Phase-separated super-enhancers confer an innate radioresistance on genomic DNA.

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

Generality Assessment of a Model Considering Heterogeneous Cancer Cells for Predicting Tumor Control Probability for Stereotactic Body Radiation Therapy Against Non-Small Cell Lung Cancer.

The generality of a model for predicting tumor control probability from in vitro clonogenic survival considering of cancer stem-like cells, the so-called integrated microdosimetric-kinetic model, is presented by comparing the model to public data on stereotactic body radiation therapy for non-small cell lung cancer cells.

Involvement of retinoic acid­inducible gene­I in radiation­induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated ß-galactosidase (SA-ß-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-ß-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.

ΔNp63 Regulates Radioresistance in Human Head and Neck Squamous Carcinoma Cells.

Radiation therapy is commonly used to treat head and neck squamous cell carcinoma (HNSCC); however, recurrence results from the development of radioresistant cancer cells. Therefore, it is necessary to identify the underlying mechanisms of radioresistance in HNSCC. Previously, we showed that the inhibition of karyopherin-ß1 (KPNB1), a factor in the nuclear transport system, enhances radiation-induced cytotoxicity, specifically in HNSCC cells, and decreases the localization of SCC-specific transcription factor ΔNp63. This suggests that ΔNp63 may be a KPNB1-carrying nucleoprotein that regulates radioresistance in HNSCC. Here, we determined whether ΔNp63 is involved in the radioresistance of HNSCC cells. Cell survival was measured by a colony formation assay. Apoptosis was assessed by annexin V staining and cleaved caspase-3 expression. The results indicate that ΔNp63 knockdown decreased the survival of irradiated HNSCC cells, increased radiation-induced annexin V+ cells, and cleaved caspase-3 expression. These results show that ΔNp63 is involved in the radioresistance of HNSCC cells. We further investigated which specific karyopherin-α (KPNA) molecules, partners of KPNB1 for nuclear transport, are involved in nuclear ΔNp63 expression. The analysis of nuclear ΔNp63 protein expression suggests that KPNA1 is involved in nuclear ΔNp63 expression. Taken together, our results suggest that ΔNp63 is a KPNB1-carrying nucleoprotein that regulates radioresistance in HNSCC.

Prediction of hub genes and key pathways associated with the radiation response of human hematopoietic stem/progenitor cells using integrated bioinformatics methods.

Hematopoietic stem cells (HSCs) are indispensable for the maintenance of the entire blood program through cytokine response. However, HSCs have high radiosensitivity, which is often a problem during radiation therapy and nuclear accidents. Although our previous study has reported that the combination cytokine treatment (interleukin-3, stem cell factor, and thrombopoietin) improves the survival of human hematopoietic stem/progenitor cells (HSPCs) after radiation, the mechanism by which cytokines contribute to the survival of HSPCs is largely unclear. To address this issue, the present study characterized the effect of cytokines on the radiation-induced gene expression profile of human CD34+ HSPCs and explored the hub genes that play key pathways associated with the radiation response using a cDNA microarray, a protein-protein interaction-MCODE module analysis and Cytohubba plugin tool in Cytoscape. This study identified 2,733 differentially expressed genes (DEGs) and five hub genes (TOP2A, EZH2, HSPA8, GART, HDAC1) in response to radiation in only the presence of cytokines. Furthermore, functional enrichment analysis found that hub genes and top DEGs based on fold change were enriched in the chromosome organization and organelle organization. The present findings may help predict the radiation response and improve our understanding of this response of human HSPCs.

Translational study for stereotactic body radiotherapy against non-small cell lung cancer, including oligometastases, considering cancer stem-like cells enable predicting clinical outcome from in vitro data.

BACKGROUND: Curative effects of stereotactic body radiotherapy (SBRT) for non-small cell lung cancer (NSCLC) have been evaluated using various biophysical models. Because such model parameters are empirically determined based on clinical experience, there is a large gap between in vitro and clinical studies. In this study, considering the heterogeneous cell population, we performed a translational study to realize the possible linkage based on a modeling approach. METHODS: We modeled cell-killing and tumor control probability (TCP) considering two populations: progeny and cancer stem-like cells. The model parameters were determined from in vitro survival data of A549 and EBC-1 cells. Based on the cellular parameters, we predicted TCP and compared it with the corresponding clinical data from 553 patients collected at Hirosaki University Hospital. RESULTS: Using an all-in-one developed model, the so-called integrated microdosimetric-kinetic (IMK) model, we successfully reproduced both in vitro survival after acute irradiation and the 3-year TCP with various fractionation schemes (6-10 Gy per fraction). From the conventional prediction without considering cancer stem cells (CSCs), this study revealed that radioresistant CSCs play a key role in the linkage between in vitro and clinical outcomes. CONCLUSIONS: This modeling study provides a possible generalized biophysical model that enables precise estimation of SBRT worldwide.

