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PDF file - 869K, Effect of DNA-PKcs or ATM inhibition on p-T2609 in NSCLCs. h pretreatment with 10 muM NU7441 or 10 muM KU55933. Square boxes represent cropped images shown in Figure 6C

MT-EGFR expression in HBEC cells is associated with dramatically elevated levels of replication factors.

PDF file - 900K, Radiation-induced T2609 phosphorylation in NSCLCs. Immuno-fluorescence detection of DNA-PKcs phosphorylation in indicated NSCLCs showing p-T2609 immunofluorescence (green) in nuclei (blue). Cropped images shown in Figure 6B

PDF file - 286K, Alterations in ATM or DNA-PKcs do not affect IR-induced nuclear import. Deficiency or inhibition of either ATM or DNA-PKcs is associated with an absence of EGFR-DNA-PKcs binding. The data in S1A eliminate the possibility that this is due to a potential abrogation of IR-induced nuclear translocation of EGFR which could prevent access to the predominantly nuclear DNA-PKcs. (A) WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from V3, V3-WT DNA-PKCS and V3-7A cells(A) or AT5 and 1BR3 fibroblast cells (B) expressing wild type, L858R or gammaE746-E750 EGFR showing EGFR, cytoplasmic marker, calnexin, and nuclear marker, lamin B. With exposure to 4 Gy, wild type but not L858R or gammaE746-E750 EGFR is seen in nuclear fractions of cells that lack DNA-PKcs or ATM. (C) DNA-PKcs or ATM inhibition does not affect EGFR nuclear import. WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from HBEC cells expressing wild type EGFR that were pretreated for 2 hours with vehicle or DNA-PKcs inhibitor, NU7441, (top panel) or ATM inhibitor, KU55933, (bottom panel). (D) Mutant forms of EGFR are also defective in binding DNA-PKcs. The data in S1A and S1B show that IR-induced nuclear translocation is completely abrogated in L858R or gammaE746-E750 forms of EGFR, which could prevent interactions with the predominantly nuclear DNA-PKcs. Here we use in vitro binding assay to demonstrate that relative to wild type EGFR, the L858R or gammaE746-E750 are also intrinsically defective in physical association with purified DNA-PKcs. In vitro binding assay: wild type, L858R or gammaE746-E750 forms of EGFR were immuno-precipitated with anti-V5 antibody from whole cell lysates from un-irradiated HBEC cells. Lane 1 from left shows no non-specific binding to beads. Antibody-bound EGFR was thoroughly washed. Lane 2 from left shows no evidence of endogenous bound protein. Antibody bound EGFR was incubated with purified human DNA-PKcs in the presence or absence of calf thymus DNA and immuno-precipitated. DNA-PKcs was detected by western blot assay. Lane 3 and 4 show that purified DNA-PKcs physically bound wild type but not the L858R or gammaE746-E750 forms of EGFR and the binding was slightly enhanced with presence of sheared calf thymus DNA.

The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non–small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the ataxia telangiectasia–mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at seven key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR-mediated radiation response and elucidates mechanisms underlying mutant EGFR-associated radiosensitivity in NSCLCs. Mol Cancer Res; 10(10); 1359–68. ©2012 AACR.

Supplementary Figure Legends.

PDF file - 607K, Effect of EGFR mutation status on DNA-Pkcs phosphorylation. Immuno-fluorescence detection of DNA-PKcs phosphorylation in HBEC cells stably expressing wild type, L858R or gammaE746-E750 forms of EGFR at various time-points following 4 Gy IR. Top panel: Representative images with anti-p-T2609 and anti-p-S2056 DNA-PKcs antibodies showing nuclei (blue) and phosphorylated DNA-PKcs (green). Cropped images shown in Figure 6A.

PDF file - 544K, Proximity ligation assay (PLA): Effect of DNA-Pkcs and ATM inhibition on EGFR-DNA-PKcs binding.NSCLCs or HBEC-3KT cells expressing wild type or gammaE746-E750 forms of EGFR following a 2 h pretreatment with vehicle, 10 muM NU7441 or 10 muM KU55933 were mock irradiated or exposed to 4 Gy IR and fixed at 60 minutes. Representative images of nuclei (blue) showing EGFR-DNA-PKcs complexes (intense red dots). Cropped images shown in Figure 4C

PDF file - 774K, Proximity ligation assay (PLA): To detect radiation-induced PP2A-DNA-PKcs binding. NSCLCs and HBEC cells expressing WT or gammaE746-E750 EGFR fixed 1 h following 0 Gy or 4 Gy IR. Left panel: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red: PLA fluorescence). Square boxes represent cropped images shown in Figure 7C

PDF file - 729K, Proximity ligation assay (PLA): Effect of T2609A and S2056A on EGFR-DNA-PKcs binding. V3-WT DNA-PKCS, V3-S2056A and V3-T2609A CHO cells expressing wild type EGFR which were either mock-irradiated or exposed to 4 Gy IR. Left panel: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red PLA fluorescence dots) after processing for PLA. Square boxes represent cropped images shown in Figure 5C.

