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Background Epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitor (TKI) has demonstrated a promising therapeutic response in lung adenocarcinoma patients with EGFR gene mutations. However, the predictive factors for this therapy have not been established, except for the EGFR gene mutation status of carcinoma cells. Methods We first performed microarray analysis in EGFR-TKI–sensitive lung adenocarcinoma cell lines. The results indicated anterior gradient 2 (AGR2) as a potential surrogate marker of EGFR-TKI. Therefore, we then evaluated the correlation between the status of AGR2 immunoreactivity and clinicopathological factors including overall survival (OS), progression-free survival (PFS) and clinical response to EGFR-TKI, in 147 cases of surgically resected lung adenocarcinoma. The biological significance of AGR2 was further evaluated by transfecting small interfering RNA (siRNA) against AGR2 in these cells. Results The status of AGR2 immunoreactivity was significantly higher in lung adenocarcinoma cases with EGFR gene mutations than in those with the wild type (pConclusions AGR2 could serve as an adjunctive surrogate protein marker possibly reflecting EGFR gene mutations in lung adenocarcinoma patients. Results from in vitro analysis indicated that AGR2 could be a potential clinical biomarker of EGFR-TKI therapeutic sensitivity in lung adenocarcinoma cells.

Introduction: Autoimmune pulmonary alveolar proteinosis (APAP), caused by macrophage dysfunction due to the anti-granulocyte-macrophage colony-stimulating factor autoantibody, is known to be complicated with opportunistic infection. We have already reported frequency of infection was 5.3% (Inoue Y, AJRCCM 2008). Subjects and Methods: Questionnaire about APAP cases complicated with infections, was mailed to 195 hospitals in Japan and was returned from 27 hospitals. Subjects of this study were APAP cases complicated with infections managed in the 27 hospitals. Pathogens, site of infection, and clinical course were examined. Results: Frequency of infection was 27 cases (7.2%) out of 375 APAP cases. Lung comorbidities were COPD (n=3), pneumoconiosis (n=1) and interstitial pneumonia (n=2), and three cases were treated with corticosteroids and/or immunosuppressant. Pathogen was bacteria (n=15), fungus (n=5), mycobacterium (n=6), Nocardia (n=2) and virus (n=1). Tuberculous lymphadenitis (n=2), meningeal cryptococcosis (n=1), aspergillus sinusitis (n=1), herpes simplex virus encephalitis (n=1) occurred out of the lung. Diagnosis of APAP preceded incidence of infection in 20 cases (74%). Disease severity of 6 cases (22.2%) improved after the occurrence of infection. Whole lung lavage could not be performed in 2 cases with infection, one case of which died with respiratory failure. Conclusions: Frequency of infection in APAP cases were similar to the previous report. Infection might improve or deteriorate the clinical course of APAP. Financial support: This study was partially granted from AMED; DLD/14526278 to Y. Inoue and T. Arai and PAP/14526182 to Y. Inoue.

Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare disease characterized by the excessive accumulation of surfactant proteins within the alveolar spaces and by higher titers of autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) in the serum and bronchoalveolar lavage fluid. The antibodies inhibit the maturation and phagocytosis of alveolar macrophages. Although the standard therapy for aPAP has been whole-lung lavage (WLL), this procedure is invasive and needs to be repeated for several years. GM-CSF inhalation therapy is a new procedure for treating aPAP and can induce remission with less invasiveness, although it is generally less effective in severe cases. We evaluated five cases with remarkable improvement by using sequential GM-CSF inhalation therapy after WLL; however, the treatment failed when this therapy preceded WLL. Therefore, sequential GM-CSF inhalation after WLL may reinforce the efficiency of WLL in patients with severe aPAP.

In this study, we found that there were significant positive correlations between the catch per unit effort (CPUE, a squid abundance index) for the neon flying squid (Ommastrephes bartramii) winter–spring cohort and the satellite-derived chlorophyll a concentrations in their spawning grounds located at 140–160°E where 21°C

Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) that has been approved for the treatment of metastatic non-small cell lung cancer (NSCLC) with EGFR mutation, and positive for the secondary T790M mutation of EGFR. In a preclinical study, it also showed efficacy against leptomeningeal carcinomatosis (LMC) derived from NSCLC resistant to first-generation and second-generation EGFR-TKIs. We report the case of a patient aged 55 years with symptomatic LMC derived from NSCLC. We detected T790M mutation of EGFR by liquid biopsy with blood sample. He showed a clinical and radiographic response to osimertinib.

