cell carcinomas, which account for over 90% of all cancers in the head and neck. Researchers in South Korea led by Sang Yoon Kim and Seong Who Kim at the University of Ulsan, Seoul, investigated the role of the proteins Sirt6, Sirt1 and MDM2 in controlling the death of cancer cells caused by chemicals called reactive oxygen species (ROS). The effects of Sirt6 and Sirt1 combine to regulate ROS-induced cancer cell death. Sirt6 controls the activity of MDM2, stimulating ROS production. Sirt6 also influences MDM2 to suppress Sirt1 activity, thereby also promoting cancer cell death. Drugs affecting these three proteins could offer new approaches to anti-cancer therapy.

Background/aim Head and neck squamous cell carcinoma (HNSCC) is an aggressive head and neck malignancy. The aim of this study was to elucidate the role of periostin (POSTN) in the epithelial-to-mesenchymal transition (EMT) process mediating the acquisition of radioresistance in HNSCC. Materials and methods The expression levels of EMT hallmark genes including POSTN and Erk/Akt signaling pathways were compared between radiosensitive and radioresistant HNSCC cells. Results POSTN mRNA expression was higher in radioresistant HNSCC cells, and silencing POSTN significantly impaired their invasiveness under the effect of EMT process represented by up-regulation of mesenchymal markers and down-regulation of an epithelial marker. Expression levels of Erk and Akt were higher in radioresistant cells. Conclusion POSTN in association with the Erk and Akt signaling pathways was up-regulated during the EMT process, leading to the conversion of radiosensitive to radioresistant HNSCC cells. POSTN may be a key marker for predicting the radioresistance and therapeutic target of HNSCC.

The role of p53 in genotoxic therapy-induced metabolic shift in cancers is not yet known. In this study, we investigated the role of p53 in the glycolytic shift in head and neck squamous cell carcinoma cell lines following irradiation. Isogenic p53-null radioresistant cancer cells established through cumulative irradiation showed decreased oxygen consumption and increased glycolysis with compromised mitochondria, corresponding with their enhanced sensitivity to drugs that target glycolysis. In contrast, radioresistant cancer cells with wild-type p53 preserved their primary metabolic profile with intact mitophagic processes and maintained their mitochondrial integrity. Moreover, we identified a previously unappreciated link between p53 and mitophagy, which limited the glycolytic shift through the BNIP3-dependent clearance of abnormal mitochondria. Thus, drugs targeting glycolysis could be used as an alternative strategy for overcoming radioresistant cancers, and the p53 status could be used as a biomarker for selecting participants for clinical trials.

EPHA3, a member of the EPH family, is overexpressed in various cancers. We demonstrated previously that EPHA3 is associated with radiation resistance in head and neck cancer via the PTEN/Akt/EMT pathway, the inhibition of EPHA3 significantly enhances the efficacy of radiotherapy in vitro and in vivo. In this study, we investigated the mechanisms of PTEN regulation through EPHA3-related signaling. Increased DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, along with increased histone H3 lysine 27 trimethylation (H3K27me3) levels, correlated with decreased levels of PTEN in radioresistant head and neck cancer cells. Furthermore, PTEN is regulated in two ways: DNMT1-mediated DNA methylation, and EZH2-mediated histone methylation through EPHA3/C-myc signaling. Our results suggest that EPHA3 could display a novel regulatory mechanism for the epigenetic regulation of PTEN in radioresistant head and neck cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted attention as a potential candidate for cancer therapy. However, many primary cancers are resistant to TRAIL, even when combined with standard chemotherapy. The mechanism of TRAIL resistance in cancer cells has not been fully elucidated. The TRAIL death receptor (DR) 3&prime, untranslated region (3&prime, UTR) is reported to contain AU-rich elements (AREs) that are important for regulating DR mRNA stability. However, the mechanisms by which DR mRNA stability is determined by its 3&prime, UTR are unknown. We demonstrate that tristetraprolin (TTP), an ARE-binding protein, has a critical function of regulating DR mRNA stability. DR4 mRNA contains three AREs and DR5 mRNA contains four AREs in 3&prime, UTR. TTP bound to all three AREs in DR4 and ARE3 in DR5 and enhanced decay of DR4/5 mRNA. TTP overexpression in colon cancer cells changed the TRAIL-sensitive cancer cells to TRAIL-resistant cells, and down-regulation of TTP increased TRAIL sensitivity via DR4/5 expression. Therefore, this study provides a molecular mechanism for enhanced levels of TRAIL DRs in cancer cells and a biological basis for posttranscriptional modification of TRAIL DRs. In addition, TTP status might be a biomarker for predicting TRAIL response when a TRAIL-based treatment is used for cancer.

The manner in which p53 maintains redox homeostasis and the means by which two key metabolic elements, glucose and glutamine, contribute to p53-dependent redox stability remain unclear. To elucidate the manner in which p53 deals with glucose-deprived, reactive oxygen species (ROS)-prone conditions in this regard, two isogenic cancer subclones (HN3R-A and HN3R-B) bearing distinct p53 mutations as an in vitro model of intratumoral p53 heterogeneity were identified. Following cumulative irradiation, the subclones showed a similar metabolic shift to aerobic glycolysis and increasing NADPH biogenesis for cellular defense against oxidative damage irrespective of p53 status. The radioresistant cancer cells became more sensitive to glycolysis-targeting drugs. However, in glucose-deprived and ROS-prone conditions, HN3R-B, the subclone with the original p53 increased the utilization of glutamine by GLS2, thereby maintaining redox homeostasis and ATP. Conversely, HN3R-A, the p53-deficient radioresistant subclone displayed an impairment in glutamine usage and high susceptibility to metabolic stresses as well as ROS-inducing agents despite the increased ROS scavenging system. Collectively, our findings suggest that p53 governs the alternative utilization of metabolic ingredients, such as glucose and glutamine, in ROS-prone conditions. Thus, p53 status may be an important biomarker for selecting cancer treatment strategies, including metabolic drugs and ROS-inducing agents, for recurrent cancers after radiotherapy.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive head and neck malignancy. The aim of this study was to elucidate the role of periostin (POSTN) in the epithelial-to-mesenchymal transition (EMT) process mediating the acquisition of radioresistance in HNSCC.The expression levels of EMT hallmark genes including POSTN and Erk/Akt signaling pathways were compared between radiosensitive and radioresistant HNSCC cells.POSTN mRNA expression was higher in radioresistant HNSCC cells, and silencing POSTN significantly impaired their invasiveness under the effect of EMT process represented by up-regulation of mesenchymal markers and down-regulation of an epithelial marker. Expression levels of Erk and Akt were higher in radioresistant cells.POSTN in association with the Erk and Akt signaling pathways was up-regulated during the EMT process, leading to the conversion of radiosensitive to radioresistant HNSCC cells. POSTN may be a key marker for predicting the radioresistance and therapeutic target of HNSCC.