【Background】 Resolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA). 【Methods】 Receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. 【Results】 RvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells. 【Conclusion】 RvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non–small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX–associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition. Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503–16. ©2017 AACR.

Background: Chronic obstructive pulmonary disease is a smoking-related disease, and is categorized into the emphysema and airway dominant phenotypes. We examined the relationship between emphysematous changes and epidermal growth factor receptor (EGFR) mutation status in patients with lung adenocarcinoma. Patients and Methods: The medical records for 250 patients with lung adenocarcinoma were retrospectively reviewed. All patients were categorized into the emphysema or non-emphysema group. Results: Wild-type EGFR was detected in 136 (54%) and mutant EGFR in 48 (19%). Emphysematous changes were observed in 87 (36%) patients. EGFR mutation was highly frequent in the non-emphysema group (p=0.0014). Multivariate logistic regression analysis showed that emphysema was an independent risk factor for reduced frequency of EGFR mutation (Odds Ratio=3.47, p=0.005). Conclusion: Our data showed a relationship between emphysematous changes and EGFR mutation status. There might be mutually exclusive genetic risk factors for carcinogenesis and development of emphysematous changes.

Background Oxidative stress plays an important role in the pathogenesis of bronchial asthma. Antioxidant nutrition and supplementation have been used to reduce oxidative stress. However, a clinical trial with antioxidant supplementation showed no beneficial effects in patients with asthma. On the other hand, physical activity is related to the prognosis of chronic obstructive pulmonary disease (COPD) and is also related to oxidant status. We investigated the relationships between oxidative stress, serum levels of vitamins, dietary vitamin intake, daily activities, and pulmonary functions in patients with asthma. Methods Eighteen patients with bronchial asthma were enrolled in this study. Reactive oxidative stress was assessed by measuring organic hydroperoxides (diacron reactive oxygen metabolites: dROM) in sera and by measuring H2O2 levels in exhaled breath condensates. The biological antioxidant capacity in serum was evaluated by measuring antioxidant potential capacity against ferric ion. We also assessed pulmonary functions, fraction of exhaled nitric oxide, serum levels of vitamins, dietary vitamin intake, and physical activities. Results There were no relationships between the index of oxidative stress (dROM and H2O2 in exhaled breathe condensates) and pulmonary functions, serum levels of vitamins, daily vitamin intakes, and activity levels in patients with asthma. Conclusion The status of transient oxidative stress may not be related to daily activities, vitamin levels, and pulmonary functions in patients with asthma in a real-life setting. However, our results were obtained in the short-term period from a small number of subjects, so a large longitudinal study is required to ascertain the relationships between oxidative stress, physical activity and vitamin intake in patients with asthma.

Immune checkpoint inhibitors (ICIs), including nivolumab, have exhibited substantial benefits in the treatment of several types of cancers. However, treatment with ICIs is often accompanied by immune-related adverse events (irAEs), and a clear understanding of the precise indications and management of irAEs is important for harnessing the full potential of these agents. While skin- or gastrointestinal-associated irAEs have been relatively well studied, there are few reports regarding nivolumab-induced cholangitis. We retrospectively reviewed data from patients with advanced or recurrent non-small cell lung cancer who were treated with nivolumab between December 2015 and December 2018 at Tottori University in Japan. Among the 59 patients, we identified four patients who experienced nivolumab-induced cholangitis. Of these four patients, stable disease (SD) was observed in two patients (50%), while partial response (PR) was achieved in two patients (50%) under nivolumab treatment. Patients were treated with corticosteroid alone (n=2) or in combination with mycophenolate mofetil (MMF) (n=2); these treatments resulted in improvements in nivolumab-induced cholangitis in three patients. In conclusion, the present retrospective study identified four cases of nivolumab-induced cholangitis. The combination of corticosteroid and MMF was effective in two cases with grade 4 nivolumab-induced cholangitis. Further reports are needed to establish the optimal management of patients with this irAE.

We report the case ofan elderly patient with squamous cell carcinoma(SCC)ofthe breast, which showed negative conversion after endocrine therapy. An 82-year-old woman with a diagnosis ofER -positive SCC(cT1N0M0, StageⅠ)received primary endocrine therapy with 5 hormonal medicines. Following the endocrine therapy, she underwent mastectomy and axillary node resection. Histological examination confirmed a diagnosis of ER-negative SCC. At 10 months after the operation, she was doing well with no evidence ofdisease without postoperative adjuvant therapy. Thus, clinicians should be aware that the ER status may change after primary endocrine therapy. For elderly patients with breast cancer, it is important to be aware that primary endocrine therapy can become ineffective due to ER-negative conversion and aging due to prolonged treatment.