DAP3-mediated cell cycle regulation and its association with radioresistance in human lung adenocarcinoma cell lines.

Mitochondria play important roles in the cellular response to various types of stress, including that triggered by ionizing radiation. We have previously reported that the mitochondrial ribosomal protein death-associated protein 3 (DAP3) regulates the radioresistance of human lung adenocarcinoma (LUAD) cell lines A549 and H1299. However, the underlying mechanism of this regulation remains to be elucidated. To this end, we have herein investigated the role of DAP3 in the cell cycle regulation after irradiation. Notably, the DAP3 knockdown attenuated the radiation-induced increase of the G2/M cell population. Furthermore, western blotting analysis has revealed that the DAP3 knockdown decreased the expression of proteins related to the G2/M arrest, such as those of the phosphorylated cdc2 (Tyr15) and the phosphorylated checkpoint kinase 1 (Ser296), in irradiated A549 cells and H1299 cells. Moreover, by using a chk1 inhibitor, we were able to demonstrate that chk1 is involved in the radiation-induced G2/M arrest in both A549 and H1299 cells. Notably, the chk1 inhibitor was able to enhance the radiosensitivity of H1299 cells, while both chk1 inhibitor-abolished G2 arrest and inhibition of chk2-mediated events such as downregulation of radiation-induced p21 expression were required for enhancing radiosensitivity of A549 cells. Collectively, our findings reveal a novel role of DAP3 to regulate G2/M arrest through pchk1 in irradiated LUAD cells and suggest that chk1-mediated G2/M arrest regulates the radioresistance of H1299 cells, whereas both the chk1-mediated G2/M arrest and the chk2-mediated events contribute to the radioresistance of A549 cells.

Cytotoxic Activity of Unique Synthesized Five-membered Heterocyclic Compounds Coordinated with Tiopronin Monovalent.

BACKGROUND: We recently synthesized a compound in which 5-mercapto-1-methyltetrazole (MM4) was coordinated to tiopronin monovalent (TPN-Au(I)) and reported its cytotoxic activity against human leukemia cells in vitro. OBJECTIVE: We further synthesized other heterocyclic compounds coordinated with TPN-Au(I) and assessed their cytotoxic activity against hepatocellular carcinoma HepG2 and lung cancer cell line H1299 in vitro. METHODS: Seven kinds of compounds were synthesized by introducing a five-membered heterocyclic compound into TPN-Au(I). The number of viable cells was counted by a trypan blue dye exclusion assay. Fluorescence conjugated-Annexin V and propidium iodide were used for the apoptosis analysis. RESULTS: Seven compounds were successfully synthesized. Among these compounds, TPN-Au(I)-MTZ (3- mercapto-1,2,4-triazole), TPN-Au(I)-MMT (2-mercapto-5-methyl-1,3,4-thiadiazole), and TPN-Au(I)-MMTT (2-mercapto-5-methylthio-1,3,4-thiadiazole) effectively suppressed the proliferation and induced apoptosis in HepG2 cells. In addition, TPN-Au(I)-MMTT and TPN-Au(I)-MMT also showed effective cytotoxicity against H1299 cells. CONCLUSION: The present results showed that introduction of some five-membered heterocyclic compounds, especially MMT and MMTT, to TPN-Au(I) improved the cytotoxicity against solid cancer cells.

cGAS Regulates the Radioresistance of Human Head and Neck Squamous Cell Carcinoma Cells.

Cyclic GMP-AMP synthase (cGAS) plays an important role in biological responses to pathogens. The activation of the cGAS pathway in immune cells is known to induce antitumor effects, but the role of cGAS in cancer cells remains poorly understood. In silico analysis using public databases suggested that high cGAS expression in head and neck squamous cell carcinoma (HNSCC) is indicative of a poor prognosis for HNSCC patients. We therefore investigated the role of cGAS in malignancies and the cellular radiation response of human HNSCC cells (SAS and Ca9-22) in vitro, because radiotherapy is one of the treatments most commonly used for HNSCC. Although cGAS knockdown failed to suppress the proliferation of non-irradiated HNSCC cells, it enhanced the radiosensitivity of HNSCC cells. The administration of the cGAS agonist increased the radioresistance of HNSCC cells. cGAS knockdown increased radiation-induced mitotic catastrophe, apoptosis, or cellular senescence, depending on the cell line, and this cell line-dependent response might be due to different responses of p21 after irradiation. Collectively, our findings indicate that the cGAS pathway regulates the radioresistance of HNSCC cells.