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway.

Background/aim Recurrent laryngeal cancer often shows an aggressive phenotype after radiotherapy and does not respond to conventional therapeutic strategies. In this study, we investigated the contribution of furin to cellular invasiveness in radio-resistant laryngeal cancer. Materials and methods Using previously established AMC-HN-3 and AMC-HN-8 cell lines from laryngeal carcinoma patients, recurrent laryngeal cancer models were generated by cumulative irradiation (AMC-HN-3-70Gy and AMC-HN-8-70Gy). Immunocytochemistry and western blotting were used to determine the epithelial-mesenchymal transition (EMT). Invasion capacity was assessed using an in vitro invasion assay. Zymography was used to assess metalloproteinase-2 (MMP-2) activity. Tumor xenografts were developed to compare growth rate and furin expression in vivo. Furin expression in 35 patients (45 samples) with salvage total laryngectomy after radiation-based treatment was assessed by laryngeal cancer tissue microarray. Results Both AMC-HN-3-70Gy and AMC-HN-8-70Gy cell lines underwent EMT following radiation. However, AMC-HN-3-70Gy cells showed increased cellular invasiveness, whereas AMC-HN-8-70Gy cells showed no difference. AMC-HN-3-70Gy cells also exhibited elevated furin expression with up-regulated expression of the active form of membrane type 1-matrix metalloproteinase (MT1-MMP)/MMP-2, whereas AMC-HN-8-70Gy cells did not show significant changes. After administration of a furin inhibitor (chloromethyl ketone (CMK)), AMC-HN-3-70Gy cells showed a significant decrease in MT1-MMP/MMP-2 expression and cellular invasiveness. Nine of 22 samples (40.9%) from salvage total laryngectomy and one of 13 pre-radiation samples (7.7%) had high furin expression. Post-radiation, furin expression increased in seven of 10 patients whose pre- and post-radiation samples were available; all-cancer mortality (three patients) was observed in this group. Conclusion Together with EMT, furin activity may serve as an indicator of an aggressive cancer phenotype, suggesting that furin is a potentially useful target for recurrent laryngeal cancer.

Background We attempted to elucidate the mechanism of cell death after radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant head and neck cancer cells. Methods The most radioresistant cancer cell line (AMC-HN-9) was selected for study. Targeted silencing of β-catenin was used on siRNAs. Sensitivity to radiation was examined using clonogenic and methylthiazol tetrazolium (MTT) assays. Results A combination of irradiation plus β-catenin silencing led to a significant reduction in the inherent radioresistance of AMC-HN-9 cells. Although expression of Ku70/80 was upregulated in AMC-HN-9 cells after irradiation, Ku70/80 was dramatically decreased in a combination of irradiation and β-catenin silencing. Interestingly, irradiation-induced Ku70/80 was completely prevented by β-catenin silencing-induced LKB1/AMP-activated protein kinase (LKB1/AMPK) signal. Conclusion The LKB1/AMPK pathway might relay the signal between the Wnt/β-catenin pathway and the Ku70/Ku80 DNA repair machinery, and play a decisive role in fine-tuning the responses of cancer cells to irradiation. © 2015 Wiley Periodicals, Inc. Head Neck, 2015

Objectives: PI3K/Akt/mTOR pathway is commonly activated in most cancers and is correlated with resistance to anticancer therapies such as radiotherapy. Therefore, PI3K is an attractive target for treating PI3K-associated cancers. Material and Methods: We investigated the basal expression and the expression after treatment of PI3K inhibitor or Src inhibitor of PI3K/Akt pathway-related proteins in AMC-HN3, AMC-HN3R, HN30 and HN31 cells by performing immunoblotting analysis. The sensitivity to PI3K inhibitors or Src inhibitor was analyzed by MTT assay and clonogenic assay. To determine the antitumoral activity of combination treatment with PI3K inhibitor and Src inhibitor, we used using xenograft mouse model. Results: We found that PI3K regulatory subunit p85 was predominantly phosphorylated in radioresistant head and neck cancer cell line (HN31), which showed resistance to PI3K inhibitors. Next, we investigated mechanism through which PI3K p85 phosphorylation modulated response to PI3K inhibitors. Of note, constitutive activation of Src was found in HN31 cells and upon PI3K inhibitor treatment, restoration of p-Src was occurred. Src inhibitor improved the efficacy of PI3K inhibitor treatment and suppressed the reactivation of both Src and PI3K p85 in HN31 cells. Furthermore, downregulation of PI3K p85 expression by using a specific siRNA suppressed Src phosphorylation. Conclusions: Together, our results imply the novel role of the PI3K regulatory subunit p85 in the development of resistance to PI3K inhibitors and suggest the presence of a regulatory loop between PI3K p85 and Src in radioresistant head and neck cancers with constitutively active PI3K/Akt pathway.

Clinical efficacy of immune checkpoint blockade is often enhanced by their co-administration with immunogenic chemotherapy. Here a novel chemo-immunotherapy combination is reported that successfully overcomes the immunosuppressive tumor microenvironment to elicit an effective anti-tumor immune response. The CD47-SIRPα innate immune checkpoint was blocked by surface engineering human ferritin with a SIRPα variant, and this FHSIRPα efficiently inhibited the “don’t-eat-me” signal of cancer cells, thereby enhancing cancer cell phagocytosis in a colon carcinoma (CT26) model in immunocompetent mice. Doxorubicin prodrug, EMC-DEVD-S-DOX, that binds to circulating albumin following intravenous administration showed enhanced half-life, and the active drug was released via caspase-3 at tumor site following radiation treatment. Radiation-induced apoptosis of cancer cells resulted in release of active drug doxorubicin and upregulation of CD47 on cancer cell surface, sensitizing cancer cells to FHSIRPα treatment. The induction of immunogenic cell death (ICD) by the prodrug was confirmed in vitro by upregulation of calreticulin, translocation of HMGB1 and increased release of chaperone proteins in CT26.CL25 mouse colon cancer cells by flow cytometric analysis and confocal microscopy. The ICD effect of the prodrug correlated well in vivo and increased phagocytosis of tumor cells by antigen-presenting cells (APCs) in immunocompetent mice as analyzed by flow cytometry of tumor tissue and draining lymph nodes. Tumor infiltrates analyzed by flow cytometry also showed enrichment of APCs; and T cell-mediated elimination of immunogenic tumors was confirmed by the tumor-specific activation of T cells ex vivo as detected by secretion of IFNγ. Whereas FHSIRPα monotherapy showed modest tumor growth inhibition, combination with a doxorubicin prodrug resulted in effective inhibition of tumor growth by 95.2% (p Citation Format: Na Kyeong Lee, Jeong Uk Choi, Jung Je Park, Ji-Hyun Seo, Mi Ra Kim, Hyo Won Chang, Sang Yoon Kim, In-San Kim, Youngro Byun. Nanocage therapeutics FHSIRPα mediating immune checkpoint blockade awakens immunity against cancer with an albumin-binding caspase-cleavable doxorubicin prodrug EMC-DEVD-S-DOX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2251.

