Clinical characteristics and outcome of lung cancer in patients with fibrosing interstitial lung disease.

BACKGROUND: Lung cancer (LC) is an important comorbidity of interstitial lung disease (ILD) and has a poor prognosis. The clinical characteristics and outcome of each ILD subtype in LC patients have not been sufficiently investigated. Therefore, this study aimed to evaluate the difference between idiopathic pulmonary fibrosis (IPF) and non-IPF ILD as well as prognostic factors in patients with ILD-LC. METHODS: The medical records of 163 patients diagnosed with ILD-LC at Asan Medical Center from January 2018 to May 2023 were retrospectively reviewed. Baseline characteristics and clinical outcomes were compared between the IPF-LC and non-IPF ILD-LC groups, and prognostic factors were analyzed using the Cox proportional-hazard model. RESULTS: The median follow-up period was 11 months after the cancer diagnosis. No statistically significant differences were observed in clinical characteristics and mortality rates (median survival: 26 vs. 20 months, p = 0.530) between the groups. The independent prognostic factors in patients with ILD-LC were higher level of Krebs von den Lungen-6 (≥ 1000 U/mL, hazard ratio [HR] 1.970, 95% confidence interval [CI] 1.026-3.783, p = 0.025) and advanced clinical stage of LC (compared with stage I, HR 3.876 for stage II, p = 0.025, HR 5.092 for stage III, p = 0.002, and HR 5.626 for stage IV, p = 0.002). In terms of treatment, surgery was the significant factor for survival (HR 0.235; 95% CI 0.106-0.520; p < 0.001). CONCLUSIONS: No survival difference was observed between IPF-LC and non-IPF ILD-LC patients. A higher level of Krebs von den Lungen-6 may act as a prognostic marker in ILD-LC patients.

AXL receptor tyrosine kinase inhibition improves the anti-tumor effects of CD8+ T cells by inducing CD103+ dendritic cell-mediated T cell priming.

AXL is a member of TAM receptor family and has been highlighted as a potential target for cancer treatment. Accumulating evidence has uncovered the critical role of the AXL signaling pathway in tumor growth, metastasis, and resistance against anti-cancer drugs, as well as its association with cancer immune escape. However, the function of AXL as a manipulator of the immune system in the tumor microenvironment (TME) remains unclear. Therefore, in this study, we investigated the impact of AXL on immune cells in the TME of a syngeneic tumor model using AXL knockout (AXL-/-) mice. Compared to AXL wild-type (AXL+/+) mice, tumor growth was significantly suppressed in AXL-/- mice, and an induced population of tumor-infiltrated CD8+ T cells and CD103+ dendritic cells (DCs) was observed. The change of CD8+ T cells and CD103+ DCs was also confirmed in tumor-draining lymph nodes (TdLN). In addition, the clonal expansion of OVA-specific CD8+ T cells was dominant in AXL-/- mice. Finally, anti-PD-1 treatment evidenced synergistic anti-cancer effects in AXL-/- mice. Overall, our data indicate that AXL signaling may inhibit the clonal expansion of tumor-specific CD8+ T cells through the regulation of the migration of CD8+ T cells and DCs in TME. Thus, AXL may be a powerful molecular target to improve anti-cancer effects through single or combined therapy with immune checkpoint inhibitors (ICI).

Stratifying non-small cell lung cancer patients using an inverse of the treatment decision rules: validation using electronic health records with application to an administrative database.

BACKGROUND: To validate a stratification method using an inverse of treatment decision rules that can classify non-small cell lung cancer (NSCLC) patients in real-world treatment records. METHODS: (1) To validate the index classifier against the TNM 7th edition, we analyzed electronic health records of NSCLC patients diagnosed from 2011 to 2015 in a tertiary referral hospital in Seoul, Korea. Predictive accuracy, stage-specific sensitivity, specificity, positive predictive value, negative predictive value, F1 score, and c-statistic were measured. (2) To apply the index classifier in an administrative database, we analyzed NSCLC patients in Korean National Health Insurance Database, 2002-2013. Differential survival rates among the classes were examined with the log-rank test, and class-specific survival rates were compared with the reference survival rates. RESULTS: (1) In the validation study (N = 1375), the overall accuracy was 93.8% (95% CI: 92.5-95.0%). Stage-specific c-statistic was the highest for stage I (0.97, 95% CI: 0.96-0.98) and the lowest for stage III (0.82, 95% CI: 0.77-0.87). (2) In the application study (N = 71,593), the index classifier showed a tendency for differentiating survival probabilities among classes. Compared to the reference TNM survival rates, the index classification under-estimated the survival probability for stages IA, IIIB, and IV, and over-estimated it for stages IIA and IIB. CONCLUSION: The inverse of the treatment decision rules has a potential to supplement a routinely collected database with information encoded in the treatment decision rules to classify NSCLC patients. It requires further validation and replication in multiple clinical settings.

