Radioresistance In Lung Cancer Research Articles

Deregulated BCL-2 family proteins impact on repair of DNA double-strand breaks and are targets to overcome radioresistance in lung cancer.

DNA damage-induced cell death is a major effector mechanism of radiotherapy. Aberrant expression of anti-apoptotic BCL-2 family proteins is frequently observed in lung cancers. Against this background, we studied radioresistance mediated by BCL-2 family proteins at the mechanistic level and its potential as target for radiochemotherapy. Lung cancer models stably expressing BCL-xL or MCL-1 were irradiated to study cell death, clonogenic survival, and DNA repair kinetics invitro, and growth suppression of established tumors invivo. Additionally, endogenous BCL-xL and MCL-1 were targeted by shRNA or pharmacologic agents prior to irradiation. Radiation exposure induced apoptosis at negligible levels. Yet, anti-apoptotic BCL-xL and MCL-1 expression conferred short-term and long-term radioresistance invitro and invivo. Radioresistance correlated with pertubations in homologous recombination repair and repair of DNA double-strand breaks by error-prone, alternative end-joining. Notably, genetic or pharmacologic targeting of BCL-xL or MCL-1 effectively sensitized lung cancer cells to radiotherapy. In addition to directly suppressing apoptosis, BCL-2 family proteins confer long-term survival benefits to irradiated cancer cells associated with utilization of error-prone repair pathways. Targeting BCL-xL and MCL-1 is an attractive strategy for improving lung cancer radiotherapy.

Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca2+) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca2+) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca2+ concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca2+ concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca2+ could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

IL-6 signaling promotes DNA repair and prevents apoptosis in CD133+ stem-like cells of lung cancer after radiation.

BackgroundLocal tumor control by standard fractionated radiotherapy (RT) remains poor because of tumor resistance to radiation (radioresistance). It has been suggested that cancer stem cells (CSCs) are more radioresistant than non-CSCs. In previous studies, we have shown IL-6 promotes self-renewal of CD133+ CSC-like cells. In this study, we investigated whether IL-6 plays roles not only in promoting self-renewal of CD133+ cells after radiation, but also in conferring radioresistance of CD133+ cells in NSCLC.Materials and methodsTo compare radiation sensitivity of CSCs and non-CSCs, CD133+ CSC-like and CD133- cell populations were isolated from two NSCLC cell lines, A549 and H157, by immunomagnetic separation and their sensitivities to ionizing radiation were investigated using the clonogenic survival assay. To further study the IL-6 effect on the radiosensitivity of CD133+ CSC-like cells, CD133+ cells were isolated from A549IL-6si/sc and H157IL-6si/sc cells whose intracellular IL-6 levels were manipulated via the lentiviral transduction with IL-6siRNA. Post-irradiation DNA damage was analyzed by γ-H2AX staining and Comet assay. Molecular mechanisms by which IL-6 regulates the molecules associated with DNA repair and anti-apoptosis after radiation were analyzed by Western blot and immunofluoresecence (IF) staining analyses.ResultsNSCLC CD133+ CSC-like cells were enriched upon radiation. Survival of NSCLC CD133+ cells after radiation was higher than that of CD133- cells. Survival of IL-6 expressing NSC LC CD133+ cells (sc) was higher than that of IL-6 knocked-down cells (IL-6si) after radiation. IL-6 played a role in protecting NSCLC CD133+ cells from radiation-induced DNA damage and apoptosis.ConclusionsIL-6 signaling promotes DNA repair while protecting CD133+ CSC-like cells from apoptotic death after radiation for lung cancer. A combined therapy of radiation and agents that inhibit IL-6 signaling (or its downstream signaling) is suggested to reduce CSC-mediated radioresistance in lung cancer.