P53 is an important biomarker in response to genotoxic stress, but it is also known to plays a key role in the regulation of metabolic homeostasis. The loss of p53 is a well-established contributor to the malignant transformation and glycolytic phenotype acquisition of cells during cancer development. However, the role of p53 in genotoxic therapy-induced metabolic shift in cancers remains unclear. Here, we attempted to elucidate how p53 participates in the glycolytic shift of head and neck cancer cell lines following irradiation. We established a stable radioresistant head and neck cancer cells (HN30-R; p53 wild type and UMSCC1-R; p53 null type) through cumulative irradiation and then analyzed their glucose metabolic profiles and mitochondria respiration. As a result, the metabolic analysis revealed no changes glycolysis of HN30-R cells, but UMSCC1-R cells exhibited increased glycolysis through increased glucose uptake and lactate production and glycolytic intermediates as well as related glycolytic enzymes, compared to UMSCC1 cells. Also, we confirmed that the mitochondrial respiration was reduced by the maximal respiration parameters of oxygen consumption rate (OCR) and that abnormal mitochondria were accumulated by electron microscopy in UMSCC1-R cells. Thus, UMSCC1-R cells exhibited an increased sensitivity to glycolysis-targeting drugs such a hexokinases inhibitor (2-deoxy-D-glucoes; 2-DG) and a lactate dehydrogenase-A inhibitor (AT101), but HN30-R cells did not shown any changes. Moreover, we identified that mitophagy limits glycolytic shift through the p53-dependent clearance of abnormal mitochondria. Taken together, these results suggest that p53 null type cells increased aerobic glycolysis to overcome the accumulation of abnormal mitochondria in radioresistnat cells. Conversely, p53 wild type cells inhibited the glycolytic shift by regulating a integrity through p53-dependent mitophagy. Thus, glycolysis-targeted drugs could be an alternative strategy for overcoming recurrent cancers after radiotherapy, and p53 status could be a biomarker for selecting participants for clinical trials. Citation Format: Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Hae Yun Nam, Myungjin Lee, Won Hyeok Lee, Song Hee Kim, Da Seul Seong, Myung Woul Han, Jong Cheol Lee, Jung Je Park, Ji-hyun Seo, Seong Who Kim, Sang Yoon Kim. P53-dependent mitophagy controls glycolytic shift in radioresistant head and neck cancer cells [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 2405.

Summary Tumor hypoxia is associated with poorer outcome in patients with head and neck carcinomas, but little is known about hypoxia biomarkers in oral tongue cancer. We evaluated whether hypoxia biomarkers and clinicopathologic variables were prognostic predictors in patients with T2-staged squamous cell carcinoma (SCC) of the oral tongue. Tissue microarrays were constructed from formalin-fixed tumor blocks of 43 patients with T2-staged tongue SCCs treated by surgical resection and neck dissection. Tissue samples were stained with monoclonal antibodies to hypoxia-inducible factor (HIF)-1α, HIF-2α, carbonic anhydrase (CA)-9, glucose transporter (GLUT)-1, and erythropoietin receptor (EPOR). Locoregional control and survival rates were calculated by the Kaplan-Meier method, and prognostic factors were calculated from uni- and multivariate analyses. Tumor thickness was correlated with expression of CA-9 and GLUT-1 and nodal classification was correlated with GLUT-1 expression. The nodal metastasis rate was 51%, and the 5-year locoregional control and disease-specific survival (DSS) rates were 59% and 69%, respectively. Univariate analysis showed that HIF-1α and EPOR expression were significantly related to DSS. Multivariate analysis showed that EPOR expression was an independent predictor of DSS ( P = 0.030). EPOR expression may be an independent predictor for DSS in patients with T2-staged SCC of the oral tongue.

We found four proteins in the AMC-HN-9 cells that might perform important functions in radio-resistance. These results also suggest that this cell line may provide us with important information about cancer cells regarding the roles of reactive oxygen species (ROS and antioxidants) in irradiation.Radiation susceptibility can be determined by the expression of some proteins. To identify such proteins involved in radiation susceptibility, we analyzed two cell lines with different radiation sensitivity.The AMC-HN-3 and -9 cell lines established from head and neck cancer patients were employed. They were irradiated with 4 Gy and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was carried out. Then matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was performed to identify 20 proteins. Western blotting was used to confirm the significantly expressed proteins.After 2-D PAGE, five spots were differentially expressed in the AMC-HN-9. Heat shock protein 27 kDa (HSP27), peroxiredoxin (Prx) II, glutathione S-transferase pi (GSTP), and an unknown protein were detected through MALDI-TOF MS. By using Western blotting, remarkable increments of the band intensity of HSP27, GSTP, Prx II, and Prx IV were confirmed in the AMC-HN-9 cell line.

It has been proposed that Wnt signaling pathway may be a key radioprotective mechanism in irradiated cancer cells; however, the specific radioresistance mechanisms remain not to be fully clarified. Here we elucidate a novel signaling pathway of radioresistance in head and neck cancer (HNC) cell lines involving interactions among the Wnt signaling pathway, cyclooxygenase-2 (COX-2) and Ku expression. Activation of the Wnt signaling pathway by (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) resulted in beta-catenin cytoplasmic accumulation and translocation to the nucleus, upregulated Ku expression and increased radioresistance in the COX-2-expressing HNC cell line. In contrast, Wnt singaling activation by BIO had no effects on Ku expression and radiosensitivity in a HNC cell line negative for COX-2. Interactions between Wnt singaling and Ku were indirectly regulated by COX-2. Blockage of COX-2 signaling led to the suppression of beta-catenin-induced Ku expression, and to consequent recovery of the radiosensitivity in HNC cells. Our results conclusively suggest that beta-catenin plays a pivotal role in the regulation of Ku expression via the proposed COX-2 intracellular pathway, thus supporting a novel radioresistance mechanism of HNC.