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

Isolation and screening of indigenous microalgae species for domestic and livestock wastewater treatment, biodiesel production, and carbon sequestration.

The use of indigenous microalgae strains for locally generated domestic (DWW) and livestock wastewater (LWW) treatment is essential for effective and economical applications. Phototrophic microalgae-based biofuel production also contributes to carbon sequestration via CO2 fixation. However, simultaneous consideration of both isolation and screening procedures for locally collected indigenous microalgae strains is not common in the literature. We aimed to isolate indigenous microalgae strains from locally collected samples on coastlines and islands in South Korea. Among five isolated strains, Chlorella sorokiniana JD1-1 was selected for DWW and LWW treatment due to its ability to grow in waste resources. This strain showed a higher specific growth rate in DWW than artificial growth medium (BG-11) with a range of 0.137-0.154 d-1. During cultivation, 96.5%-97.1% of total nitrogen in DWW and 89.2% in LWW was removed. Over 99% of total phosphorus in DWW and 96.4% in LWW was also removed. Finally, isolated C. sorokiniana JD1-1 was able to fix CO2 within a range of 0.0646-0.1043 g CO2 L-1 d-1. These results support the domestic applications of carbon sequestration-efficient microalgae in the waste-to-energy nexus.

Optimizing palliative chemotherapy for advanced invasive mucinous adenocarcinoma of the lung.

BACKGROUND: A primary pulmonary invasive mucinous adenocarcinoma (IMA) is a rare subtype of invasive adenocarcinoma of the lung. The prognosis of advanced IMA depending on chemotherapy regimen has not been fully investigated. Here, we compared the clinical outcomes of patients with advanced IMA treated with different palliative chemotherapies that included novel therapeutics. METHODS: This single-center retrospective study included a total of 79 patients diagnosed with IMA and treated with palliative chemotherapy. The primary outcome was the comparison of overall survival according to palliative chemotherapy type. Risk factors associated with death were evaluated as a secondary outcome. RESULTS: The study cohort of 79 patients comprised 27 progressive or recurrent cases and 52 initial metastatic patients. Thirteen patients (16.5%) received targeted therapy and 18 cases (22.8%) received immunotherapy. When we compared the survival outcomes of the different treatment regimens, patients with IMA treated by immunotherapy (undefined vs. non-immunotherapy 17.0 months, p < 0.001) had better overall survival rates. However, there was no difference in the prognosis between the cases treated with a targeted therapy (35.6 vs. non-targeted therapy 17.0 months, p = 0.211). None of the conventional regimens produced a better outcome. By multivariable analysis, immunotherapy (HR 0.28; 95% CI 0.11-0.74; P = 0.008) was found to be an independent prognostic factor for death. CONCLUSIONS: This study suggests that immunotherapy for patients with advanced IMA may provide favorable outcomes than other chemotherapy options.

Establishment and Long-Term Expansion of Small Cell Lung Cancer Patient-Derived Tumor Organoids.

Differential chemo-sensitivity of cancer cells, which is attributed to the cellular heterogeneity and phenotypic variation of cancer cells, is considered to be the main reason for tumor recurrence after chemotherapy. Here, we generated small cell lung cancer patient-derived tumor organoids and subjected them to long-term expansion with the addition of WNT3A or R-spondin1. We confirmed that the organoids have similar genetic profiles, molecular characteristics, and morphological architectures to the corresponding patient tumor tissue during and after long-term expansion. Interestingly, the cellular heterogeneity of organoids is reflected in their differential response to cisplatin or etoposide. We propose to utilize the organoids as small cell lung cancer patient avatar models that would be ideal for investigating the mechanisms underlying tumor recurrence after chemotherapy, and would ultimately help to develop personalized medicine.