Loss of transcription factor Bach2 results in a pulmonary alveolar proteinosis-like accumulation of surfactant proteins in the lungs due to altered function of alveolar macrophages., Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), chiefly due to disruptions in the signaling of granulocyte macrophage colony–stimulating factor (GM-CSF). We found that mice deficient for the B lymphoid transcription repressor BTB and CNC homology 2 (Bach2) developed PAP-like accumulation of surfactant proteins in the lungs. Bach2 was expressed in AMs, and Bach2-deficient AMs showed alterations in lipid handling in comparison with wild-type (WT) cells. Although Bach2-deficient AMs showed a normal expression of the genes involved in the GM-CSF signaling, they showed an altered expression of the genes involved in chemotaxis, lipid metabolism, and alternative M2 macrophage activation with increased expression of Ym1 and arginase-1, and the M2 regulator Irf4. Peritoneal Bach2-deficient macrophages showed increased Ym1 expression when stimulated with interleukin-4. More eosinophils were present in the lung and peritoneal cavity of Bach2-deficient mice compared with WT mice. The PAP-like lesions in Bach2-deficient mice were relieved by WT bone marrow transplantation even after their development, confirming the hematopoietic origin of the lesions. These results indicate that Bach2 is required for the functional maturation of AMs and pulmonary homeostasis, independently of the GM-CSF signaling.

Background: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. Methods: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. Results: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). Conclusion: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.

Previous studies have demonstrated that mesenchymal stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1). This study shows that MSC-derived STC1 promotes survival of lung cancer cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more glycolytic metabolic profile. MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1-dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1-treated A549 cells displayed decreased levels of ROS, mitochondrial membrane potential (MMP), and increased lactate production, all of which were dependent on the upregulation of UCP2. Our data suggest that MSCs can promote cell survival by regulating mitochondrial respiration via STC1.

Airway surface fluids are mainly secreted from submucosal glands, and play important roles in the defense of airways via the up-regulation of mucociliary transport, resulting in an exclusion of many microbes or foreign substances. Although there are many articles concerning the importance of Toll-like receptors (TLRs) in airway immune systems, whether TLRs directly cooperate with tracheal submucosal glands to increase secretion remains unknown. We investigated the effects of ligands of the three TLR subtypes (TLR2, TLR3, and TLR4) on the physiologic secretion of electrolytes by using a patch-clamp technique. Among these TLRs, only the TLR4 ligand, LPS, showed potentiating effects on acetylcholine (ACh)-induced ionic currents in a dose-dependent manner. These potentiating effects were completely abolished by pretreatment with a specific TLR4 antagonist or the anti-TLR4 antibody. LPS per se exerted no appreciable effect on baseline currents. Next, we demonstrated the abundant expression of TLR4 in submucosal gland acinar cells by using immunofluorescent staining and RT-PCR. Furthermore, we revealed that both nitric oxide synthase inhibitors and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) inhibitors abolished the LPS-induced potentiating effects completely. Analyses of fluorescence intensities, using an intracellular nitric oxide (NO) indicator, demonstrated that LPS could further increase the ACh-induced synthesis of NO. These findings suggest that TLR4 takes part in airway mucosal defense systems as a unique exogenous potentiator of electrolyte-water secretion from submucosal gland acinar cells, and that NO/cGMP/cGK signaling is involved in this rapid TLR4 signaling pathway.

A 59-year-old man presented with a skin eruption and bilateral swelling of the legs. Soon after the initial presentation, he developed acute respiratory distress syndrome (ARDS) with miliary lung nodules. Culture of samples from the skin ulcers, sputum, and bronchoalveolar lavage fluid all revealed Mycobacterium szulgai infection. The patient was successfully treated with antituberculosis drugs. M. szulgai infection is very rarely reported worldwide, and disseminated infection usually occurs in immunocompromised patients. However, the present patient was a non-immunocompromised case, although he was a hepatitis B virus carrier. While the progression to ARDS from M. tuberculosis infection is well known, this is the first case of M. szulgai infection progressing to ARDS.

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory infiltration of leukocytes, lung injury induced by reactive oxygen species (ROS), in particular superoxide anion, and fibrosis (collagen deposition). No treatment has been shown to improve definitively the prognosis for IPF patients. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) has overcome clinical limitations of SOD, including low tissue affinity and low stability in plasma. In this study, we examined the effect of PC-SOD on bleomycin-induced pulmonary fibrosis. Severity of the bleomycin-induced fibrosis in mice was assessed by various methods, including determination of hydroxyproline levels in lung tissue. Intravenous administration of PC-SOD suppressed the bleomycin-induced increase in the number of leukocytes in bronchoalveolar lavage fluid. Bleomycin-induced collagen deposition and increased hydroxyproline levels in the lung were also suppressed in animals treated with PC-SOD, suggesting that PC-SOD suppresses bleomycin-induced pulmonary fibrosis. The dose-response profile of PC-SOD was bell-shaped, but concurrent administration of catalase restored the ameliorative effect at high doses of PC-SOD. Intratracheal administration or inhalation of PC-SOD also attenuated the bleomycin-induced inflammatory response and fibrosis. The bell-shaped dose-response profile of PC-SOD was not observed for these routes of administration. We consider that, compared with intravenous administration, inhalation of PC-SOD may be a more therapeutically beneficial route of administration due to the higher safety and quality of life of the patient treated with this drug.

Background: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. Methods: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. Results: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005–6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium -positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005–6 period were combined with those from 2007 (p = 0.003). Conclusion: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium .

背景．上皮増殖因子受容体（EGFR）チロシンキナーゼ阻害剤であるゲフィチニブは，活性型EGFR遺伝子変異を有する非小細胞肺癌患者に対して著明な抗腫瘍効果をもたらすが，後に生じる耐性化への対策は十分に検討されていない．方法．2004年6月∼2007年6月の間に，当施設で活性型EGFR遺伝子変異陽性と診断され，ゲフィチニブ治療が開始された進行非小細胞肺癌患者を対象に，増悪形式に関連した臨床的特徴をレトロスペクティブに解析した．結果．51例にゲフィチニブ治療が行われ，奏効率，病勢制御率は各々71%（36/51），86%（44/51）であった．病勢制御できた44例のうち，2008年4月末時点で33例に増悪を認め（無増悪生存期間中央値14ヶ月），21例は胸郭内，12例は遠隔臓器での増悪（うち10例は脳転移）であった．脳転移増悪例の過半数では放射線治療とともにゲフィチニブが3ヶ月以上継続され，生存期間中央値は28.2ヶ月と極めて良好であった．耐性遺伝子変異T790Mは全て原発巣近傍から検出された．結論．ゲフィチニブ治療例においては異なる増悪形式が認められ，それぞれの機序をふまえた治療法の開発が望まれる．

Nrf2 activation promotes resistance to chemical carcinogenesis in animal models, but activating mutations in Nrf2 also confer malignant characters to human cells by activating antioxidative/detoxifying enzymes and metabolic reprogramming. In this study, we examined how these contradictory activities of Nrf2, cancer chemoprevention and cancer cell growth enhancement, can be reconciled in an established mouse model of urethane-induced lung carcinogenesis. Using Keap1-knockdown (kd) mice, which express high levels of Nrf2, we found that urethane was rapidly excreted into the urine, consistent with an upregulation in the expression of urethane detoxification genes. Consequently, urethane-induced tumors were significantly smaller and less frequent in Keap1-kd mice than in wild-type mice. In contrast, tumor cells derived from Keap1-kd mice and transplanted into nude mice exhibited higher tumorigenicity compared with cells derived from wild-type mice. To identify the factors contributing to the tumor growth phenotype in the transplantation model, we performed a microarray analysis and found that many antioxidative stress genes were upregulated in the Keap1-kd–derived tumors. Therefore, we suggest that Nrf2 activation in cancer cells enhances their tumorigenicity, but global Nrf2 activation, as in Keap1-kd mice, simultaneously enhances anticancer immunity, thereby suppressing the growth potential of Keap1-kd tumors. Our findings provide relevant insight into the dual role of Nrf2 in cancer and warrant further studies of Nrf2 function during different stages of carcinogenesis. Cancer Res; 76(10); 3088–96. ©2016 AACR.

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-β1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-β1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-β1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial–mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

We examined therapeutic gene transfer of human hepatocyte growth factor (hHGF) to alveolar septa in mouse bleomycin-induced lung fibrosis using macroaggregated albumin-polyethylenimine complex (MAA-PEI). Intravenous administration of MAA-PEI along with 1 μg pCAG.hHGF to C57BL/6 mice increased the uptake of plasmids into alveolar capillary endothelial cells and epithelial cells, prolonged hHGF expression in the lung, and induced a level of hHGF expression equal to that seen with 10 μg of hHGF-expression plasmids alone. The exogenous source of hHGF gene expression increased the endogenous mouse HGF in the lungs and significantly decreased TNF-α, IL-6, and collagen synthesis after bleomycin injury. Because GFP-labeled bone marrow-derived stem cells after bleomycin injury were reduced in number by HGF, the primary mechanism of HGF is likely to be the prevention of apoptosis, as has been suggested by in vitro experiments. This novel HGF gene transfer method to alveolar septa with nonstimulatory MAA-PEI conjugates may have promising clinical applications.

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-β, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 × l09plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 ± 0.02 to 0.96 ± 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.

Secretory leukoprotease inhibitor (SLPI) protects tissue against the destructive action of neutrophil elastase at the site of inflammation. Recent studies on new functions of SLPI have demonstrated that SLPI may play a larger role in innate immunity than merely as a protease inhibitor. To clarify the functions of SLPI in bacterial infections, we generated SLPI-deficient mice (SLPI−/− mice) and analyzed their response to experimental endotoxin shock induced by lipopolysaccharide (LPS). SLPI−/− mice showed a higher mortality from endotoxin shock than did wild type mice. This may be explained in part by our observation that SLPI−/− macro-phages show higher interleukin 6 and high-mobility group (HMG)-1 production and nuclear factor κB activities after LPS treatment than do SLPI+/+ macrophages. SLPI also affects B cell function. SLPI−/− B cells show more proliferation and IgM production after LPS treatment than SLPI+/+ B cells. Our results suggest that SLPI attenuates excessive inflammatory responses and thus assures balanced functioning of innate immunity.

