Hee Jung Yang

PAK1 tyrosine phosphorylation is required to induce epithelial-mesenchymal transition and radioresistance in lung cancer cells

The p21-activated Ser/Thr kinase 1 (PAK1) kinase has an essential role in tumorigenesis and cell survival in many cancers, but its regulation is not fully understood. In this study, we showed that in response to irradiation of lung cancer cells, PAK1 was upregulated, tyrosine phosphorylated, and translocated to the nucleus. Tyrosine phosphorylation relied upon JAK2 kinase activity and was essential for PAK1 protein stability and binding to Snail. This radiation-induced JAK2-PAK1-Snail signaling pathway increased epithelial-mesenchymal transition (EMT) by regulating epithelial and mesenchymal cell markers. Notably, JAK2 inhibitors mediated radiosensitization and EMT blockade in a mouse xenograft model of lung cancer. Taken together, our findings offered evidence that JAK2 phosphorylates and stabilizes functions of PAK1 that promote EMT and radioresistance in lung cancer cells, with additional implications for the use of JAK2 inhibitors as radiosensitizers in lung cancer treatment.

Phosphorylation of Ribosomal Protein S3 and Antiapoptotic TRAF2 Protein Mediates Radioresistance in Non-small Cell Lung Cancer Cells

Background: Radioresistance is a critical factor restricting efficacy of radiotherapy. Results: Phosphorylation of both rpS3 and TRAF2 induces dissociation of rpS3-TRAF2 complex and influences radioresistance through activation of NF-κB pathway. Conclusion: Phosphorylation of rpS3 and TRAF2 is a key control point of radioresistance in NSCLC cells. Significant: Our findings reveal a novel radioresistance mechanism through functional orchestration of rpS3, TRAF2, and NF-κB in NSCLC cells. Radioresistance is considered as a main factor restricting efficacy of radiotherapy. However, the exact molecular mechanism of radioresistance has not been explained yet. In this study, to elucidate radioresistance mechanism in lung cancer, we compared radiation responses in two types of non-small cell lung cancer (NSCLC) cells with different radiosensitivity and identified key molecules conferring radioresistance. In radioresistant NSCLC cells, ionizing radiation (IR) led to casein kinase 2α (CK2α)- and PKC-mediated phosphorylation of rpS3 and TRAF2, respectively, which induced dissociation of rpS3-TRAF2 complex and NF-κB activation, resulting in significant up-regulation of prosurvival genes (cIAP1, cIAP2, and survivin). Also, dissociated phospho-rpS3 translocated into nucleus and bound with NF-κB complex (p65 and p50), contributing to p65 DNA binding property and specificity. However, in radiosensitive NSCLC cells, IR-mediated rpS3 phosphorylation was not detected due to the absence of CK2α overexpression. Consequently, IR-induced rpS3-TRAF2 complex dissociation, NF-κB activation, and prosurvival gene expression were not presented. Taken together, our findings revealed a novel radioresistance mechanism through functional orchestration of rpS3, TRAF2, and NF-κB in NSCLC cells. Moreover, we provided the first evidence for the function of rpS3 as a new TRAF2-binding protein and demonstrated that phosphorylation of both rpS3 and TRAF2 is a key control point of radioresistance in NSCLC cells. These results suggest that regulation of rpS3 and TRAF2 in combination with radiotherapy could have high pharmacological therapeutic potency for radioresistance of NSCLC.

PIM1 kinase inhibitors induce radiosensitization in non-small cell lung cancer cells

Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the potential involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Consequently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic proteins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradiation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance.

PIM1-activated PRAS40 regulates radioresistance in non-small cell lung cancer cells through interplay with FOXO3a, 14-3-3 and protein phosphatases.

Resistance of cancer cells to ionizing radiation plays an important role in the clinical setting of lung cancer treatment. To date, however, the exact molecular mechanism of radiosensitivity has not been well explained. In this study, we compared radioresistance in two types of non-small cell lung cancer (NSCLC) cells, NCI-H460 and A549, and investigated the signaling pathways that confer radioresistance. In radioresistant cells, exposure to radiation led to overexpression of PIM1 and reduction of protein phosphatases (PP2A and PP5), which induced translocation of PIM1 into the nucleus. Increased nuclear PIM1 phosphorylated PRAS40. Consequently, pPRAS40 made a trimeric complex with 14-3-3 and AKT-activated pFOXO3a, which then moved rapidly to the cytoplasm. Cytoplasmic retention of FOXO3a was associated with downregulation of proapoptotic genes and possibly radioresistance. On the other hand, no suppressive effect of radiation on protein phosphatases was detected and, concomitantly, protein phosphatases downregulated PIM1 in radiosensitive cells. In this setting, PIM1-activated pPRAS40, AKT-activated pFOXO3a, and their complex formation with 14-3-3 could be key regulators of the radiation-induced radioresistance in NSCLC cells.

