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Targeting CREB-binding protein overrides LPS induced radioresistance in non-small cell lung cancer cell lines.

ABSTRACT: Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an active TLR-4 pathway. Proteome profiling array revealed that LPS combined with irradiation resulted in a strong phosphorylation of cAMP response element-binding protein (CREB). Inhibition of CREB binding protein (CBP) by the specific inhibitor ICG-001 not only abrogated the LPS-induced radioresistance but even led to an increase in radiosensitivity. The sensitization caused by ICG-001 could be attributed to a reduction of DNA double-strand break (DSB) repair. It is shown that in NSCLC cells LPS leads to a CREB dependent radioresistance which is, however, reversible through CBP inhibition by the specific inhibitor ICG-001. These findings indicate that the combined treatment with radiation and CBP inhibition may improve survival of NSCLC patients suffering from pulmonary infections.

SUBMITTER: Gokyildirim MY

PROVIDER: S-EPMC6034744 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Targeting CREB-binding protein overrides LPS induced radioresistance in non-small cell lung cancer cell lines.

Gökyildirim Mira Y MY Grandel Ulrich U Hattar Katja K Dahlem Gabriele G Schuetz Elena E Leinberger Florian H FH Eberle Fabian F Sibelius Ulf U Grimminger Friedrich F Seeger Werner W Engenhart-Cabillic Rita R Dikomey Ekkehard E Subtil Florentine S B FSB

Oncotarget 20180622 48

Non-small cell lung cancer (NSCLC) has a very poor prognosis even when treated with the best therapies available today often including radiation. NSCLC is frequently complicated by pulmonary infections which appear to impair prognosis as well as therapy, whereby the underlying mechanisms are still not known. It was investigated here, whether the bacterial lipopolysaccharides (LPS) might alter the tumor cell radiosensitivity. LPS were found to induce a radioresistance but solely in cells with an ...[more]

PMID: 29989005

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Project description:Purpose:Radiotherapy is a major treatment method for patients with non-small cell lung cancer (NSCLC). However, the presence of radioresistant cancer stem cells (CSCs) may be associated with disease relapse or a poor outcome after radiotherapy. Voltage-gated calcium channel ?2?1 subunit (encoded by the gene CACNA2D1) isoform 5 is a marker of CSCs in hepatocellular carcinoma. This study aimed to investigate the radiosensitivity of ?2?1-high cells in NSCLC cell lines. Materials and methods:NSCLC cell lines A549, H1975, H1299, and PC9 were used. CACNA2D1-knockdown and CACNA2D1-overexpressing cell lines were established by lentiviral infection. Colony formation assay was performed to determine radiosensitivity. Sphere formation assay in serum-free medium was performed to evaluate self-renewal capacity. Proteins associated with DNA damage repair were analyzed by immunofluorescence or Western blot. The monoclonal antibody of ?2?1 was applied alone or in combination with radiation either in vitro or in vivo to determine the anti-tumor effect of the antibody. Results:?2?1-high cells showed greater sphere-forming efficiency than ?2?1-low cells and were relatively resistant to radiation. CACNA2D1 knockdown in A549 cells enhanced radiosensitivity, whereas CACNA2D1 overexpression in PC9 and H1975 cells reduced radiosensitivity, suggesting that ?2?1 imparted radioresistance to NSCLC cells. Analysis of proteins involved in DNA damage repair suggested that ?2?1 enhanced the efficiency of DNA damage repair. The monoclonal antibody of ?2?1 had a synergistic effect with that of radiation to block the self-renewal of ?2?1-high cells and enhanced the radiosensitivity of ?2?1-positive cells in colony formation assays. The combination of the ?2?1 antibody with radiation repressed A549 xenograft growth in vivo. Conclusion:?2?1 enhances radioresistance in cancer stem-like cells in NSCLC. The ?2?1 monoclonal antibody sensitizes ?2?1-high cells to radiation, suggesting that the antibody may be used to improve the treatment outcome when combined with radiation in NSCLC.

Project description:The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional level of Hypoxia Inducible Factor (HIF) dependent transcriptional regulation has emerged involving modulation of a specific set of miRNAs including miR-210. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. We therefore hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed Non-Small Cell Lung Cancer (NSCLC)-derived cell lines (A549 and H1975) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). MiR-210 expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. The cells were subjected to radiation levels ranging from 0 to 10Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of resistance than control cells in hypoxia. pmiR-210 cells under hypoxia showed a low mortality rate due to a decrease in apoptosis and an ability to grow even at 10Gy. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we show here that genomic double strand breaks (DSB) foci disappeared faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells (pmiR-210/HIF-1-) abolished radioresistance of cells showing that this mechanism was dependent upon HIF-1. In conclusion, miR-210 appears to be a major component in the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could even be used by tumor cells in conditions where hypoxia may not be present anymore and strongly suggests that strategies targeting miR-210 would enhance tumor radiosensitization. To identify the set of transcripts targeted by miR-210, we overexpressed has-miR-210 or a control miRNA (ce-miR-67) in human lung adenocarcinoma A549 cells by transduction using lentivirus. The resulting pmiR-67 and pmiR-210 that stably overexpress miR-210 were selected by puromycin. RNA samples were harvested at 2 different times following cell plating (24 hours or 48 hours). Experiments were performed in dye-swap.

Dataset Information

Targeting CREB-binding protein overrides LPS induced radioresistance in non-small cell lung cancer cell lines.

Publications

Targeting CREB-binding protein overrides LPS induced radioresistance in non-small cell lung cancer cell lines.

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