Ganglion is a common benign lesion that usually arises adjacent to the joints or tendons of the hand. However, an intratendinous ganglion is a rare condition. We report two cases of intratendinous ganglion of the extensor tendon of the hand which were treated with excision.

We attempted to elucidate the mechanism of cell death after radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant head and neck cancer cells.The most radioresistant cancer cell line (AMC-HN-9) was selected for study. Targeted silencing of β-catenin was used on siRNAs. Sensitivity to radiation was examined using clonogenic and methylthiazol tetrazolium (MTT) assays.A combination of irradiation plus β-catenin silencing led to a significant reduction in the inherent radioresistance of AMC-HN-9 cells. Although expression of Ku70/80 was upregulated in AMC-HN-9 cells after irradiation, Ku70/80 was dramatically decreased in a combination of irradiation and β-catenin silencing. Interestingly, irradiation-induced Ku70/80 was completely prevented by β-catenin silencing-induced LKB1/AMP-activated protein kinase (LKB1/AMPK) signal.The LKB1/AMPK pathway might relay the signal between the Wnt/β-catenin pathway and the Ku70/Ku80 DNA repair machinery, and play a decisive role in fine-tuning the responses of cancer cells to irradiation. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1909-E1917, 2016.

The Ku protein is essential for the repair of a majority of DNA double-strand breaks in mammalian cells. The purpose of this study was to investigate the relationship between the expression of Ku70/80 and sensitivity to radiation in cancer cell lines of the head and neck. The sensitivity to radiation in various head and neck cancer cell lines (AMC-HN-1 to -9) was analyzed by colony forming assay. Of the nine cell lines examined, the most radiosensitive cell line (AMC-HN-3) and the most radioresistant cell line (AMC-HN-9) were selected for this experiments. The expression of Ku70/80 was examined after irradiation using real time PCR, Western blotting and immunofluorescence in two different cell lines. Cell cycle distribution after irradiation were analysed. A differential radioresponse was demonstrated by expression of Ku70/80 in AMC-HN-3 and AMC-HN-9 cells. While the expression of Ku70 was slightly increased in the radioresistant AMC-HN-9 cell line, the expression of Ku80 was remarkably increased, suggesting a correlation between Ku80 expression and radiation resistance. Overexpression of Ku80 plays an important role in the repair of DNA damage induced by radiation. Ku80 expression may provide an effective predictive assay of radiosensitivity in head and neck cancers.

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L benchscale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett packard HP 5890 II gas chromatograph. As such, the bacterium was identified as aStenotrophomonas species and designated asStenotrophomonas sp. OK-5.

The presence of p53 mutation was associated with poor survival in various tumors. The involvement of p53 in apoptosis and cell-cycle control makes it a plausible biomarker of prognosis . Classifying TP53 mutations in HNSCC described disruptive TP53 mutation in either the L2 or L3 loop of the DNA binding domain, resulting in polarity change within the protein, or stop cordon. Disruptive TP53 mutation in HNSCC tumors predicts for locoregional recurrence, due to increased radioresistance via the inhibition of senescence. Senescence induction contributes to cancer therapy responses and is crucial for p53-mediated tumor suppression. However, whether p53 inactivation actively suppresses senescence induction has been unclear. Here, we found that overexpression CIP2A (cancerous inhibitor of protein phosphatase 2A) positively correlated with presence of p53 mutation in head and neck cancer and mediates radioresistance through suppression of radiation induced senescence. And we demonstrated that rapamycin could induce senescence via CIP2A downregulation and increase radiosensitivity in p53 disruptive mutation cell line. This is the first investigation to analyze the role of CI2A in resistance to the radiotherapy of head and neck cancer. As a consequence, a greater understanding of radioresistance mechanisms through our results in head and neck cancer with p53 mutation will enable the rational design of combination regimens and sequential treatment algorithms to improve clinical outcomes and points to the usefulness of CIP2A as a biomarker to predict clinical response to rapamycin in head and neck cancer. Citation Format: Song Hee Kim, Won hyeok Lee, Myung Jin Lee, Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Jung Je Park, Myung Woul Han, Sang Yoon Kim. CIP2A can be a therapeutic target of rapamycin in radioresistant head and neck cancer with P53 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2017-5476

Although intramedullary nailing is an ideal treatment for subtrochanteric femoral fractures, it is technically challenging in fractures extending into the nail entry area and/or involving the lesser trochanter. Although the application of circumferential wire may facilitate reduction in these situations, its use remains controversial due to possible blood supply disturbances to underlying bone. In the present study, we evaluated complex subtrochanteric fractures treated by percutaneous cerclage wiring followed by intramedullary (IM) nailing for anatomical fracture reduction and union. Twelve patients (mean age 48.3 years) with an unstable subtrochanteric fracture were prospectively treated. Indications of percutaneous cerclage wiring followed by IM nailing were a fracture extending proximally into the nail entry area deemed difficult to treat by anatomical reconstruction by IM nailing or a fracture with long oblique or spiral component. One or two cerclage wires were percutaneously applied for the temporary reduction of main fragments, and then, a cephalo-medullary or a reconstruction nail was fixed. We assessed radiologic results (union time, alignment), functional results, and complications. All 12 cases healed, without a bone graft, at an average of 19.1 weeks after surgery (range 16–24). In 11 cases, acceptable alignment was achieved (mean, valgus 0.3° extension 0.6°) with minimal leg-length discrepancy; the other exhibited 1 cm of shortening. All patients were able to return to pre-injury activity levels, and median Merle d’Aubigne score was 16.9 (15–18). No infection or implant-related complication was encountered to latest follow-up (minimum 12 months postoperatively). Temporary reduction by percutaneous wiring offers a means of satisfactory nailing in difficult subtrochanteric femoral fractures, and affords anatomical reconstruction and favorable bony union.