Cytotoxic Activity of a Unique Monomeric Heterogeneous Two-Coordinate Ligand Monovalent Gold Complex with Tiopronin and a Heterocyclic Mercapto-Tetrazole Compound.

BACKGROUND: The importance of the role of NF-κB is recognized in situations such as malignant transformation and metastasis of cancer, and it has been suggested that inhibiting this role can be one of the cancer treatment strategies. Gold preparations such as auranofin are known to have an indirect NF-κB inhibitory effect. OBJECTIVE: We synthesized a novel gold complex [tiopronin monovalent gold-5-mercapto- 1-methyl tetrazole, abbreviated as TPN-Au(I)-MM4], with different physical properties and chemical structure from auranofin, and evaluated its cytotoxic activity and radiation sensitizing effect on human THP1 cells. METHODS: The number of viable cells was counted by the trypan blue dye exclusion method. The cell death evaluation was performed by FITC-Annexin V+ and PI staining. In investigating the radiation sensitizing effect of TPN-Au(I)-MM4, this compound [10 or 25 µM] was added into the culture medium 1 h before X-ray irradiation. RESULTS: In the cells treated with 25 µM TPN-Au(I)-MM4 for 72 h, a decrease in the proliferation of THP1 cells was observed [The relative values of viable cells in the control group and the 25 µM treatment group were approximately 6.8 and 4.2, respectively]. In the combination of 25 µM of the compound treatment and X-ray irradiation, an increase of approximately 3.0-fold was observed in 2 Gy irradiation and approximately 1.4-fold in 4 Gy irradiation as in comparison to the case of irradiation alone. CONCLUSION: These results suggest that TPN-Au(I)-MM4 reduces the proliferation of THP1 cells through the induction of cell death, and the combined use of TPN-Au(I)-MM4 and X-ray irradiation shows effective cytotoxicity against THP1 cells.

Discovery of a Novel Small-molecule Interleukin-6 Inhibitor Through Virtual Screening Using Artificial Intelligence.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine involved in various cell functions and diseases. Thus far, several IL-6 inhibitors, such as humanized monoclonal antibody have been used to block excessive IL-6 signaling causing autoimmune and inflammatory diseases. However, anti-IL-6 and anti-IL-6 receptor monoclonal antibodies have some clinical disadvantages, such as a high cost, unfavorable injection route, and tendency to mask infectious diseases. While a small-molecule IL-6 inhibitor would help mitigate these issues, none are currently available. OBJECTIVE: The present study evaluated the biological activities of identified compounds on IL-6 stimulus. METHODS: We virtually screened potential IL-6 binders from a compound library using INTerprotein's Engine for New Drug Design (INTENDD®) followed by the identification of more potent IL-6 binders with artificial intelligence (AI)-guided INTENDD®. The biological activities of the identified compounds were assessed with the IL-6-dependent cell line 7TD1. RESULTS: The compounds showed the suppression of IL-6-dependent cell growth in a dose-dependent manner. Furthermore, the identified compound inhibited expression of IL-6-induced phosphorylation of signal transducer and activator of transcription 3 in a dose-dependent manner. CONCLUSION: Our screening compound demonstrated an inhibitory effect on IL-6 stimulus. These findings may serve as a basis for the further development of small-molecule IL-6 inhibitors.

The Effect of High-Dose-Rate Pulsed Radiation on the Survival of Clinically Relevant Radioresistant Cells.

We demonstrated that low dose pulsed radiation (0.25 Gy) at a high-dose-rate, even for very short intervals (10 s), decreases cell survival to a greater extent than single exposure to a similar total dose and dose rate. The objective of this study was to clarify whether high-dose-rate pulsed radiation is effective against SAS-R, a clinically relevant radioresistant cell line. Cell survival following high-dose-rate pulsed radiation was evaluated via a colony assay. Flow cytometry was utilized to evaluate γH2AX, a molecular marker of DNA double-strand breaks and delayed reactive oxygen species (ROS) associated with radiation-induced apoptosis. Increased cytotoxicity was observed in SAS-R and parent SAS cells in response to high dose rate pulsed radiation compared to single dose, as determined by colony assays. Residual γH2AX in both cells subjected to high-dose-rate pulsed radiation showed a tendency to increase, with a significant increase observed in SAS cells at 72 h. In addition, high-dose-rate pulsed radiation increased delayed ROS more than the single exposure did. These results indicate that high-dose-rate pulsed radiation was associated with residual γH2AX and delayed ROS, and high-dose-rate pulsed radiation may be used as an effective radiotherapy procedure against radioresistant cells.