Nuclear receptors and their ligands are known to play very important roles in lung development. Among these receptors, retinoid receptors, members of the steroid/thyroid hormone receptor superfamily, are classified into retinoic acid receptor (RAR) isoforms alpha, beta, and gamma and retinoid X receptor (RXR) isoforms alpha, beta, and gamma. In addition, isoforms I and II of the orphan receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) have been shown to negatively regulate the activation of retinoid receptors. Both of these receptors have been shown to regulate lung development in the mouse. In the present study we utilized immunohistochemistry and real-time quantitative PCR to examine the expression of RAR-alpha, -beta and -gamma, RXR-alpha, -beta and -gamma and COUP-TFII in the human fetal lung at 13-16 gestational weeks, a very critical stage of human pulmonary development, in order to study possible roles in pulmonary morphogenesis by comparing these findings with those of the adult lung. RXR-gamma immunoreactivity was detected at both proximal (epithelia and mesenchyme of the trachea and bronchi associated with cartilage) and distal (epithelia and mesenchyme of smaller distal bronchi) sites in the fetal lung, but was markedly weaker in the adult lung. RAR-beta immunoreactivity was detected in distal mesenchymal cells of the fetal lung, but was not discernible in distal mesenchymal cells in the adult lung (bronchioles, alveolar ducts and alveolus). Relatively intense RAR-gamma immunoreactivity was detected in the chondrocytes of bronchial cells. COUP-TFII immunoreactivity was detected with a similar pattern to that of RAR-beta. Real-time quantitative PCR analyses revealed that mRNA levels of RXR-gamma at proximal and distal sites (ratio of fetal lung/adult lung: 3.4+/-0.05-fold and 3.1+/-0.03-fold respectively; P

Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane.

The development of therapeutic resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs, ie erlotinib or gefitinib) has been the major clinical problem when treating lung adenocarcinoma patients with these agents. However, its mechanisms have not necessarily been well studied to this date. Autophagy has been recently considered to play pivotal roles in escaping from the effects of anti-neoplastic agents. Therefore, in this study, we examined its roles in the development of resistance to EGFR-TKIs in lung adenocarcinoma. We first established erlotinib-resistant cell lines (PC9/ER) from parental PC9 cells by exposing the cells to erlotinib. In PC9/ER, autophagy-related LC3A expression came to be up-regulated and constitutive activation of LC3A-mediated autophagy became more pronounced through the process of acquiring therapeutic resistance. In addition, inhibition of LC3A or autophagy restores sensitivity to EGFR-TKIs in PC9/ER. LC3A was also activated at the transcriptional level in de novo resistant cells via demethylation of the MAP1LC3A gene. We then evaluated the status of LC3A in 169 lung adenocarcinoma patients using immunohistochemistry. LC3A immunoreactivity was only detected in carcinoma cells (89/169 patients), not in non-tumoural cells. In addition, LC3A immunoreactivity was significantly correlated with progression-free survival (p = 0.0039) and overall survival (p = 0.0040) of 35 patients treated with EGFR-TKIs. The results of our present study demonstrated that LC3A-mediated autophagy in carcinoma cells was involved in the development of resistance to EGFR-TKIs, and that LC3A could serve as a promising therapeutic target for overcoming resistance to EGFR-TKIs and a novel predictor of response to EGFR-TKIs in lung adenocarcinoma patients. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Limited information on the degree of irregularity of branching patterns of a bronchial tree may obscure the cause of heterogeneous distribution of the lesions in a variety of lung diseases. We reconstructed three dimensional (3-D) images from hilum to terminal bronchioles of a mouse lung, and defined the irregularity of airway branching by diameter-based morphometric analysis. The relative diameter ratios of a daughter to the parent branch (D1/D0) and those of a minor to major daughter branches (D1/D2) were calculated, and irregular dichotomies were found to be distributed in 48% of bifurcations. D1/D0 is well correlated with D1/D2, and is proved to indicate regular and irregular branching, as well as D1/D2. Irregular branches with D1/D0 smaller than 0.4 correspond to typical lateral branches, taking off from major bronchi. Our novel 3-D morphometric analysis showed the first portrayal of the 3-D structures of mouse bronchial airways, which provides a quantitative description of branching patterns leading to the correlation with distribution of lesions in the diseased lung.

The possible roles of airway branching patterns on the pathogenesis of lung diseases, especially on the heterogeneous distribution of the lesions, were examined through three-dimensional (3-D) morphometric analysis of a mouse lung injury, induced by bleomycin. On serial sections of a mouse lung damaged by subcutaneous injection of bleomycin, we performed a computer-assisted reconstruction of the lung for visualizing the relation of airways with the distribution of the lesions, and defined the features of bronchial routes to each lesion by four parameters: LTB; the distance from the hilum, Ng; generation numbers, RD(min); irregularity of airway branching, and thetaTB; the grade of airway recurrence. Among these four parameters, only thetaTB was found to correlate with the severity of lesions (p0.05 by an ANOVA test), which was proved by a posthoc test (p0.0001). These results showing the acini supplied by recurrent branches are more prone to be damaged than those by non-recurrent branches suggest that branching patterns may underlie the heterogeneous distribution of lesions in diffuse interstitial lung diseases.

