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author:("Ji, honglian")
1.  Spectrum of LKB1, EGFR, and KRAS Mutations in Chinese Lung Adenocarcinomas 
Introduction
Somatic LKB1 mutations are found in lung adenocarcinomas at different frequencies in Caucasian and East Asian (Japanese and Korean) populations. This study was designed to characterize the frequency of LKB1 mutations, their relationship to EGFR and KRAS mutations, and their associated clinicopathologic characteristics in Chinese patients.
Methods
Two hundred thirty-nine lung adenocarcinomas consecutively collected from October 2007 to July 2009 were dissected into 3 to 4 small (3 mm) pieces for histopathological analyses of tumor content. Genomic DNA and/or cDNA from 86 samples with more than 70% tumor content were used for sequencing of LKB1 (exons 1–9), EGFR (exons 18–21), and KRAS (exon 2). LKB1 germline mutation status was determined by sequencing of genomic DNA from matched histologically distant lung tissues that are histologically normal.
Results
6.9% of lung adenocarcinomas harbored LKB1 somatic mutations. A total of 10.5% of patients had an LKB1 germline polymorphism, F354L. Interestingly, in two of these patients, tumors displayed loss of heterozygosity at this allele. EGFR kinase domain and KRAS mutations were found in 66.3% and 2.3% of Chinese lung adenocarcinomas, respectively. Concurrent LKB1 and EGFR somatic mutations were observed in one patient. Both KRAS-mutant tumors harbored LKB1 mutations.
Conclusions
These data provide important clinical and molecular characteristics of lung adenocarcinomas from Chinese patients.
doi:10.1097/JTO.0b013e3181e05016
PMCID: PMC4009449  PMID: 20559149
Chinese lung adenocarcinoma; LKB1; EGFR; KRAS; Mutation
2.  The RNA-Binding Protein QKI Suppresses Cancer-Associated Aberrant Splicing 
PLoS Genetics  2014;10(4):e1004289.
Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.
Author Summary
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicates that splicing program is frequently deregulated during tumorigenesis, and cancer cells favor to produce protein isoforms that can promote growth and survival. Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide. Although a number of lung cancer-related splicing events have been detected in several genome-wide analyses, much less is known about how aberrant splicing takes place in lung cancer and how it contributes to tumor development. In this study, we characterized the RNA-binding protein QKI as a new critical regulator of alternative splicing in lung cancer and as a potential marker for prognosis. Genome-wide analysis of QKI-dependent splicing by RNA-Seq identified some cancer-associated splicing changes as its targets. Our results demonstrate that QKI-5 inhibits cancer cell proliferation and prevents inappropriate activation of the Notch signaling pathway by regulating its key target, NUMB. We further showed that QKI-5 represses the inclusion of NUMB alternative exon through competing with a core splicing factor SF1. In summary, our data indicate that down-regulation of QKI causes aberrant splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated repression of Notch signaling.
doi:10.1371/journal.pgen.1004289
PMCID: PMC3983035  PMID: 24722255
3.  Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome 
Cancer letters  2013;342(1):36-42.
Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.
doi:10.1016/j.canlet.2013.08.032
PMCID: PMC3981830  PMID: 23981578
P53 V157D; PMS2 R20Q; Germline mutation; Familial cancer syndrome; Co-existing
4.  Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling 
eLife  2013;2:e00999.
Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped–Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation.
DOI: http://dx.doi.org/10.7554/eLife.00999.001
eLife digest
Most tissues can generate new cells to repair damage or replace worn-out cells. The new cells are often generated from stem cells—cells that can either reproduce themselves or mature into other types of cells. In the fruit-fly Drosophila, for example, intestinal stem cells in the midgut are capable of producing more stem cells or they can differentiate to produce immature cells called enteroblasts that go on to become either enterocytes (the cells that line the gut) or enteroendocrine cells (which secrete hormones).
Researchers have identified a number of signalling pathways that are involved in the proliferation and differentiation of intestinal stem cells in the midgut of fruit flies. These include the Hippo pathway, which is important for regulating both cell proliferation and programmed cell death (apoptosis). Activation of the Hippo protein triggers a cascade of signals that culminate in the regulation of many of the genes involved in cell proliferation, division and apoptosis.
Another process that is important for controlling the proliferation and differentiation of cells is chromatin remodelling. Chromatin is the ‘packaging’ that keeps DNA tightly wound within the cell nucleus, and remodelling refers to the structural changes that allow proteins called transcription factors to reach the genes and transcribe them into messenger RNA (which then leaves the nucleus to generate the protein).