Effects and Related Mechanisms of the Senolytic Agent ABT-263 on the Survival of Irradiated A549 and Ca9-22 Cancer Cells.

Senolytic agents eliminate senescent cells and are expected to reduce senescent cell-mediated adverse effects in cancer therapy. However, the effects of senolytic agents on the survival of irradiated cancer cells remain unknown. Here, the effects of the senolytic agent ABT-263 on the survival of irradiated A549 and Ca9-22 cancer cells were investigated. ABT-263 was added to the culture medium after irradiation. SA-ß-gal activity and cell size, which are hallmarks of cell senescence, were evaluated using a flow cytometer. The colony-forming assay and annexin V staining were performed to test cell survival. We first confirmed that radiation increased the proportion of cells with high SA-ß-gal activity and that ABT-263 decreased it. Of note, ABT-263 decreased the survival of irradiated cancer cells and increased the proportion of radiation-induced annexin V+ cells. Furthermore, the caspase inhibitor suppressed the ABT-263-induced decrease in the survival of irradiated cells. Intriguingly, ABT-263 decreased the proportion of SA-ß-gal low-activity/large cells in the irradiated A549 cells, which was recovered by the caspase inhibitor. Together, these findings suggest that populations maintaining the ability to proliferate existed among the irradiated cancer cells showing senescence-related features and that ABT-263 eliminated the population, which led to decreased survival of irradiated cancer cells.

KPNB1 Inhibitor Importazole Reduces Ionizing Radiation-Increased Cell Surface PD-L1 Expression by Modulating Expression and Nuclear Import of IRF1.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that negatively regulates anti-tumor immunity. Recent reports indicate that anti-cancer treatments, such as radiation therapy, increase PD-L1 expression on the surface of tumor cells. We previously reported that the nuclear transport receptor karyopherin-ß1 (KPNB1) is involved in radiation-increased PD-L1 expression on head-and-neck squamous cell carcinoma cells. However, the mechanisms underlying KPNB1-mediated, radiation-increased PD-L1 expression remain unknown. Thus, the mechanisms of radiation-increased, KPNB1-mediated PD-L1 expression were investigated by focusing on the transcription factor interferon regulatory factor 1 (IRF1), which is reported to regulate PD-L1 expression. Western blot analysis showed that radiation increased IRF1 expression. In addition, flow cytometry showed that IRF1 knockdown decreased cell surface PD-L1 expression of irradiated cells but had a limited effect on non-irradiated cells. These findings suggest that the upregulation of IRF1 after irradiation is required for radiation-increased PD-L1 expression. Notably, immunofluorescence and western blot analyses revealed that KPNB1 inhibitor importazole not only diffused nuclear localization of IRF1 but also decreased IRF1 upregulation by irradiation, which attenuated radiation-increased PD-L1 expression. Taken together, these findings suggest that KPNB1 mediates radiation-increased cell surface PD-L1 expression through both upregulation and nuclear import of IRF1.

Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules In Vitro.

The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm2 significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.

DAP3 Is Involved in Modulation of Cellular Radiation Response by RIG-I-Like Receptor Agonist in Human Lung Adenocarcinoma Cells.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) mediate anti-viral response through mitochondria. In addition, RLR activation induces anti-tumor effects on various cancers. We previously reported that the RLR agonist Poly(I:C)-HMW/LyoVec™ (Poly(I:C)) enhanced radiosensitivity and that cotreatment with Poly(I:C) and ionizing radiation (IR) more than additively increased cell death in lung adenocarcinoma cells, indicating that Poly(I:C) modulates the cellular radiation response. However, it remains unclear how mitochondria are involved in the modulation of this response. Here, we investigated the involvement of mitochondrial dynamics and mitochondrial ribosome protein death-associated protein 3 (DAP3) in the modulation of cellular radiation response by Poly(I:C) in A549 and H1299 human lung adenocarcinoma cell lines. Western blotting revealed that Poly(I:C) decreased the expression of mitochondrial dynamics-related proteins and DAP3. In addition, siRNA experiments showed that DAP3, and not mitochondrial dynamics, is involved in the resistance of lung adenocarcinoma cells to IR-induced cell death. Finally, we revealed that a more-than-additive effect of cotreatment with Poly(I:C) and IR on increasing cell death was diluted by DAP3-knockdown because of an increase in cell death induced by IR alone. Together, our findings suggest that RLR agonist Poly(I:C) modulates the cellular radiation response of lung adenocarcinoma cells by downregulating DAP3 expression.