Papillary serous carcinoma of the peritoneum (PSCP) is a primary neoplasm of peritoneal origin, and is histologically difficult to differentiate from papillary serous carcinoma of the ovary (PSCO). PSCP is frequently accompanied by many peritoneal tumors, and has been managed as a disseminated disease. In previous reports, however, the clonality of the tumors has not been fully discussed. Recently, the significant roles of the p53 and BRCA1 genes in PSCP have been reported. In this study, we investigated immunohistochemical staining for p53 proteins, and investigated p53 gene mutations, using DNA sequencing analysis, to clarify the clonality of PSCP tumors. Immunohistochemically, all the tumor samples demonstrated nuclear overexpression of p53 proteins, and the DNA sequencing analysis of the p53 gene showed diverse point mutations at codons 167 and 192 in two of four anatomically different tumors. In conclusion, the possibility of polyclonality of PSCP tumors is suggested.

The heterogeneity of tumor cells is frequently observed in lung cancer, but the clonality of these cells has not yet been established. The distinct components of 12 lung adenosquamous carcinomas were compared by genetic alterations of p53 and K-ras, chromosomal abnormalities at 9p21 and 9q31-32, and immunohistochemical reactions. The immunoreactivity of p53 was consistent in both adenocarcinomatous and squamous cell carcinomatous components as well as in the transitional areas, retaining the morphological characteristics of the distinct components. The same p53 mutation was found in both components of each tumor with p53 overexpression. No K-ras mutations were detected in any of the tumors examined. Three of the four tumors with chromosomal abnormalities detected, one at 9p21 and two at 9q31-32, had coincident abnormalities between the distinct components, whereas one tumor deleted homozygously at 9p21 (D9S259) in the adenocarcinomatous component with loss of heterozygosity in the other component. The expression of squamous cell carcinoma-related antigen in adenocarcinomatous components was significantly higher than that of lung adenocarcinomas (57 +/- 5.8% vs. 1.0 +/- 0.5%, P < 0.0001), whereas Mucin 1 expression is less in these components (9.0 +/- 4.9% vs. 55 +/- 8.2%, P = 0.003). These results suggest monoclonal transition from squamous cell carcinoma to adenocarcinoma in lung adenosquamous carcinoma.

Uterine sarcomas are unusual neoplasms of the female genital tract whose molecular etiology is largely unknown. We examined 20 leiomyomas as well as 23 uterine leiomyosarcomas for the presence of mutations in the Ki-ras and p53 genes, and overexpression of the MDM2 gene. Codons 12, 13, and 61 from the Ki-ras gene were characterized for the presence of mutations by restriction enzyme polymorphisms using mismatched primers and nucleic acid sequencing as appropriate. Activated Ki-ras genes were identified in 3/20 leiomyomas and 0/23 leiomyosarcomas. The p53 gene was analyzed by SSCP, nucleic acid sequencing, and immunohistochemical staining. None of 20 leiomyomas and 6/23 leiomyosarcomas exhibited p53 abnormalities. The SSCP/sequencing results did not consistently correlate with the IHC staining. MDM2 overexpression occurred in 0/20 leiomyomas and 3/23 sarcomas. Clinical correlation suggested that tumors with p53 mutations have a higher histologic grade or stage at presentation. We conclude that leiomyomas and leiomyosarcomas have different patterns of molecular alterations and are separate biologic entities. In addition, p53 and MDM2 overexpression may play a role in the development of a subset of leiomyosarcomas.

A 55-year-old non-smoking woman was admitted to our hospital for re-evaluation of unimproved peripheral ground-glass opacities despite prednisolone and cyclosporine treatment. She was diagnosed with autoimmune pulmonary alveolar proteinosis (PAP) based on transbronchial lung biopsy and granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody testing. GM-CSF inhalation therapy markedly improved the opacities. Bilateral, centrally located lung opacities are typical in PAP, however 10 PAP cases with peripheral infiltration were reported in Japan recently, of which GM-CSF antibody was positive in six. To avoid inappropriate immunosuppressant treatment, PAP should be considered in the differential diagnosis of such peripheral opacities. GM-CSF antibody might be useful for diagnosis.

The Runx1 transcription factor is abundantly expressed in naive T cells but rapidly downregulated in activated T cells, suggesting that it plays an important role in a naive stage. In the current study, Runx1−/−Bcl2tg mice harboring Runx1-deleted CD4+ T cells developed a fatal autoimmune lung disease. CD4+ T cells from these mice were spontaneously activated, preferentially homed to the lung, and expressed various cytokines, including IL-17 and IL-21. Among these, the deregulation of IL-21 transcription was likely to be associated with Runx binding sites located in an IL-21 intron. IL-17 produced in Runx1-deleted cells mobilized innate immune responses, such as those promoted by neutrophils and monocytes, whereas IL-21 triggered humoral responses, such as plasma cells. Thus, at an initial stage, peribronchovascular regions in the lung were infiltrated by CD4+ lymphocytes, whereas at a terminal stage, interstitial regions were massively occupied by immune cells, and alveolar spaces were filled with granular exudates that resembled pulmonary alveolar proteinosis in humans. Mice suffered from respiratory failure, as well as systemic inflammatory responses. Our data indicate that Runx1 plays an essential role in repressing the transcription of cytokine genes in naive CD4+ T cells and, thereby, maintains cell quiescence.