Background Precision medicine based on driver oncogenes is now developed for non-small cell lung cancer. Recent approval of next-generation sequencing (NGS)-based companion diagnostics has increased the need for the use of high-quality specimens for the diagnosis. Methods From August 2015 to May 2018, 82 patients who were enrolled in a nation-wide genome screening, LC-SCRUM-Japan, from our institution were examined for the success rates of genetic analysis according to sample types. A NGS analysis, Oncomine Comprehensive Assay, was performed using DNA and RNA extracted from lung cancer specimens. Results The success rates of DNA and RNA analyses were 100% (82/82 cases) and 78% (64/82), respectively. Of the 82 samples analyzed, 80 (98%) were tissues and two (2%) were pleural effusions. The success rates of RNA analysis in the 80 tissue samples were 45/60 (73%) in bronchoscopic byopsy, 10/10(100%) in operation, 5/6(83%) in Percutaneous biopsy and 3/4(75%) in others. Of the 60 bronchoscopic samples, the success rates were 72% (18/25) in EBUS-TBNA, 88% (7/8) in EBUS-GS (large diameter), 69% (11/16) in EBUS-GS (small diameter) and 82% (9/11) in direct vision biopsy. In the 82 cases analyzed, a total of 52 actionable gene alterations (18 KRAS mut, 6 EGFR mut, 6 MET amp/fus, 5 RET fus, 5 ALK fus, 4 PIK3CA mut, 2 ERBB2 mut, 2 ROS1 fus, 2 FGFR1 amp, 1 BRAF mut, 1 FGFR3 fus) were detected in 48 cases (59%). Twelve of the 48 (15%) were registered in ongoing clinical trials of targeted drugs. Conclusions All the samples were available for DNA analysis of the NGS. The success rates of RNA analysis were lower in samples obtained from EBUS-TBNA and EBUS-GS (small diameter), suggesting that the success rates depend on the sample size.

Objectives Zoledronate (ZOL) is usually used for prevention of skeletal-related events in cancer patients with bone metastases. The first administration of ZOL is occasionally associated with development of acute-phase reaction (APR), which is due to activation of γδ T cells. ZOL-related APR was associated with better overall survival (OS) of patients with non-small cell lung cancer (NSCLC) in our previous retrospective study. However, it remains to be clarified whether γδ T cells are more activated in patients who experienced ZOL-related APR, and whether γδ T cell activation is involved in prolongation of OS. Materials and Methods Twenty-three patients with advanced NSCLC were recruited between 2012 and 2014 in this study. We administered ZOL to participants with standard care. The patient characteristics, change in γδ T cell counts and cytokines, OS, and skeletal-related event-free survival were compared between patients with APR (APR group) and those without APR (non-APR group). Results Ten patients (43.5%) experienced a ZOL-related APR. The number of γδ T cells at baseline in the APR group was significantly higher than that in the non-APR group. Serum interleukin-6 and tumor necrosis factor-α in the APR group were significantly increased, but no change in the number of γδ T cells was observed after the first administration of ZOL in both groups. OS in the APR group was significantly longer than that in the non-APR group (median survival time: 23.1 vs. 14.5 months, p Conclusion We showed that APR is related to higher numbers of γδ T cells at baseline and increased cytokines after the first ZOL administration, but not to proliferative responses of γδ T cells. In addition, better OS was observed in the APR group. Therefore, the number of γδ T cells might be a prognostic marker in patients with NSCLC.

We report the case of a patient with triple negative breast cancer(TNBC)who showed isolated brain metastasis relatively soon after pathological complete response(pCR)to neoadjuvant chemotherapy. A 45-year-old woman with a diagnosis of TNBC(T2N1M0, Stage II B)received neoadjuvant chemotherapy with 5-FU/epirubicin/cyclophosphamide(FEC), followed by docetaxel. After the neoadjuvant chemotherapy, she underwent mastectomy and axillary lymph node dissection. Histological examination of the resected specimens revealed pCR. Brain metastasis, however, developed 7 months after the resection. She underwent total removal of the brain tumor and 50 Gy irradiation to the right frontal lobe. Histological examination confirmed a diagnosis of metastasis from TNBC. She is doing well with no evidence of disease 81 months after resection of the brain metastasis. This case and a review of the literature suggest that the clinician should be aware that brain metastasis from breast cancer may develop even after achieving pCR to neoadjuvant chemotherapy. Surgical resection followed by radiotherapy may provide a survival benefit for selected patients with isolated brain metastasis from breast cancer.

Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.