Karyopherin-ß1 Regulates Radioresistance and Radiation-Increased Programmed Death-Ligand 1 Expression in Human Head and Neck Squamous Cell Carcinoma Cell Lines.

Nuclear transport receptors, such as karyopherin-ß1 (KPNB1), play important roles in the nuclear-cytoplasmic transport of macromolecules. Recent evidence indicates the involvement of nuclear transport receptors in the progression of cancer, making these receptors promising targets for the treatment of cancer. Here, we investigated the anticancer effects of KPNB1 blockage or in combination with ionizing radiation on human head and neck squamous cell carcinoma (HNSCC). HNSCC cell line SAS and Ca9-22 cells were used in this study. Importazole, an inhibitor of KPNB1, or knockdown of KPNB1 by siRNA transfection were applied for the blockage of KPNB1 functions. The roles of KPNB1 on apoptosis induction and cell surface expression levels of programmed death-ligand 1 (PD-L1) in irradiated HNSCC cells were investigated. The major findings of this study are that (i) blockage of KPNB1 specifically enhanced the radiation-induced apoptosis and radiosensitivity of HNSCC cells; (ii) importazole elevated p53-upregulated modulator of apoptosis (PUMA) expression via blocking the nuclear import of SCC-specific oncogene ΔNp63 in HNSCC cells; and (iii) blockage of KPNB1 attenuated the upregulation of cell surface PD-L1 expression on irradiated HNSCC cells. Taken together, these results suggest that co-treatment with KPNB1 blockage and ionizing radiation is a promising strategy for the treatment of HNSCC.

Fas Ligand Enhances Apoptosis of Human Lung Cancer Cells Cotreated with RIG-I-like Receptor Agonist and Radiation.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play key roles in the antiviral response, but recent works show that RLR activation elicits anticancer activity as well, including apoptosis. Previously, we demonstrated that the anticancer activity of the RLR agonist Poly(I:C)-HMW/LyoVec™ [Poly(I:C)-HMW] against human lung cancer cells was enhanced by cotreatment with ionizing radiation (IR). In addition, cotreatment with Poly(I:C)-HMW and IR induced apoptosis in a Fas-independent manner, and increased Fas expression on the cell surface. OBJECTIVE: The current study investigated the resultant hypothesis that Fas ligand (FasL) may enhance apoptosis in lung cancer cells cotreated with Poly(I:C)-HMW+IR. METHODS: FasL was added into culture medium at 24 h following cotreatment with Poly(I:C)- HMW+IR, after upregulation of cell surface Fas expression on human lung cancer cells A549 and H1299 have already been discussed. RESULTS: FasL enhanced the apoptosis of A549 and H1299 cells treated with Poly(I:C)-HMW+IR. Similarly, IR alone - and not Poly(I:C)-HMW - resulted in the upregulation of cell surface Fas expression followed by a high response to FasL-induced apoptosis, thus suggesting that the high sensitivity of cells treated with Poly(I:C)-HMW+IR to FasL-induced apoptosis resulted from the cellular response to IR. Finally, knockdown of Fas by siRNA confirmed that the high response of treated cells to FasL-induced apoptosis is dependent on Fas expression. CONCLUSION: In summary, the present study indicates that upregulated Fas expression following cotreatment with Poly(I:C)-HMW and IR is responsive to FasL-induced apoptosis, and a combination of RLR agonist, IR, and FasL could be a potential promising cancer therapy.

Disease-specific alteration of karyopherin-α subtype establishes feed-forward oncogenic signaling in head and neck squamous cell carcinoma.

Nuclear import, mediated in part by karyopherin-α (KPNA)/importin-α subtypes, regulates transcription factor access to the genome and determines cell fate. However, the cancer-specific changes of KPNA subtypes and the relevancy in cancer biology remain largely unknown. Here, we report that KPNA4, encoding karyopherin-α4 (KPNA4), is exclusively amplified and overexpressed in head and neck of squamous cell carcinoma (HNSCC). Depletion of KPNA4 attenuated nuclear localization signal-dependent transport activity and suppressed malignant phenotypes and induced epidermal differentiation. Mechanistically, KPNA4-mediated nuclear transport of Ras-responsive element-binding protein (RREB1), which sustains Ras/ERK pathway signaling through repressing miR-143/145 expression. Notably, MAPK signaling enhanced trafficking activity of KPNA4 via phosphorylation of KPNA4 at Ser60. These data reveal that KPNA4 establishes a feed-forward cascade that potentiates Ras/ERK signaling in HNSCC.