Now, Jin et al. have explored how the Hippo pathway and chromatin remodelling work together to regulate of stem cells. Using a technique called RNA interference to block the expression of various genes in intestinal stem cells and enteroblasts, Jin et al. found that a protein called Brahma—which is an essential part of a chromatin-remodelling complex—must be present for the stem cells to multiply normally.
Jin et al. also showed how the Hippo signalling pathway interacts with chromatin remodelling. Activation of the Hippo pathway inhibits gene expression by preventing two other proteins, Yorkie and Scalloped, from forming a complex in the nucleus. The new work shows that Brahma interacts physically with the Yorkie and Scalloped proteins to regulate the proliferation of the intestinal stem cells. It also shows that the Hippo protein regulates the activity of the Brahma protein by inducing a process called caspase-dependent cleavage. Because many of the proteins involved in these pathways are evolutionarily conserved and expressed in a variety of tissues, these findings may have implications for stem cell function and tissue repair in many species.
DOI: http://dx.doi.org/10.7554/eLife.00999.002
doi:10.7554/eLife.00999
PMCID: PMC3796317  PMID: 24137538
Hippo signaling; brahma; midgut; D. melanogaster
5.  Detecting somatic point mutations in cancer genome sequencing data: a comparison of mutation callers 
Genome Medicine  2013;5(10):91.
Background
Driven by high throughput next generation sequencing technologies and the pressing need to decipher cancer genomes, computational approaches for detecting somatic single nucleotide variants (sSNVs) have undergone dramatic improvements during the past 2 years. The recently developed tools typically compare a tumor sample directly with a matched normal sample at each variant locus in order to increase the accuracy of sSNV calling. These programs also address the detection of sSNVs at low allele frequencies, allowing for the study of tumor heterogeneity, cancer subclones, and mutation evolution in cancer development.
Methods
We used whole genome sequencing (Illumina Genome Analyzer IIx platform) of a melanoma sample and matched blood, whole exome sequencing (Illumina HiSeq 2000 platform) of 18 lung tumor-normal pairs and seven lung cancer cell lines to evaluate six tools for sSNV detection: EBCall, JointSNVMix, MuTect, SomaticSniper, Strelka, and VarScan 2, with a focus on MuTect and VarScan 2, two widely used publicly available software tools. Default/suggested parameters were used to run these tools. The missense sSNVs detected in these samples were validated through PCR and direct sequencing of genomic DNA from the samples. We also simulated 10 tumor-normal pairs to explore the ability of these programs to detect low allelic-frequency sSNVs.
Results
Out of the 237 sSNVs successfully validated in our cancer samples, VarScan 2 and MuTect detected the most of any tools (that is, 204 and 192, respectively). MuTect identified 11 more low-coverage validated sSNVs than VarScan 2, but missed 11 more sSNVs with alternate alleles in normal samples than VarScan 2. When examining the false calls of each tool using 169 invalidated sSNVs, we observed >63% false calls detected in the lung cancer cell lines had alternate alleles in normal samples. Additionally, from our simulation data, VarScan 2 identified more sSNVs than other tools, while MuTect characterized most low allelic-fraction sSNVs.
Conclusions
Our study explored the typical false-positive and false-negative detections that arise from the use of sSNV-calling tools. Our results suggest that despite recent progress, these tools have significant room for improvement, especially in the discrimination of low coverage/allelic-frequency sSNVs and sSNVs with alternate alleles in normal samples.
doi:10.1186/gm495
PMCID: PMC3971343  PMID: 24112718
6.  The CRTC1-NEDD9 Signaling Axis Mediates Lung Cancer Progression Caused by LKB1 Loss 
Cancer research  2012;72(24):6502-6511.
Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.
doi:10.1158/0008-5472.CAN-12-1909
PMCID: PMC3755891  PMID: 23074285
7.  Par-1 Regulates Tissue Growth by Influencing Hippo Phosphorylation Status and Hippo-Salvador Association 
PLoS Biology  2013;11(8):e1001620.
Par-1 regulates the Hippo signaling pathway in Drosophila melanogaster by modifying the phosphorylation status of Hippo and also by inhibiting the interaction of Hippo and Salvador.
The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.
