Hyo Won Chang

Wnt signaling controls radiosensitivity via cyclooxygenase-2-mediated Ku expression in head and neck cancer

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 β‐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 β‐catenin‐induced Ku expression, and to consequent recovery of the radiosensitivity in HNC cells. Our results conclusively suggest that β‐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.

Radioresistant Cancer Cells Can Be Conditioned to Enter Senescence by mTOR Inhibition

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.

Peroxiredoxin IV Protects Cells From Radiation-Induced Apoptosis in Head-and-Neck Squamous Cell Carcinoma

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.

Prolonged autophagy by MTOR inhibitor leads radioresistant cancer cells into senescence

Radiotherapy is one of the well-established therapeutic modalities for cancer treatment. However, the emergence of cells refractory to radiation is a major obstacle to successful treatment with radiotherapy. Many reports suggest that inhibitors targeting the mechanistic target of rapamycin (MTOR) can sensitize cancer cells to the effect of radiation, although by which mechanism MTOR inhibitors enhance the efficacy of radiation toward cancer cells remains to be elucidated. Our studies indicate that a potent and persistent activation of autophagy via inhibition of the MTOR pathway, even in cancer cells where autophagy is occurring, can trigger premature senescence, cellular proliferation arrest. Combined treatment of MTOR inhibitor and radiation induce heterochromatin formation, an irreversible growth arrest and an increase of senescence-associated GLB1 (β-galactosidase) activity, which appear to result from a constant activation of TP53 and a restoration in the activity of retinoblastoma 1 protein (RB1)-E2F1. Thus, this study provides evidence that promoting cellular senescence via inhibition of the MTOR pathway may serve as an avenue to augment radiosensitivity in cancer cells that initiate an autophagy-survival mode to radiotherapy.

Proteomic analysis of two head and neck cancer cell lines presenting different radiation sensitivity

Conclusion. 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. Objective. 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. Materials and methods. 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. Results. 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.

Abstract 1226: p53 signaling pathway acts as rescue mechanism to β1 integrin silencing in head and neck cancer cell line

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL β1 integrins 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

EphA3 maintains radioresistance in head and neck cancers through epithelial mesenchymal transition

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.

Abstract 81: Rapamycin increases radiosensitivity of cancer cells by induction of premature senescence.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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