Abstract 3326: Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers

Abstract Therapies for poor prognosis cancers, such as lung cancer and glioblastoma, are limited due to radio-resistance and tumor recurrence. Development of molecular targeted therapy can serve as a potential method to improve the efficacy of radiation therapy in both glioblastoma and lung cancer. Ionizing radiation (IR) can activate a series of pro-survival pathways which contributes to the pathogenesis of cancer cells. Among these pathways, cytosolic phospholipase A2 (cPLA2) is an integral component which is activated by IR. Following activation, cPLA2 cleaves arachidonic acid to form phosphatidylcholine (PC) and yields lysophosphatidylcholine (LPC). Autotaxin (ATX) then converts LPC to lysophosphatidic acid (LPA). LPA acts through G-protein-coupled receptors (GPCR) such as LPA1, LPA2, and LPA3, influencing various fundamental cellular functions. Specifically, these receptors can modulate the ability of cancer cells to proliferate, differentiate, and survive. Earlier we have shown that inhibition of ATX decreases invasion and enhances radiation sensitivity of cancer cells. In the present study we investigated the role of LPA receptors and its effect on radioresistance in human A549, murine Lewis Lung Carcinoma (LLC), and rat C6 glioma cells. We analyzed the expression levels of the three LPA receptors in various cancer cell lines including lung, brain, breast and pancreatic cancer cells. We found high expression of LPA1 in LLC, C6 and A549, which we thus chose for further study. Using inhibitors of LPA1/LPA3, Ki16425 and VPC 12249, we determined the effect of LPA1 inhibition on the AKT and ERK activation. Inhibition of LPA1 attenuated phosphorylation of both AKT and ERK in irradiated lung cancer cells. This implicated LPA1 in the regulation of pro-survival signaling, and we hypothesized that LPA1 regulation could influence radioresistance in lung cancer and glioma cells. We targeted LPA1 using siRNA in LLC, C6 and A549 cells which led to an average of 50% reduction in clonogenic survival and cell proliferation after irradiation. Similar results were obtained when LLC and A549 cells were treated with 10μM Ki16425 or 10μM VPC 12249. In addition, LLC and A549 cells which were treated with combinations of radiation and LPA1 inhibitors and analyzed using cell invasion assays showed a 70% reduction in cancer cell invasion. These results indicate that LPA1 could serve as a novel target for cancer treatment due to its ability to enhance the efficacy of radiotherapy in multiple cancer cell lines. Citation Format: Arpine Khudanyan, David Dadey, Rowan Karvas, Rama Kotipatruni, Dennis Hallahan, Dinesh Thotala. Targeting lysophosphatidic acid receptor 1 (LPAR1) radiosensitizes poor prognosis cancers. [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 3326. doi:10.1158/1538-7445.AM2015-3326

Abnormal Expression of miR-21 and miR-95 in Cancer Stem-Like Cells is Associated with Radioresistance of Lung Cancer

This study demonstrated that miR-21 and miR-95 expression were significantly higher in the ALDH1+CD133+subpopulation than in the ALDH1―CD133― subpopulation of lung cancer cells. Combined delivery of anti-miR-21 and anti-miR-95 by calcium phosphate nanoparticles significantly inhibited tumor growth in a xenograft tumor model and sensitized radiotherapy. The anti-miRNAs significantly reduced miR-21 and miR-95 levels, increased PTEN, SNX1, and SGPP1 protein expression, but reduced Akt Ser473 and Thr308 phosphorylation. ALDH1+CD133+ subpopulation of NSCLC tumor cells confers radioresistance due to high expression of miR-21 and miR-95. Targeting inhibition of miR-21 and miR-95 can inhibit tumor growth through elevating PTEN, SNX1, and SGPP1 expression and inhibiting Akt phosphorylation.

Regulation of miRNAs affects radiobiological response of lung cancer stem cells.