We have demonstrated that multipotent stromal cells (MSCs) promote cell survival though upregulation and secretion of stanniocalcin-1 (STC1) (Stem Cells 2009; 27: 670-81). This study demonstrates that MSC derived STC1 promotes survival of lung cancer A549 cells through the uncoupling of oxidative phosphorylation, the reduction of intracellular reactive oxygen species (ROS) and concomitant shift towards a more glycolytic and more oxygen consumptive metabolic profile (known as the Uncoupling Respiration). MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549s in an STC1 dependent manner. Knock down of UCP2 reduced the ability of MSCs to reduce cell death in the A549 population. Our data suggest that MSCs promote cell survival by regulating the uncoupling respiration in an STC1 dependent manner. Furthermore, STC1 may provide promising avenues for treatment of reactive oxygen species and metabolic disorders.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)- and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.

The pathogenesis of acute exacerbation of idiopathic pulmonary fibrosis (IPF) remains to be elucidated. To evaluate the roles of inflammatory mediators in acute exacerbation, the concentrations of high mobility group protein B1 (HMGB1), a chief mediator of acute lung injury, and 18 inflammatory cytokines were measured in the bronchoalveolar lavage fluid, serially sampled from seven IPF patients after the onset of acute exacerbation. HMGB1 gradually increased in the alveolar fluid after the onset of acute exacerbation, in positive correlation with monocytes chemotactic protein-1 (MCP-1), a potent fibrogenic mediator. In the lung tissues of eight IPF patients autopsied after acute exacerbation, intense cytoplasmic staining for HMGB1 was observed in the alveolar epithelial cells in alveolar capillary augmented lesions, where the capillary endothelial cells remarkably reduced the expression of thrombomodulin, an intrinsic antagonist of HMGB1. These results suggest pathogenic roles for HMGB1 and MCP-1 in the late phase of acute exacerbation of IPF.

The Nrf2 transcription factor is crucial for regulating the cellular defense against various carcinogens. However, relationship between host Nrf2 and cancer metastasis remains unexplored. To address this issue, we examined susceptibility of Nrf2-deficient mice to pulmonary cancer metastasis following implantation of the mouse Lewis lung carcinoma (3LL) cell line. Nrf2-deficient mice reproducibly exhibited a higher number of pulmonary metastatic nodules than wild-type mice did. The lung and bone marrow (BM) of cancer-bearing Nrf2-deficient mice contained increased numbers of inflammatory cells, including myeloid-derived suppressor cells (MDSCs), a potent population of immunosuppressive cells. MDSCs can attenuate CD8(+) T-cell immunity through modification of the T-cell receptor complex exploiting reactive oxygen species (ROS). MDSCs of Nrf2-deficient mice retained elevated levels of ROS relative to wild-type mice. BM transplantation experiments revealed functional disturbance in the hematopoietic and immune systems of Nrf2-deficient mice. Wild-type recipient mice with Nrf2-deficient BM cells showed increased levels of lung metastasis after cancer cell inoculation. These mice exhibited high-level accumulation of ROS in MDSCs, which showed very good coincidence to the decrease of splenic CD8(+) T-cells. In contrast, Keap1-knockdown mutant mice harboring high-level Nrf2 expression displayed increased resistance against the cancer cell metastasis to the lung, accompanied by a decrease in ROS in the MDSCs fraction. Our results thus reveal a novel function for Nrf2 in the prevention of cancer metastasis, presumably by its ability to preserve the redox balance in the hematopoietic and immune systems.

Moesin belongs to the ezrin/radixin/moesin (ERM) protein family and participates in cellular functions, such as morphogenesis and motility, by cross-linking between the actin cytoskeleton and plasma membranes. Although moesin seems necessary for tissue construction and repair, its function at the whole body level remains elusive, perhaps because of redundancy among ERM proteins. To determine the role played by moesin in the modulation of pulmonary alveolar structure associated with lung injury and repair, we examined the morphological changes in the lung and the effect of bleomycin-induced lung injury and fibrosis in moesin-deficient ( Msn−/ Y) and control wild-type mice ( Msn+/ Y). Immunohistochemical analysis revealed that moesin was specifically localized in the distal lung epithelium, where ezrin and radixin were faintly detectable in Msn+/ Ymice. Compared with Msn+/ Ymice, Msn−/ Ymice displayed abnormalities of alveolar architecture and, when treated with bleomycin, developed more prominent lung injury and fibrosis and lower body weight and survival rate. Furthermore, Msn−/ Ymice had abnormal cytokine and chemokine gene expression as shown by real-time PCR. This is the first report of a functional involvement of moesin in the regulation of lung inflammation and repair. Our observations show that moesin critically regulates the preservation of alveolar structure and lung homeostasis.