Investigation of Radiation-induced Transcriptome Profile of Radioresistant Non-small Cell Lung Cancer A549 Cells Using RNA-seq

Radioresistance is a main impediment to effective radiotherapy for non-small cell lung cancer (NSCLC). Despite several experimental and clinical studies of resistance to radiation, the precise mechanism of radioresistance in NSCLC cells and tissues still remains unclear. This result could be explained by limitation of previous researches such as a partial understanding of the cellular radioresistance mechanism at a single molecule level. In this study, we aimed to investigate extensive radiation responses in radioresistant NSCLC cells and to identify radioresistance-associating factors. For the first time, using RNA-seq, a massive sequencing-based approach, we examined whole-transcriptome alteration in radioresistant NSCLC A549 cells under irradiation, and verified significant radiation-altered genes and their chromosome distribution patterns. Also, bioinformatic approaches (GO analysis and IPA) were performed to characterize the radiation responses in radioresistant A549 cells. We found that epithelial–mesenchymal transition (EMT), migration and inflammatory processes could be meaningfully related to regulation of radiation responses in radioresistant A549 cells. Based on the results of bioinformatic analysis for the radiation-induced transcriptome alteration, we selected seven significant radiation-altered genes (SESN2, FN1, TRAF4, CDKN1A, COX-2, DDB2 and FDXR) and then compared radiation effects in two types of NSCLC cells with different radiosensitivity (radioresistant A549 cells and radiosensitive NCI-H460 cells). Interestingly, under irradiation, COX-2 showed the most significant difference in mRNA and protein expression between A549 and NCI-H460 cells. IR-induced increase of COX-2 expression was appeared only in radioresistant A549 cells. Collectively, we suggest that COX-2 (also known as prostaglandin-endoperoxide synthase 2 (PTGS2)) could have possibility as a putative biomarker for radioresistance in NSCLC cells.

Inhibitory effect of traditional oriental medicine-derived monoamine oxidase B inhibitor on radioresistance of non-small cell lung cancer.

Increased survival of cancer cells mediated by high levels of ionizing radiation (IR) reduces the effectiveness of radiation therapy for non-small cell lung cancer (NSCLC). In the present study, danshensu which is a selected component of traditional oriental medicine (TOM) compound was found to reduce the radioresistance of NSCLC by inhibiting the nuclear factor-κB (NF-κB) pathway. Of the various TOM compounds reported to inhibit the IR activation of NF-κB, danshensu was chosen as a final candidate based on the results of structural comparisons with human metabolites and monoamine oxidase B (MAOB) was identified as the putative target enzyme. Danshensu decreased the activation of NF-κB by inhibiting MAOB activity in A549 and NCI-H1299 NSCLC cells. Moreover, it suppressed IR-induced epithelial-to-mesenchymal transition, expressions of NF-κB-regulated prosurvival and proinflammatory genes, and in vivo radioresistance of mouse xenograft models. Taken together, this study shows that danshensu significantly reduces MAOB activity and attenuates NF-κB signaling to elicit the radiosensitization of NSCLC.

Inhibition of hedgehog signalling attenuates UVB-induced skin photoageing

The hedgehog (Hh) signalling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis and progression of a variety of aggressive human cancers including skin cancer. Despite its importance, little is known about its role in photoageing, a type of UV‐induced skin lesions. In this study, we investigated the involvement of Hh signalling in the photoageing process as well as the use of an Hh‐regulating alkaloid compound as a novel therapeutic drug to regulate photoageing in keratinocytes. We found that UVB induced Hh signalling by the expression of Hh ligands and Hh‐mediated transcription factors, respectively. Moreover, UVB‐induced Hh activation relied on mitogen‐activated protein kinase (p38, ERK and JNK) activity and inflammatory responses (upregulation of COX‐2, IL‐1β, IL‐6 and TNF‐α), resulting in premature senescence and photoageing in vitro and in vivo. Notably, a selected Hh inhibitor, evodiamine, mediated photoageing blockade in a mouse skin model. Taken together, our findings demonstrated that Hh signalling is associated with UVB‐induced photoageing, while pharmacological inhibition of Hh signalling significantly reduced experimental photoageing, indicating its potential for use as a therapeutic target for this disease.