Radiotherapy (RT) is a key therapeutic strategy for lung cancer, the most common cause of cancer-related deaths worldwide, but radioresistance often occurs and leads to failure of RT. It is therefore important to clarify the mechanism underlying radioresistance in lung cancer. Cancer stem cells (CSCs) are considered the fundamental reason for radioresistance. MicroRNAs (miRNAs) have been regarded as important regulatory molecules of CSCs, carcinogenesis, and treatment response of cancers. It is crucial to clarify how regulation of miRNAs affects repair of DNA damage, redistribution, repopulation, reoxygenation, and radiosensitivity (5R) of lung cancer stem cells (LCSCs). A thorough understanding of the regulation of miRNAs affecting 5R of LCSCs has potential impact on identifying novel targets and thus may improve the efficacy of lung cancer radiotherapy.

Disruption of STAT3 by Niclosamide Reverses Radioresistance of Human Lung Cancer

A major challenge affecting the outcomes of patients with lung cancer is the development of acquired radioresistance. However, the mechanisms underlying the development of resistance to therapy are not fully understood. Here, we discovered that ionizing radiation induces phosphorylation of Janus-associated kinase (JAK)-2 and STAT3 in association with increased levels of Bcl2/Bcl-XL in various human lung cancer cells. To uncover new mechanism(s) of radioresistance of lung cancer, we established lung cancer cell model systems with acquired radioresistance. As compared with radiosensitive parental lung cancer cells (i.e., A549, H358, and H157), the JAK2/STAT3/Bcl2/Bcl-XL survival pathway is significantly more activated in acquired radioresistant lung cancer cells (i.e., A549-IRR, H358-IRR, and H157-IRR). Higher levels of STAT3 were found to be accumulated in the nucleus of radioresistant lung cancer cells. Niclosamide, a potent STAT3 inhibitor, can reduce STAT3 nuclear localization in radioresistant lung cancer cells. Intriguingly, either inhibition of STAT3 activity by niclosamide or depletion of STAT3 by RNA interference reverses radioresistance in vitro. Niclosamide alone or in combination with radiation overcame radioresistance in lung cancer xenografts. These findings uncover a novel mechanism of radioresistance and provide a more effective approach to overcome radioresistance by blocking the STAT3/Bcl2/Bcl-XL survival signaling pathway, which may potentially improve lung cancer outcome, especially for those patients who have resistance to radiotherapy.

Novel Small-Molecule Inhibitors of Bcl-XL to Treat Lung Cancer

Bcl-XL is a major antiapoptotic protein in the Bcl-2 family whose overexpression is more widely observed in human lung cancer cells than that of Bcl-2, suggesting that Bcl-XL is more biologically relevant and therefore a better therapeutic target for lung cancer. Here, we screened small molecules that selectively target the BH3 domain (aa 90-98) binding pocket of Bcl-XL using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We identified two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells compared with normal human bronchial epithelial cells. Fluorescence polarization assay reveals that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein but not Bcl2, Bcl-w, Bfl-1/A1, or Mcl-1 in vitro with high binding affinities. Treatment of cells with BXI-72 results in disruption of Bcl-XL/Bak or Bcl-XL/Bax interaction, oligomerization of Bak, and cytochrome c release from mitochondria. Importantly, BXI-61 and BXI-72 exhibit more potent efficacy against human lung cancer than ABT-737 but less degree in platelet reduction in vivo. BXI-72 overcomes acquired radioresistance of lung cancer. On the basis of our findings, the development of BXI(s) as a new class of anticancer agents is warranted and represents a novel strategy for improving lung cancer outcome.

Inhibition of SENP1 induces radiosensitization in lung cancer cells

Lung cancer is one of the most common and lethal types of malignancy. To date, radiotherapy and chemotherapy have been used as the two major treatment methods. However, radioresistance of lung cancer remains a therapeutic hindrance. The aim of this study was to identify whether small ubiquitin-related modifier (SUMO)-specific protease 1 (SENP1) is a marker of radioresistance that may serve as a target for enhancing the efficacy of lung carcinoma radiotherapy. SENP1 was observed to be overexpressed in lung cancer tissues, and the modulation of SENP1 expression was demonstrated to significantly affect the proliferation of lung cancer cells. Moreover, silencing the expression of SENP1 using small interfering RNA (siRNA) significantly sensitized lung cancer cells to radiation. Mechanically, it was demonstrated that SENP1 depletion significantly enhanced ionizing radiation (IR)-induced cell cycle arrest, γ-H2AX expression and apoptosis. Thus, these data suggest that SENP1 may be a desirable drug target for lung carcinoma radiotherapy.