Autophagy is frequently activated in radioresistant cancer cells. In the present study, we evaluated the role of autophagy and transforming growth factor-activated kinase 1 (TAK1) in radioresistance.TAK1 phosphorylation in MDA-MB231 breast cancer cells was evaluated by western blotting. The regulatory effects of the TAK1 inhibitor and autophagy inhibitor were assessed by cell morphology, cell survival and induction of apoptosis.Radiation induced the phosphorylation of TAK1, whereas the inhibition of TAK1 activity enhanced the cytotoxicity of radiation in MDA-MB231 cells. Autophagy inhibitors significantly enhanced radiation-induced apoptosis of MDA-MB231 cells. This augmentation in radiosensitivity seemed to result from the suppression of TAK1 activation.Inhibition of autophagy enhanced radiosensitivity through suppression of radiation-induced TAK1 activation, suggesting that the modulation of TAK1-induced autophagy may be a good therapeutic strategy to treat radioresistant breast cancer.

There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis.Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line.HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine.Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

Autophagy is frequently activated in radioresistant cancer cells where it provides a cell survival strategy. The mTOR inhibitor rapamycin activates autophagy but paradoxically it also enhances radiosensitivity. In this study, we investigated the mechanisms of these opposing actions in radiation-resistant glioma or parotid carcinoma cells. Radiation treatment transiently enhanced autophagic flux for a period of 72 hours in these cells and treatment with rapamycin or the mTOR inhibitor PP242 potentiated this effect. However, these treatments also increased heterochromatin formation, irreversible growth arrest, and premature senescence, as defined by expression of senescence-associated β-galactosidase activity. This augmentation in radiosensitivity seemed to result from a restoration in the activity of the tumor suppressor RB and a suppression of RB-mediated E2F target genes. In tumor xenografts, we showed that administering rapamycin delayed tumor regrowth after irradiation and increased senescence-associated β-galactosidase staining in the tumor. Our findings suggest that a potent and persistent activation of autophagy by mTOR inhibitors, even in cancer cells where autophagy is occurring, can trigger premature senescence as a method to restore radiosensitivity. Cancer Res; 73(14); 4267–77. ©2013 AACR.

Radiotherapy is a well-established therapeutic modality used in treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after treatment by radiation. Thus, special emphasis has been given to the discovery of effective radiosensitizers. EphA3 receptors functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains to be elucidated. Here, we established the stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and identified that EphA3 was overexpressed predominantly in the AMC HN3R cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we identified that EphA3 was overexpressed in recurred laryngeal cancer specimen after radiation therapy. And we found that EphA3 mediated the tumor invasiveness and migration and EMT (epithelial mesenchymal transition) related protein expression in AMC HN3R cancer cell line. To investigate the role of EphA3 in modulating the radiosensitivity, we observed the change of survival fraction after transfection EphA3 siRNA. And we found that inhibition of EphA3 enhances radiosensitivity in AMC HN 3R cell line. In conclusion, these results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and can play a crucial role in development radioresistance in head and neck cancers through regulation of EMT pathway. Citation Format: Myung Woul Han, Song Hee Kim, Hyo Won Chang, Hae Yun Nam, Myungjin Lee, Gui Chul Kim, Yoon sun Lee, Mi Ra Kim. EphA3 maintains radioresistance in head and neck cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1170.

The Wnt/β-catenin pathway regulates the viability and radiosensitivity of head and neck squamous cancer cells (HNSCC). Increased β-catenin predisposes HNSCC patients to poor prognosis and survival. This study was conducted to determine the mechanism by which β-catenin regulates the viability of HNSCC. AMC-HN-3, -HN-8, UM-SCC-38, and -SCC-47 cells, which were established from human head and neck cancer specimens, and underwent cell death following β-catenin silencing. β-Catenin silencing significantly induced G1 arrest and increased the expression of Bax and active caspase-3, which demonstrates the sequential activation of apoptotic cascades following treatment of HNSCC with targeted siRNA. Intriguingly, β-catenin silencing also induced autophagy. Here, we confirm that the number of autophagic vacuoles and the expression of type II light chain 3 were increased in cells that were treated with β-catenin siRNA. These cell death modes are most likely due to the activation of LKB1-dependent AMPK following β-catenin silencing. The activated LKB1/AMPK pathway in AMC-HN-3 cells caused G1 arrest by phosphorylating p53 and suppressing mTOR signaling. In addition, treating AMC-HN-3 cells with LKB1 siRNA preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, these results imply that following β-catenin silencing, HNSCC undergo both apoptotic and autophagic cell death that are under the control of LKB1/AMPK. To the best of our knowledge, these results suggest for the first time that novel crosstalk between β-catenin and the LKB1/AMPK pathway regulates the viability of HNSCC. This study thus presents new insights into our understanding of the cellular and molecular mechanisms involved in β-catenin silencing-induced cell death.

The issue of whether aberrant expression of β1-integrin is associated with cancer progression and development of resistance to cytotoxic therapy is of considerable interest. Studies to date have shown that the anchorage-independent survival of cancer is attributed, in part, to epithelial-to-mesenchymal transition (EMT). Here, we have reported a novel alternative mechanism of anchorage-independent survival of cancer cells. Cell lines derived from head and neck cancer patients (AMC-HN-3 and AMC-HN-9) and the well-known EMT cancer cell line, MDA-MB231, were examined. The EMT features of AMC-HN-9 cells were comparable to those of MDA-MB231, whereas AMC-HN-3 cells showed no EMT characteristics. Although the pattern and degree of β1-integrin expression were similar in all three cell lines, sensitivities of the cells to β1-integrin knockdown with small interfering RNA (siRNA) were different. Cancer cells with no EMT features underwent cell death to a more significant extent following β1-integrin silencing than those with EMT. Intriguingly, we observed reactive activation of the p53–p21 pathway after β1-integrin silencing in AMC-HN-9 cells lacking an apparent cell death response. Simultaneous knockdown of wild-type p53 and β1-integrin in this cell line promoted cell death. Our data collectively indicate that β1-integrin-related cell death is closely associated with EMT phenotypes and activation of the p53–p21 pathway is partly involved in the acquisition of resistance to apoptosis induced by β1-integrin silencing. Further clarification of the mechanisms underlying p53 integration with β1-integrin signaling may facilitate the development of novel anti-cancer strategies.