High-mobility group box chromosomal protein 1 (HMGB1) has recently been shown to be an important late mediator of endotoxin shock, intraabdominal sepsis, and acute lung injury, and a promising therapeutic target of severe sepsis. We sought to investigate the effect of antibodies to HMGB1 on severe sepsis in a rat cecal ligation and puncture (CLP) model. Adult male Sprague-Dawley rats underwent CLP and then were randomly divided into two groups: treatment with anti-HMGB1 polyclonal antibodies, and non-immune IgG-treated controls. The serum HMGB1 concentrations were measured at ten time points (preoperatively, and postoperatively at 4, 8, 20, 32, and 48 h and at 3, 4, 5, and 6 days). Hematoxylin-eosin staining, elastica-Masson staining, and immunohistochemical staining for HMGB1 were performed on the cecum and the lung to assess pathological changes 24 h after the CLP procedure. Treatment with anti-HMGB1 antibodies significantly increased survival [55% (anti-HMGB1) vs. 9% (controls); P< 0.01]. The serum HMGB1 concentrations at postoperative hours 20 and 32 of the anti-HMGB1 antibody-treated animals were significantly lower than those of the controls (P

Proinflammatory cytokines play an important role in ventilator-induced lung injury (VILI). High-mobility group box-1 (HMGB1) is a macrophage-derived proinflammatory cytokine that can cause lung injury.This study tested the hypothesis that HMGB1 is released in intact lungs ventilated with large Vt. A second objective was to identify the source of HMGB1. A third objective was to examine the effects of blocking HMGB1 on the subsequent development of VILI.Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained from rabbits mechanically ventilated for 4 h with a small (8 ml/kg) versus a large (30 ml/kg) Vt. BALF was also obtained from rabbits with intratracheal instillation of anti-HMGB1 antibody before the initiation of large Vt ventilation.The concentrations of HMGB1 in BALF were fivefold higher in the large than in the small Vt group. Immunohistochemistry and immunofluorescence studies revealed expression of HMGB1 in the cytoplasm of macrophages and neutrophils in lungs ventilated with large Vt. Blocking HMGB1 improved oxygenation, limited microvascular permeability and neutrophil influx into the alveolar lumen, and decreased concentrations of tumor necrosis factor-alpha in BALF.These observations suggest that HMGB1 could be one of the deteriorating factors in the development of VILI.

Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor-beta (TGF-beta) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF-beta and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly-ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF-beta receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF-beta receptors. In contrast to Smad7, expression of Arkadia is down-regulated by TGF-beta. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF-beta and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF-beta superfamily signalling under both physiological and pathological conditions.

The bioactive and potent androgen 5alpha-dihydrotestosterone (DHT) has been postulated to be involved in the development of branching morphogenesis in the human fetal lung, but its expression has not been examined. We therefore examined the expression of the androgen receptor (AR) and 5alpha-reductases (type 1 and type 2), which catalyse the conversion of testosterone into DHT, in the human fetal lung using immunohistochemistry and reverse transcription-PCR (RT-PCR). Immunoreactive AR was detected predominantly in the nuclei of epithelial cells of the budding component of the early gestational fetal lung. 5alpha-Reductase type 1 immunoreactivity was detected in the cytoplasm of epithelial cells, whereas immunoreactivity for 5alpha-reductase type 2 was not detected in the samples of human fetal lung examined. RT-PCR also confirmed the presence of AR and 5alpha-reductase in all fetal lung and epithelial cell lines. The results of our present study suggest that DHT may play an important role in epithelial cells, which might include precursor cells, in which both AR and 5alpha-reductases are expressed during branching morphogenesis of the human fetal lung.

Urocortin (Ucn) is a new member of the corticotropin-releasing factor (CRF) neuropeptide family and has positive inotropic actions and protective effects against ischemia in the rat heart. Ucn binds with very high affinity to both CRF receptor type 1 (CRF-R1) and CRF receptor type 2 (CRF-R2). However, to date, endogenous ligand(s) for CRF receptors expressed in the human heart have yet to be elucidated. In this study, we therefore examined the expression of Ucn and CRF receptors in human heart obtained at autopsy by RT-PCR, immunohistochemistry, and RIA. RT-PCR analysis demonstrated that Ucn and CRF-R2alpha mRNAs were detected in all four chambers. CRF-R1 mRNA was weakly present in some left atria, left ventricles, and in one right ventricle. CRF-R2beta mRNA was detected predominantly in the left atrium. CRF mRNA was not detected in any of the four chambers. Immunostaining for both Ucn and CRF receptors was detected in cardiac myocytes in all four chambers. Ucn-like immunoreactivity was detected in all four chambers by RIA, with the highest concentrations in the left ventricle (1.90 +/- 0.5 pmol/g wet weight, mean +/- SEM; n = 4). On the other hand, CRF-like immunoreactivity was very low or undetectable in the human heart. Sephadex G-50 column chromatography demonstrated that most of the Ucn-like immunoreactivity in the human heart was eluting earlier than the standard Ucn, with one minor peak in the position for Ucn. Ucn immunoreactivity was not detected in skeletal muscle by immunohistochemistry or RIA. These results suggest that Ucn is produced in the human heart and stored there mainly in the larger molecular weight forms. Endogenously produced Ucn may therefore exert its effects mostly through CRF-R2 in an autocrine and/or paracrine manner in the human heart.