How Cancer Patients Perceive Clinical Trials (CTs) in the Era of CTs: Current Perception and Its Differences Between Common and Rare Cancers.

Perception has recently been highlighted as a critical determinant for participation in clinical trials (CTs) among cancer patients. We evaluated cancer patients' current perceptions of CTs using the PARTAKE questionnaires, focusing on differences between patients with common and rare cancers. From November 2015 to May 2017, we prospectively surveyed patients who had received anti-cancer treatment at Asan Medical Center. Among 333 respondents, 70.9% had common and 29.1% had rare cancers. In the cohort, 87.7% of patients with common cancers and 75.3% of patients with rare cancers answered that they heard of and knew about CTs. However, willingness to participate in CTs was expressed only in approximately 56% of patients, although it was significantly associated with awareness and perception. Surprisingly, patients with rare cancers when compared with patients with common cancers showed significantly lower levels of awareness and perception (64.2% vs 79.9%, p = 0.003 and 77.3% vs 91.9%, p < 0.001), and consequently less willingness to participate in CTs (47.4% vs 58.9%, p = 0.06). In addition, cancer patients still harbored fear and concerns about safety and reward of CTs, and demonstrated substantial lack of knowledge about the voluntary nature of CTs, which was more obvious in patients with rare cancers. We identified relatively modest willingness of cancer patients to participate in CTs regardless of generally favorable perception. These findings are highlighted by the more negative perception of CTs among patients with rare cancers relative to those with common cancers. Further education and encouragement by research and public entities seem essential to improve motivation of CTs in cancer patients beyond good perception, especially for patients with rare cancers.

Genomic profiles of lung cancer associated with idiopathic pulmonary fibrosis.

Little is known about the pathogenesis or molecular profiles of idiopathic pulmonary fibrosis-associated lung cancer (IPF-LC). This study was performed to investigate the genomic profiles of IPF-LC and to explore the possibility of defining potential therapeutic targets in IPF-LC. We assessed genomic profiles of IPF-LC by using targeted exome sequencing (OncoPanel version 2) in 35 matched tumour/normal pairs surgically resected between 2004 and 2014. Germline and somatic variant calling was performed with GATK HaplotypeCaller and MuTect with GATK SomaticIndelocator, respectively. Copy number analysis was conducted with CNVkit, with focal events determined by Genomic Identification of Significant Targets in Cancer 2.0, and pathway analysis (KEGG) with DAVID. Germline mutations in TERT (rs2736100, n = 33) and CDKN1A (rs2395655, n = 27) associated with idiopathic pulmonary fibrosis risk were detected in most samples. A total of 410 somatic mutations were identified, with an average of 11.7 per tumour, including 69 synonymous, 177 missense, 17 nonsense, 1 nonstop and 11 splice-site mutations, and 135 small coding indels. Spectra of the somatic mutations revealed predominant C > T transitions despite an extensive smoking history in most patients, suggesting a potential association between APOBEC-related mutagenesis and the development of IPF-LC. TP53 (22/35, 62.9%) and BRAF (6/35, 17.1%) were found to be significantly mutated in IPF-LC. Recurrent focal amplifications in three chromosomal loci (3q26.33, 7q31.2, and 12q14.3) and 9p21.3 deletion were identified, and genes associated with the JAK-STAT signalling pathway were significantly amplified in IPF-LC (P = 0.012). This study demonstrates that IPF-LC is genetically characterized by the presence of somatic mutations reflecting a variety of environmental exposures on the background of specific germline mutations, and is associated with potentially targetable alterations such as BRAF mutations. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Retrospective Study on Efficacy and Safety of Rivaroxaban and Dalteparin for Long-Term Treatment of Venous Thromboembolism in Patients with Lung Cancer.