Targeting hypoxia-inducible factor-1 (HIF-1) active cells in tumors may be an excellent strategy to improve the outcome of radiation therapy. On the basis of the reported role of YC-1 as a HIF-1 inhibitor with anti-cancer activity, we tested the therapeutic efficacy of YC-1 against radioresistance in vitro. The AMC-HN3 cancer cell line, developed from squamous cell carcinoma of the larynx, was cultured under hypoxic conditions or in the presence of cobalt chloride. Both treatments induced nuclear accumulation of HIF-1alpha protein. Cells cultured under normoxic or hypoxic conditions with and without YC-1 treatment were irradiated and analyzed using flow cytometry and clonogenic assays. In the absence of YC-1 treatment, irradiation induced a greater cytotoxic effect in normoxic cells than in cobalt-treated cells. Treatment of cobalt-treated cells with YC-1 effectively inhibited HIF-1alpha expression, and enhanced the sensitivity of cells to radiation, decreasing the surviving fraction to that of normoxic cells. Flow cytometry confirmed these results, showing that the sub-G1 fraction was increased in YC-1-treated hypoxic cells after irradiation. Our results suggest that YC-1 treatment may be an effective therapeutic strategy for overcoming the radioresistance of HIF-1alpha-expressing, hypoxic cancer cells.

Orally absorbable anticancer medications have great advantages for conventional cancer therapies to patients. Here we evaluated the potent anticancer effect of orally absorbable LHD, a chemical conjugate of low-molecular-weight heparin and deoxycholic acid, on tumor graft growth models.We characterized the angiogenic factors, such as VEGF, heparanase, and MMPs, of murine squamous cell carcinoma (SCC7), melanoma (B16F10) or lung carcinoma (LLC1). Two weeks after oral administration of LHD into these cancer-cell-bearing mice, we evaluated the antiangiogenic activity of LHD.Although all cancer cells expressed the angiogenic factors, SCC7 cells had much higher angiogenic potential and grew rapidly after implantation into mice. When orally administered, LHD delayed tumor graft growth regardless of cancer types. Particularly, LHD powerfully diminished the SCC7-derived tumor growth. Also, the expression of angiogenic factors in all kinds of tumor tissues was decreased, thereby attenuating the neovascularization in tumor tissue.Our study shows that LHD has potent anticancer and antiangiogenic effect on at least three kinds of tumor cells. LHD can be specifically used for preventing neovascularization in tumor tissue because it has therapeutical potential as an antiangiogenic drug and can be orally absorbed.

Purpose: Human peroxiredoxins (Prxs) are known as a family of thiol-specific antioxidant enzymes, among which Prx-I and -II play an important role in protecting cells from irradiation-induced cell death. It is not known whether Prx-IV also protects cells from ionizing radiation (IR). Methods and Materials: To evaluate the protective role of Prx-IV in IR, we transfected full-length Prx-IV cDNA into AMC-HN3 cells, which weakly express endogenous Prx-IV, and knocked down the expression of Prx-IV with siRNA methods using AMC-HN7 cells, which express high levels of endogenous Prx-IV. Radiosensitivity profiles in these cells were evaluated using clonogenic assay, FACS analysis, cell viability, and TUNEL assay. Results: Three Prx-IV expressing clones were isolated. Prx-IV regulated intracellular reactive oxygen species (ROS) levels and made cells more resistant to IR-induced apoptosis. Furthermore, the knockdown of Prx-IV with siRNA made cells more sensitive to IR-induced apoptosis. Conclusion: The results of these studies suggest that Prx-IV may play an important role in protecting cells from IR-induced apoptosis in head-and-neck squamous cell carcinoma.

Conventional chemotherapy requires therapy holiday due to their toxic adverse effects. However, the therapy holiday has been allowing cancer cell to relapse and caused secondary metastasis to occur in other tissue. On the contrast, metronomic maintenance chemotherapy (MMC) treats anticancer drugs frequently to reduce the adverse effects from normal tissues by maintaining low concentration in the blood. In this context, an oral route is more suitable for MMC than a parenteral route. Our oral delivery system that uses electrochemical property of bile acid derivatives increases bioavailability of doxorubicin (DOX) from below 5% to 26% by eluding p-gp. The antitumor studies showed that 5mg/kg/day oral DOX complex inhibited 70% of tumor volume in C3H mice model bearing SCC7 tumor. The H&E staining of intestines and liver and the blood analysis proved that there were no histological and hematological toxicities. In addition, low dose of the DOX complex affects cell cycle regulation to arrest cancer cells. The concentration of 10nM, 30nM of DOX restricted the cell cycle at checkpoints in G1 phase of MCF-7 cells which are p-53 wild type and checkpoints in G2-M phase of MDA-MB 231 cells which are p-53 disruptive type. When MK8776, a inhibitor of cell cycle checkpoints, is treated with the low concentration of DOX, the cancer cells went through apoptosis or senescence depending on a type of cancer. In conclusion, our oral delivery of doxorubicin can offer safe and effective chemotherapy. Note: This abstract was not presented at the meeting. Citation Format: Seho Kweon, Foyez K.A. Mahmud, Hyo Won Chang, Have Yoon Nam, Mi Ra Kim, Jung Je Park, Youngro Byun, Sang Yoon Kim. Oral delivery of doxorubicin using bile enhancer for metronomic maintenance chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2015-5351

At present, most of the anticancer drugs are given parenterally, showing drug concentration above the maximum tolerable concentration (MTC) followed by rapid elimination from the plasma that causes enhanced side effects and lower efficacy. In contrast, low dose oral maintenance therapy may increase therapeutic efficacy by increasing the exposure time to cancer cells and also reduce the side effects by maintaining the drug concentration below MTC level. Pemetrexed is a folate antagonist approved for maintenance chemotherapy in lung cancer. However, a major limitation of Pemetrexed is the poor oral bioavailability which imposes IV administration. In this context, we have developed a physical complex of Pemetrexed with lysine conjugated deoxycholic acid (DCK) in water (mole ratio 1:2) for the purpose of oral delivery. The DSC thermogram of Pemetrexed/DCK complex showed the complex formation due to disappearance of exothermic and endothermic peaks from Pemetrexed. The pharmacokinetic studies of Pemetrexed/DCK were done in SD rats and analyzed by HPLC method. In antitumor study, low dose continuous oral therapy from 2.5 mg/kg to 20 mg/kg showed substantial tumor inhibition in SCC7 tumor bearing C3H mice. We also evaluated maintenance chemotherapy as oral Pemetrexed/DCK administration in A549 tumor bearing nude mice. We found that 5 mg/kg dose of oral Pemetrexed successfully inhibited tumor progression and also showed additional antitumor effects when given with Cisplatin as maintenance chemotherapy. In the context of oral anticancer drugs, we need to consider the toxicities in GI tracts (GIT). For this purpose, apoptosis in GI tracts and morphology of different organs were assessed by TUNEL and H&E staining. One month toxicity studies have been conducted in nude mice and we found that 5 mg/kg oral dose did not produce any abnormalities in hematological and serological parameters in mice having intact morphology of GI tracts. Our studies offer safe and effective oral maintenance chemotherapy of pemetrexed for cancer patients at home. Citation Format: Foyez Mahmud, Seho Kweon, Hyo Won Chang, Hae Yoon Nam, Mi Ra Kim, Jung Je Park, Sang Yoon Kim, Youngro Byun. Metronomic maintenance chemotherapy of orally active pemetrexed for effective treatment of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4390. doi:10.1158/1538-7445.AM2015-4390