Abstract 3449: Development of small molecule Bcl-XL inhibitors for treatment of lung cancer.

Abstract Bcl-XL is a major anti-apoptotic protein in the Bcl-2 family whose overexpression is more widely observed in human lung cancer cells than that of Bcl-2, suggesting that Bcl-XL is more biologically relevant and therefore a better therapeutic target for lung cancer. Here, we screened small molecules that selectively target the BH3 domain (aa 90-98) binding pocket of Bcl-XL using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We identified two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells compared with normal human bronchial epithelial cells. Fluorescence polarization assay reveals that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein but not Bcl2 in vitro with high binding affinities. Treatment of cells with BXI-72 results in disruption of Bcl-XL/Bak interaction, oligomerization of Bak and cytochrome c release from mitochondria. These two BXI compounds potently repress lung cancer xenografts in vivo without significant normal tissue toxicity within effective doses. Importantly, BXI-72 can also overcome acquired radioresistance of lung cancer. Based on our findings, the development of BXI(s) as a new class of anticancer agents is warranted and represents a novel strategy for improving lung cancer outcome. Citation Format: Dongkyoo Park, Andrew T. Magis, Rui Li, Taofeek K. K. Owonikoko, Gabriel L. Sica, Shi-Yong Sun, Suresh S. Ramalingam, Fadlo R. Khuri, Walter J. Curran, Xingming Deng. Development of small molecule Bcl-XL inhibitors for treatment of lung cancer. [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 3449. doi:10.1158/1538-7445.AM2013-3449

Enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) by adenovirus-mediated ING4 gene therapy

Radiotherapy is the common treatment of choice for locally advanced lung cancer, but the radioresistance of lung cancer remains a significant therapeutic obstacle. We previously demonstrated that adenovirus-mediated inhibitor of growth 4 (ING4) tumor suppressor gene delivery (AdVING4) can chemosensitize human hepatocarcinoma cells to anticancer drug cisplatin (CDDP). However, its radiosensitizing effects in cancer therapy are largely elusive. In the present study, we investigated the therapeutic efficacy of AdVING4 gene therapy combined with ionizing radiotherapy for SPC-A1 human non-small-cell lung cancer (NSCLC) cells in vitro and in vivo in athymic nude mice, and also elucidated its underlying mechanisms. We found that AdVING4 gene therapy plus radiotherapy induced synergistic tumor suppression and apoptosis in in vitro SPC-A1 human NSCLC cells and in vivo SPC-A1 xenografted tumors s.c. implanted in athymic nude mice. Mechanistically, AdVING4 combined with radiation resulted in a substantial upregulation of Bax, Fas, FasL and Cleaved Caspase-3, and downregulation of Bcl-2 in SPC-A1 human NSCLC xenografted tumors. In addition, AdVING4 plus radiation synergistically reduced the tumor vessel CD34 expression and microvessel density (MVD) in vivo. Most importantly, AdVING4 potentially blocked the radiation-induced enhancement of cyclooxygenase-2 and survivin radioresistant factors, and vascular endothelial growth factor and IL-8 proangiogenic factors. The enhanced antitumor effects elicited by AdVING4 plus radiotherapy were closely associated with the cooperative activation of intrinsic and extrinsic apoptotic pathways, and synergistic inhibition of tumor angiogenesis. Thus, our results suggested that AdVING4 combined with radiotherapy may be a feasible and effective strategy for treatment of radioresistant NSCLC and other cancers.