BACKGROUND: Standard therapy for cancer-associated venous thromboembolism (VTE) is low-molecular-weight heparin. The use of direct oral anticoagulants for cancer-associated VTE has increased; however, their efficacy and safety in lung cancer patients remain unclear. OBJECTIVES: We examined the efficacy and safety of rivaroxaban compared with dalteparin for cancer-associated VTE in patients with primary lung cancer. METHODS: A single-center retrospective study of 204 patients with primary lung cancer who were prescribed rivaroxaban (n = 131) or dalteparin (n = 73) for VTE was performed. The primary endpoint was a composite event including recurrence and major or clinically relevant nonmajor bleeding. Secondary endpoints included the incidence of recurrence, major and clinically relevant nonmajor bleeding, all-cause mortality, and bleeding or pulmonary embolism-related mortality. RESULTS: The composite event occurred in 38 (29.0) and 12 (16.4%) patients in the rivaroxaban and dalteparin (p = 0.045) groups, respectively. The multivariate Cox proportional hazards model for age, Eastern Cooperative Oncology Group performance score, and bleeding risk factors revealed the rivaroxaban group showed a 1.176-fold composite event risk without statistical significance (0.595-2.324, p = 0.641). There was no statistically significant intergroup difference for the incidence of VTE recurrence (5.3% in the rivaroxaban group versus 2.7% in the dalteparin group, p = 0.495) and major or clinically relevant nonmajor bleeding (23.7% in the rivaroxaban group versus 13.7% in the dalteparin group, p = 0.089). There was no significant difference in the all-cause mortality rate (hazard ratio 0.864, 95% CI 0.624-1.196, p = 0.337). CONCLUSIONS: There was no difference in the safety and efficacy profile of rivaroxaban compared with dalteparin. Therefore, rivaroxaban may be a valuable treatment option for lung cancer-associated VTE.

Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients.

Tumor cells shed an abundance of extracellular vesicles (EVs) to body fluids containing bioactive molecules including DNA, RNA, and protein. Investigations in the field of tumor-derived EVs open a new horizon in understanding cancer biology and its potential as cancer biomarkers as well as platforms for personalized medicine. This study demonstrates that successfully isolated EVs from plasma and bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients contain DNA that can be used for EGFR genotyping through liquid biopsy. In both plasma and BALF samples, liquid biopsy results using EV DNA show higher accordance with conventional tissue biopsy compared to the liquid biopsy of cfDNA. Especially, liquid biopsy with BALF EV DNA is tissue-specific and extremely sensitive compared to using cfDNA. Furthermore, use of BALF EV DNA also demonstrates higher efficiency in comparison to tissue rebiopsy for detecting p.T790 M mutation in the patients who developed resistance to EGFR-TKIs. These finding demonstrate possibility of liquid biopsy using EV DNA potentially replacing the current diagnostic methods for more accurate, cheaper, and faster results.

Non-Small Cell Lung Cancer with Resistance to EGFR-TKI Therapy: CT Characteristics of T790M Mutation-positive Cancer.

Purpose To evaluate the clinical and CT characteristics of T790M mutation-positive non-small cell lung cancer (NSCLC) after epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy failure. Materials and Methods A retrospective study of 304 patients with NSCLC who underwent rebiopsy after first-line EGFR-TKI therapy was conducted. Rebiopsy methods included CT- or fluoroscopy-guided lung biopsies (n = 105), endobronchial US- or bronchofibroscopy-guided biopsies (n = 66), pleural fluid analysis (n = 47), other solid organ biopsies (n = 43), US-guided axillary or supraclavicular lymph node biopsies (n = 31), and cerebrospinal fluid analysis (n = 12). CT findings at the initial diagnosis and rebiopsy were analyzed. Progression-free survival, the duration from the start of TKI therapy to rebiopsy, and survival were calculated. Results At rebiopsy, 144 (47.4%) patients were T790M mutation positive. The percentages of T790M mutation-positive NSCLCs were similar in 106 patients with rebiopsy of the lungs (53 [50%] of 106) and in 77 patients with rebiopsy of the primary lung lesions (36 [47%] of 77). T790M mutation positivity was associated with peripheral tumors (odds ratio [OR], 2.6; P = .01), pleural tag (OR, 5.0; P < .001), and air bronchogram (OR, 4.0; P = .006) at CT after TKI failure. The duration from the start of TKI therapy to rebiopsy was longer in T790M mutation-positive than in T790M mutation-negative patients (20.5 vs 13.6 months; P < .001). Cumulative survival from the time of rebiopsy to the last follow-up was significantly longer in patients with T790M mutation-positive lung cancers (P = .014). However, median survival time after rebiopsy was not statistically different between patients with and those without T790M mutation. Conclusion Peripheral tumor location with vascular convergence, the presence of a pleural tag, and air bronchogram of lung lesions at CT at the time of rebiopsy were significantly associated with T790M mutation in patients with non-small cell lung cancer after first-line epidermal growth factor receptor-tyrosine kinase inhibitor therapy failure. © RSNA, 2018 Online supplemental material is available for this article.