Many solid tumors, including non-small cell lung cancers (NSCLCs), are characterized by heterogenous expression of p53 protein in the neoplastic cells. To analyse the molecular implications of this finding, we examined topographic distribution of p53 mutations using in situ polymerase chain reaction (PCR) in primary NSCLCs, showing distinct patterns of variable p53 overexpression by immunohistochemistry. Unique sets of primers for each mutation were designed, and optimal PCR conditions were determined by standard PCR using DNA from cloned mutants or cell lines established from these tumors. All tumor cell nuclei, regardless of the status of p53 overexpression, demonstrated homogeneous distribution of mutant p53 with specific primers, indicating that only subgroups of the mutated cells overexpressed p53 protein. In situ reverse transcription (RT)-PCR was applied to detect mutant mRNA in the individual tumor cells using specific primers. We found that in each case the distribution of mutant p53 mRNA coincided with that of immunohistochemical overexpression of p53 protein. Our results suggest that the regulation of mutant p53 expression, but not the genotype, is heterogeneous in the neoplastic cells. The topographic genomapping of p53 in NSCLC using in situ PCR provides a novel approach to view molecular mechanisms of lung carcinogenesis.

Concentrations of H+ and HCO3- rise in fluid lining hypercapnic airways. Effects of these ions on pulmonary endocrine cells were studied in 119 fetal rat lung organ cultures by semiquantitative staining for calcitonin gene-related peptide (CGRP)-like immunoreactive material. Intracellular CGRP was determined in cultures under "no-release" baseline conditions and after incubation in control or test media. After exposure to HCO3(-)-free medium at pH 7.4 (incubation control), CGRP fell moderately from no-release levels. Bombesin (1 ng/ml) promoted further significant loss of peptide, which was dependent on extracellular Ca2+ and inhibited by somatostatin and [D-Arg(1),D-Pro(2),D-Trp(7,9),Leu(11)]substance P, a bombesin receptor antagonist. CGRP staining of explants incubated with 24 mM HCO3- maintained no-release levels at and above pH 7.1 but decreased significantly at pH 6.8. The drop was blocked by somatostatin or exclusion of HCO3- and was not augmented by bombesin or 48 mM HCO3-. Results suggest that pulmonary endocrine cells may respond to hypercapnia by releasing bioactive peptides like CGRP, thus stimulating afferent nerves and altering patterns of ventilation and perfusion.

Background: Epidermal growth factor receptor (EGFR) mutant-lung cancer showed significant response to EGFR-tyrosine kinase inhibitor (TKI) treatment, however, treatment failure due to acquired resistance to EGFR-TKI become important clinical problem for long-term survivors. Our purpose is to develop the new treatment for EGFR-TKI resistance. Methods: We used PC9 human lung cancer cell line harboring EGFR exon 19 deletion (E746-A750). When PC9 cells were cultured with 2 μM gefitinib solved in dimethylsulfoxide (DMSO), most of cells died and surviving cells proliferated slowly. By 90 days-culture with 2 μM gefitinib, PC9 gefitinib resistant cells (PC9GR90) were established and confirmed that EGFR-T790M resistance-related mutation was negative. In PC9GR90, an immunoblot assay revealed MET (hepatocyte growth factor receptor) amplification and alternative EGFR3 tyrosine phosphorylation, which is known as one of the major resistant mechanisms. Results: When PC9GR90 were cultured with 2 μM gefitinib and one inhibitor (histone deacetylase inhibitor, insulin-like growth factor receptor inhibitor, gamma secretase inhibitor, or imatinib known as bcr-abl, c-kit and platelet derived growth factor receptor inhibitor), 10 μM imatinib treatment with gefitinib showed significant suppression of cell growth. The ratio of cell count of 2 μM gefitinib/DMSO and 10 μM imatinib with 2 μM gefitinib/DMSO were 91.7% and 6.0%, respectively. When imatinib concentration was decreased to 1 μM known as appropriate concentration for chronic myeloid leukemia treatment, there was no effect. The imatinib effect was not shown for NCI-H1975 lung cancer cells harboring both EGFR-L858R and -T790M. By immunoblot, we found EGFR3 phosphorylation was inhibited by imatinib treatment. Conclusion: It is the first report that imatinib inhibits EGFR3 phophorylation and induce cell death in the EGFR-TKI resistant lung cancer with MET amplification and EGFR3 phophorylation. Although further studies are required to apply this strategy to a clinical setting, we demonstrate a hopeful data to conquer one important resistant mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1793. doi:1538-7445.AM2012-1793