The inhibitory efficacies of new bile acid acylated-heparin derivative (heparin-DOCA) were evaluated on experimental lung metastasis. We evaluated the effect of heparin-DOCA on intercellular interactions including those between B16F10 and thrombin-activated platelets and TNF-alpha-activated HUVECs, and between B16F10 and immobilized mouse P-selectin. In addition, the inhibitory effects of heparin-DOCA on adhesion and invasion of B16F10 to Matrigel were studied. In an animal mouse study, the blood clot formation and the retention of red fluorescence protein (RFP)-B16F10 in lungs were assessed after heparin-DOCA and RFP-B16F10 intravenous administration. Furthermore, we investigated the anti-metastatic effect of heparin-DOCA against lung metastasis induced by B16F10 and SCC7. Heparin-DOCA inhibited intercellular interactions between B16F10 and activated platelets or activated HUVECs by blocking P- and E-selectin-mediated interactions. Moreover, it reduced adhesion and invasion of B16F10 to ECM, thereby affecting the reduction of early retention of B16F10 in the lung. Heparin-DOCA attenuated lung colony formation on the surfaces and in interior of the lung, and attenuated metastasis by B16F10 and SCC7. These results suggest that heparin-DOCA may have potentials as therapeutic agent that prevents tumor metastasis and progression.

In this study, the cellular responses of Stenotrophomonas sp. OK-5 to explosive 2,4,6-trinitrotoluene (TNT) have been extensively analyzed. The stress shock proteins, which might contribute to enhancing cellular resistance to TNT-mediated toxicity, were induced at different concentrations of TNT used as a substrate for cell culture of Stenotrophomonas sp. OK-5 capable of utilizing TNT. Proteomic analysis for 2-DE of soluble protein fractions from the culture of OK-5 exposed to TNT demonstrated approximately 300 spots on the silver-stained gel ranging from pH 3 to pH 10. Among them, 10 spots significantly induced and expressed in response to TNT were selected and analyzed. As the result of internal amino acid sequencing with ESI-Q TOF mass spectrometry, TNT-mediated stress shock proteins such as DnaK, OmpW, and OsmC were identified and characterized. Survival of strain OK-5 was periodically monitored in the presence of different concentrations of TNT along with the production of the stress shock proteins. Cells of strain OK-5 pre-exposed to TNT had in improved survival tolerance. Analysis of total cellular fatty acids in strain OK-5 suggested that several saturated or unsaturated fatty acids might increase or decrease under TNT-mediated stress condition. Scanning electron microscopy of cells treated with 0.8 mM TNT for 12 h revealed irregular rod shapes with wrinkled surfaces.

The Wnt/β-catenin pathway regulates the cell growth and survival following radiation in various types of cancer cells. Our previous report show that activation of the Wnt/β-catenin signaling pathway is a key radioprotective mechanism in irradiated head and neck cancer (HNC) cells. However, the molecular mechanisms by which β-catenin regulates radiation sensitivity are not clear. Here we attempted to elucidate the mechanism of cell death following radiation by studying how β-catenin silencing controls the radiation sensitivity of radioresistant HNC cells. Of nine cell lines examined, the most radioresistant cell line (AMC-HN-9) were selected for this experiments. β-catenin silencing using small interfering RNA(siRNA) down-regulated β-catenin expression up to 72 h, which was confirmed by western blot analysis. The sensitivity to radiation was anlayzed by clonogenic analysis and MTT assay. As a result, β-catenin silencing remarkably decreased the survival of irradiated AMC-HN-9 cells and the cell viability also significantly reduced more by the combination treatment with β-catenin siRNA and radiation (0.37±0.034 fold) than when treated with β-catenin siRNA or radiation alone (0.68±0.055 fold and 0.90±0.043 fold, respectively). Interestingly, whereas expression of Ku70/80 was up regulated in AMC-HN-9 cells following irradiation (4Gy), in the cells treated with combination of radiation and β-catenin siRNA, Ku70/80 expression was dramatically decreased. In additionally, when exposed to radiation after β-catenin silencing, the up-regulation of irradiation-induced Ku80 completely was prevented by β-catenin silencing-induced AMPK. Taken together, these results suggest that suppression of Ku70/80 expression through β-catenin silencing-induced AMPK is associated with its radio-sentitizing effect in AMC-HN-9 cells, thus supporting a novel radiosensitive mechanism of radioresistant HNC cells. Citation Format: Hyo Won Chang, Hae Yun Nam, Mi Ra Kim, Hyang Ju Lee, Ji Hyun Seo, So Yeon Lee, Seong Who Kim, Sang Yoon Kim. β-catenin silencing enhances radiation sensitivity through antagonizing effect of AMPK against Ku70/80 in head and neck cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2014-4904

Background: For patients with recurrent head and neck cancer after radiation therapy (RT), there are limited therapeutic selections. There is great interest in mechanisms of resistance to RT. To investigate these mechanisms, we established RT-resistant clones from head and neck squamous cell carcinoma (HNSCC) cells. Methods: To develop cell lines with acquired resistance to RT, we utilized the human HNSCC cell line AMC-HN3 and UDSCC-2. We irradiatied clinically relevant cumulative irradiation to cell lines and characterized for the molecular changes in the radioresistant lines relative to the parent cell lines. Results: A comparative analysis revealed that acquired resistance to RT was associated consistently with the overexpression of HER2 and cross-resistance to cetuximab. To examine the role of HER2, we knocked down HER2 in both radio-resistant and -sensitive parental cells and found an increase in sensitivity to cetuximab. Conversely, inhibition of HER2 in RT-resistant cells was sufficient in resensitizing them to cetuximab in vitro. Further studies indicated that HER2 may diminish sensitivity to cetuximab via activation of the phosphatidyl-inositol-3-kinase (PI3K)/AKT pathway. Conclusion: Taken together, these findings suggest a central role of HER2 in the development of acquired resistance to RT and prompt consideration to apply HER2 inhibition strategies in future clinical trials of recurrent HNSCC after RT. Citation Format: Jung Je Park, Sang Yoon Kim, Ji Hyun Seo, Hyo Won Chang, Roza Khalmuratova, Thomas E. Carey, Jin Pyeong Kim, Seung Hoon Woo. Activation of HER2 changes the cetuximab sensitivity in radioresisitant head and neck cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3940. doi:10.1158/1538-7445.AM2014-3940