Assessment of EGFR mutation status using cell-free DNA from bronchoalveolar lavage fluid.

BACKGROUND: Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. METHODS: From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. RESULTS: We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. CONCLUSIONS: cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.

GAB2 Amplification in Squamous Cell Lung Cancer of Non-Smokers.

Lung squamous cell cancer (SCC) is typically found in smokers and has a very low incidence in non-smokers, indicating differences in the tumor biology of lung SCC in smokers and non-smokers. However, the specific mutations that drive tumor growth in non-smokers have not been identified. To identify mutations in lung SCC of non-smokers, we performed a genetic analysis using arrays comparative genomic hybridization (ArrayCGH). We analyzed 19 patients with lung SCC who underwent surgical treatment between April 2005 and April 2015. Clinical characteristics were reviewed, and DNA was extracted from fresh frozen lung cancer specimens. All of copy number alterations from ArrayCGH were validated using The Cancer Genome Atlas (TCGA) copy number variation (CNV) data of lung SCC. We examined the frequency of copy number changes according to the smoking status (non-smoker [n = 8] or smoker [n = 11]). We identified 16 significantly altered regions from ArrayCGH data, three gain and four loss regions overlapped with the TCGA lung squamous cell carcinoma (LUSC) patients. Within these overlapped significant regions, we detected 15 genes that have been reported in the Cancer Gene census. We also found that the proto-oncogene GAB2 (11q14.1) was significantly amplified in non-smokers patients and vice versa in both ArrayCGH and TCGA data. Immunohistochemical analyses showed that GAB2 protein was relatively upregulated in non-smoker than smoker tissues (37.5% vs. 9.0%, P = 0.007). GAB2 amplification may have an important role in the development of lung SCC in non-smokers. GAB2 may represent a potential biomarker for lung SCC in non-smokers.

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment.

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

Hippo pathway effector YAP inhibition restores the sensitivity of EGFR-TKI in lung adenocarcinoma having primary or acquired EGFR-TKI resistance.

The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer.

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance.

Predictive factors for a long-term response duration in non-squamous cell lung cancer patients treated with pemetrexed.

BACKGROUND: Pemetrexed is widely used for the treatment of advanced non-squamous non-small-cell lung cancer (NSCLC). However, factors that can predict the benefits of pemetrexed therapy have not yet been defined. METHODS: We compared the clinical and molecule pathological characteristics of good and poor responders among a cohort of 1,848 non-squamous NSCLC patients who had received at least two cycles of pemetrexed therapy between November 2006 and February 2015. Among these cases, 92 good responders who were the top 5 % in terms of progression-free survival (PFS) and 222 poor responders who had progressive disease after only 2 cycles of therapy were selected for the analysis. RESULTS: The median PFS of the good responders was 29.9 months (range; 20.9-90.0) and the median number of cycle was 37 (range; 18-129). Although 53.5 % of patients showed stable disease (SD), this response was sustained (median PFS in SD, 29.6 months). A never-smoking status was related to better survival outcome, whereas EGFR mutation, two or more metastatic sites, and intra-abdominal metastasis were each associated with a poor PFS. ALK translocation showed a tendency for a positive impact on response to pemetrexed, whereas metastatic lesion to liver, adrenal gland or bone showed a tendency for a negative impact despite not reaching our threshold for statistical significance. CONCLUSIONS: Predictive factors, such as smoking status, the status of genetic alteration and tumor burden, should be considered when administering pemetrexed therapy for non-squamous NSCLC.

P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement.

BACKGROUND: Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement. METHODS: Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123. RESULTS: The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp. CONCLUSION: P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement.

Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.