EGFR-dependent impact of indol-3-carbinol on radiosensitivity of lung cancer cells

背景与目的吲哚-3-甲醇（indole-3-carbinol, I3C）是十字花科蔬菜中一种主要的有效植物化学物质，且具有防癌和抗癌作用。本研究旨在观察I3C是否影响表皮生长因子受体（epidermal growth factor receptor, EGFR）表达水平不同的肺癌细胞放射敏感性。方法采用MTT和克隆形成实验方法分别检测肺癌细胞的生长和存活率；siRNA转染方法降低细胞中EGFR蛋白表达水平；Western blot和RT-PCR法分别测定EGFR蛋白和mRNA的表达。结果采用无明显毒副作用的5 μmol/L剂量的I3C预处理明显降低了EGFR表达阳性的人肺腺癌H1975和人肺鳞癌H226细胞对γ-射线照射的放射敏感性，而I3C对EGFR表达阴性的人肺鳞癌NIH-H520细胞的放射敏感性则影响非常小。Western blot结果显示I3C可以增加H1975和H226细胞中EGFR蛋白的表达水平和Y845位点磷酸化水平。EGFR siRNA降低了NIH-H1975细胞中EGFR蛋白的表达，增加了细胞的放射敏感性，并有效地降低和抑制了I3C导致的细胞耐辐射效应。结论我们的研究结果首次证实I3C可以通过调节EGFR表达和磷酸化水平从而影响肺癌细胞的放射治疗敏感性，提示EGFR可能是I3C影响肺癌放射治疗敏感性的重要靶蛋白。

DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair.

The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated γ-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following γ-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent γ-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced γ-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of γ-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the quantity of γ-H2AX determines the radioresistance of cells. The DNA repair pathway is important in mediating radioresistance, and treatment with the DNA-PKcs inhibitor, NU7026 restored the acquired radiation resistance.

Abstract A1: MicroRNAs expression profile associated with radioresistance in lung cancer

Abstract Lung cancer is the neoplasia with highest incidence and mortality worldwide. One of the strategies of treatment for this tumor type is based on ionizing radiation. Radiation induces cytotoxicity through some mechanisms, including gene expression changes. Recently, microRNAs (miRNAs) have been described as one of the molecules that regulate expression genes and may contributes to radioresistance in several tumor types. Global miRNAs expression profiles of radiosensitive and radioresistant lung tumor cells may elucidate the molecular mechanism related to tumoral radioresistance. In order to identify miRNAs associated with innate and acquired radioresistance, we will evaluate the miRNAs expression profiles in two lung carcinoma cell lines and in the same cell lines with acquire radioresistance by fractionated doses radiation treatment. For establish the median lethal dose radiation, H1944 and Calu-1 lung carcinoma cell lines were treated with increasing doses of X-rays (from 2 to 12 Gy). Cell survival, proliferative ability and clonogenic potential postradiation were evaluated. Moreover, we will generate a model of acquired radioresistance by treating lung tumor cells with fractionated doses of 2 Gy X-rays up to total dose of 60 Gy for to evaluate the expression of miRNAs in cells with acquired radioresistance. Results showed differential sensitivity to radiotherapy from lung tumors cells, whit 8 Gy as median lethal dose radiation. For to evaluate the expression of miRNAs during innate response to radiotherapy, total RNA was obtained from tumor cells treated with 8 Gy of X-rays. Additionally, it has been obtained lung tumor cells clones with treatment total doses of 28 Gy in a fractionated radiation scheme. These results will allow compare miRNAs expression during immediate cell response to radiation and those whose expression will modified during fractionated radiation treatment and may be associated with radioresistance acquired, which contribute to understanding the molecular mechanism to radioresistance in lung cancer.

M867, A Novel Inhibitor of Caspase-3, Enhances the Effects of Radiation in Human Lung Cancer Models

Radioresistance of lung cancer results in an unacceptable rate of locoregional failure. Although radiation (RT) is known to induce apoptosis, our recent study showed that knockdown of pro-apoptotic proteins Bak/Bax resulted in increased autophagy and lung cancer radiosensitivity in vitro. To further explore the potential of apoptosis inhibition to enhance lung cancer therapy, we tested M867, a novel reversible caspase-3 inhibitor, in combination with RT in vivo and in vitro.