Model Systems in Radiation Biology; Implication for Preclinical Study of Radiotherapy

In radiation biology, analysis of various mechanisms in response to radiation has been accomplished with the use of model organisms. These model organisms are powerful tools for providing a biologically intact in vivo environment to assess physiological and pathophysiological processes affected by radiation. Accumulated data using these models have been applied to human clinical studies (including the evaluation of radiotherapeutic efficacy) and discovery of radiotherapy reagents. However, there are few studies to provide overall integrated information about these useful model organisms. Thus, this review summarizes the results of radiation biology studies using four well-known model organisms: yeast, Caenorhabditis elegans, Drosophila melanogaster, and mice.

Chemoprevention of Skin Cancer with Dietary Phytochemicals

Cancer is responsible for a major cause of death in human. It is calculated that more than 11 million people are diagnosed with cancer worldwide (Jemal et al., 2009). Cancer arises through accumulation of multiple genetic alterations. In skin, UV radiation-induced gene mutations have been considered as a driving force of the skin carcinogenesis. Over the past 30 years, ozone depletion has induced increase in the level of UV-B radiation at the earth’s surface. As a result, incidence of the skin cancer has been significantly increased, and it is recognized as a serious public health issue. Many researchers have studied mechanisms of UV-B radiation-induced skin cancer and strategies for skin cancer prevention and treatment. Among the various cancer therapies, chemoprevention is a pharmacological approach using natural, synthetic or biological agents that can prevent, inhibit and reverse the carcinogenic progression. Especially, dietary natural products in chemoprevention have been appreciated as credible components for the management of cancer. Epidemiological studies including more than 250 populations indicated that people who take five different kinds of fruits and vegetables a day showed about 50% decrease in cancer incidence and development than not or less eating plant foods. Based on accumulated researches, dietary plants have been believed to outstanding sources of the cancer preventive substances, and received considerable attention due to their various biological effects – anti-oxidant, antiinflammatory and anti-carcinogenic functions. Therefore, chemoprevention by dietary phytochemicals has been regarded as a new, safety and efficiency strategy for cancer treatment. This chapter gives a useful overview of recent studies in chemoprevention of skin cancer with dietary phytochemicals, and especially, focuses on UV-B radiation as a major factor of skin cancer and summarizes the UV-B radiation-induced skin carcinogenic mechanism.

TRAF4 promotes lung cancer aggressiveness by modulating tumor microenvironment in normal fibroblasts

Normal fibroblasts surrounding tumor cells play a crucial role in cancer progression through formation of the tumor microenvironment. Because factors secreted from normal fibroblasts can modulate the tumor microenvironment, it is necessary to identify key factors associated with regulation of secreted factors and to investigate the molecular mechanisms contributing to the tumor microenvironment formation process. In this study, we found that radiation induced the expression and K63-linkage poly-ubiquitination of TRAF4 in normal lung fibroblasts. The K63-linkage poly-ubiquitinated TRAF4 formed complexes with NOX2 or NOX4 by mediating phosphorylated p47-phox in normal lung fibroblasts. Moreover, we showed that TRAF4 stabilized NOX complexes by decreasing lysosomal degradation of NOX2 and NOX4 after irradiation. NOX complexes increased endosomal ROS levels that were permeable into cytoplasm, leading to NF-κB-mediated ICAM1 up-regulation. Soluble ICAM1 was subsequently secreted into conditioned media of radiation-activated normal lung fibroblasts. The conditioned media from irradiated normal fibroblasts enhanced proliferation and epithelial-mesenchymal transition of non-small cell lung cancer cells both in vitro and in vivo. These results demonstrate that TRAF4 in irradiated fibroblasts is positively associated with aggressiveness of adjacent cancer cells by altering the tumor microenvironment. Thus, we suggest that regulation of TRAF4 might be a promising strategy for cancer therapy.