Autophagy is frequently activated in radioresistant cancer cells. Rapamycin, mammalian target of rapamycin (mTOR) inhibitor, activates autophagy but enhances radiosensitivity. The mechanism of these actions by which such opposing functions coexist was investigated on radiation-resistant cancer cell lines (AMC-HN-9 and U-87) and the antitumor activity was evaluated in mice bearing xenografts of the cancer cells. Enhanced autophagic flux induced by radiation returned to untreated control levels. Treatment of the cancer cells with rapamycin leads to the potentiation and prolongation of radiation-induced autophagy, the increases in senescence-associated β-galactosidase activity, heterochromatin formation, and irreversible growth arrest. Furthermore, rapamycine resulted in a tumor regrowth delay and increased the level of β-galactosidase staining and the expression of heterochromatin markers in irradiated xenografts. These results suggest that even though autophagy is a survival mechanism in radioresistant cells, a persistent activation of autophagy by mTOR inhibitor induces premature senescence in these cells, eventually making the cells radiosensitive. Our data suggest a novel mechanism by which an inhibition of mTOR pathway increases autophagy but paradoxically increases radiosensitivity in radioresistant cancer cells. Citation Format: Hae Yun Nam, Myung Woul Han, Hyo Won Chang, Yoon Sun Lee, Myungjin Lee, Mi Ra Kim, Hyang Ju Lee, Ji Yung Jeoung, So Young Moon, Hyo Jung Kim, Sang Yoon Kim, Seong Who Kim. Rapamycin increases radiosensitivity of cancer cells by induction of premature senescence. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2013-81

β-Catenin, a multifunctional protein involved in key molecule of Wnt signaling transduction, has been well known to regulate cell proliferation, differentiation, cell-cell adhesion, survival, and apoptosis as well as plays an important role in human tumorigenesis. Our previous report showed that Wnt/β-catenin pathway controls radiosensitivity of head and neck caner (HNC) cell via cyclooxygenase-2-mediated Ku expression. However, the specific molecular mechanisms of β-catenin in tumor cell survival and death are still not fully understood, so in the present study we attempted to elucidate the mechanism of cell death in HNC cell by studying how β-catenin silencing regulates cell death through KB1/AMPK signaling. We performed β-catenin knockdown in HNC cell by using small interfering RNA. Its effect on the cell viability was confirmed by observation of cell morphologic change, electron microscopy, MTT and FACS analysis. Also, we confirmed the expression of several proteins corresponding signal pathway by western blot analysis. β-catenin silencing significantly induced G1 cell-cycle arrest and also increased the expression of Bax and the active form of caspase-3, and decreased the expression of Bcl-2, demonstrating the sequential activation of apoptotic cascades following treatment with β-catenin siRNA in HNC cell. Interestingly, β-catenin silencing induced autophagy as well as apoptosis. We confirmed that the numbers of autophagic vacuoles and the expression of LC3-II increased in cells treated with β-catenin siRNA. This is probably because LKB1-dependent AMPK is activated by β-catenin silencing in HNC cell. Activated AMPK, in β-catenin silenced AMC-HN-3 cells, causes a G1 cell cycle arrest by phosphorylated p53 as well as inhibited protein synthesis by suppressing mTOR activity. Also, Treatment with LKB1 siRNA or compound C in AMC-HN-3 cells preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, our data show that β-catenin silencing induces cell death through LKB1/AMPK signal, and as a result, apoptosis and autophagy are induced together in HNC cell, suggesting that a novel interaction between β-catenin and LKB1/AMPK signaling pathway occurs. Our study provides new insights into understanding the cellular and molecular mechanisms involving β-catenin silencing-induced cell death. 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 2283. doi:1538-7445.AM2012-2283

β1 integrin's aberrant expression has implicated a cancer progression and resistance to cytotoxic therapy. Moreover, cancer cells gained epithelial mesenchymal transition (EMT) features were more resistant to the detachment-induced cell death. In this regard, we investigated the mechanism by which β1-Integrin governs cell viability and verified whether the signaling pathway is associated with EMT features. Cancer cell lines from head and neck cancer patients (AMC-HN3 and AMC-HN9) and well-known EMT cancer, MDA-MB231, were used in this study. To determine whether three cell lines reveal the epithelial EMT features, EMT markers such as Snail, vimentin, fibronectin and E-cadherin were evaluated by western blot analysis and immunofluroscence. To knockdown β1 integrin signal, β1 integrin small interfering RNA (siRNA) was transiently transfected into all cell lines. The cell viability was examined by looking at the results of propidium iodide staining, MTT assay, changes in the mitochondrial membrane potential and cell morphology. MDA-MB231 and AMC-HN9 cells possessed EMT features whereas AMC-HN3 cells were not shown EMT phenotype. β1 integrin silencing cell increased the sub G1 from 1.0% to 29.8%, increased the percentage of cells losing their mitochondrial membrane potential from 5.3% to 65.9% as well as the number of fragmented mitochondria in AMC-HN3 cells. In contrast, MDA-MB231 cells showed more resistance to silencing of β1 integrin, lower cell death rate from 0.6% to 9.6% and less changes in the mitochondrial membrane potential from 4.34% to 34.51%. Interestingly, AMC-HN9 cells showed complete resistance to β1 integrin blocking even though FAK phosphorylation signaling was blocked in AMC-HN9 cells, same as AMC-HN3 cells, which is a different response from typical EMT featured cells. When AMC-HN9 cells were treated to inhibit β1 integrin and p53 separately, it showed no response to changes in the cellular morphology, viability and apoptosis-related signal pathway; however, when it was given the combined β1 integrin and p53, apoptosis occurred in the cell line. In this study, acquisition of the EMT features in cancer cells presented resistance to the β1 integrin inhibition. In addition, the activation of p53-p21 signaling pathway provided resistance to detachment-induced apoptosis in head and neck cancer cell line with EMT features. 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 1226. doi:10.1158/1538-7445.AM2011-1226