Author Summary
An organism's organ size is determined by cell number, the size of each cell, and the distance between cells. All of these factors are controlled by the coordination of different cell signaling pathways and other mechanisms. The Hippo signaling pathway controls organ size by restricting the number of cells that make up the organ. Malfunction of this pathway leads to abnormal overgrowth, and is involved in a large number of human diseases and cancers. We identify here a component of the Hippo pathway, Par-1, which controls tissue growth by negatively regulating the Hippo pathway. We show that overexpression or depletion of Par-1 influences tissue growth in fruit flies via Hippo signaling. Then, by genetic and biochemical experiments, we show that Par-1 interacts with Hippo, regulating the Hippo Ser30 phosphorylation status to alter Hippo activity. In addition, we found that Par-1 regulates Hippo signaling via inhibition of the Hippo-Salvador association in a kinase-dependent fashion. We predict that Par-1 is a potential oncogene and that its regulatory role in Hippo signaling could be conserved.
doi:10.1371/journal.pbio.1001620
PMCID: PMC3735459  PMID: 23940457
9.  ROS1 Rearrangements Define a Unique Molecular Class of Lung Cancers  
Journal of Clinical Oncology  2012;30(8):863-870.
Purpose
Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase gene have recently been described in a subset of non–small-cell lung cancers (NSCLCs). Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with NSCLC with ROS1 rearrangement.
Patients and Methods
Using a ROS1 fluorescent in situ hybridization (FISH) assay, we screened 1,073 patients with NSCLC and correlated ROS1 rearrangement status with clinical characteristics, overall survival, and when available, ALK rearrangement status. In vitro studies assessed the responsiveness of cells with ROS1 rearrangement to the tyrosine kinase inhibitor crizotinib. The clinical response of one patient with ROS1-rearranged NSCLC to crizotinib was investigated as part of an expanded phase I cohort.
Results
Of 1,073 tumors screened, 18 (1.7%) were ROS1 rearranged by FISH, and 31 (2.9%) were ALK rearranged. Compared with the ROS1-negative group, patients with ROS1 rearrangements were significantly younger and more likely to be never-smokers (each P < .001). All of the ROS1-positive tumors were adenocarcinomas, with a tendency toward higher grade. ROS1-positive and -negative groups showed no difference in overall survival. The HCC78 ROS1-rearranged NSCLC cell line and 293 cells transfected with CD74-ROS1 showed evidence of sensitivity to crizotinib. The patient treated with crizotinib showed tumor shrinkage, with a near complete response.
Conclusion
ROS1 rearrangement defines a molecular subset of NSCLC with distinct clinical characteristics that are similar to those observed in patients with ALK-rearranged NSCLC. Crizotinib shows in vitro activity and early evidence of clinical activity in ROS1-rearranged NSCLC.
doi:10.1200/JCO.2011.35.6345
PMCID: PMC3295572  PMID: 22215748
10.  A network-based gene-weighting approach for pathway analysis 
Cell Research  2011;22(3):565-580.
Classical algorithms aiming at identifying biological pathways significantly related to studying conditions frequently reduced pathways to gene sets, with an obvious ignorance of the constitutive non-equivalence of various genes within a defined pathway. We here designed a network-based method to determine such non-equivalence in terms of gene weights. The gene weights determined are biologically consistent and robust to network perturbations. By integrating the gene weights into the classical gene set analysis, with a subsequent correction for the “over-counting” bias associated with multi-subunit proteins, we have developed a novel gene-weighed pathway analysis approach, as implemented in an R package called “Gene Associaqtion Network-based Pathway Analysis” (GANPA). Through analysis of several microarray datasets, including the p53 dataset, asthma dataset and three breast cancer datasets, we demonstrated that our approach is biologically reliable and reproducible, and therefore helpful for microarray data interpretation and hypothesis generation.
doi:10.1038/cr.2011.149
PMCID: PMC3292304  PMID: 21894192
gene weighting; functional association network; pathway analysis; gene set analysis; gene expression microarray; multi-subunit protein
11.  Correction: Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele 
PLoS ONE  2012;7(7):10.1371/annotation/0671c124-a263-49e9-9c23-421fd125db2c.
doi:10.1371/annotation/0671c124-a263-49e9-9c23-421fd125db2c
PMCID: PMC3394811
12.  Consensus Rules in Variant Detection from Next-Generation Sequencing Data 
PLoS ONE  2012;7(6):e38470.
A critical step in detecting variants from next-generation sequencing data is post hoc filtering of putative variants called or predicted by computational tools. Here, we highlight four critical parameters that could enhance the accuracy of called single nucleotide variants and insertions/deletions: quality and deepness, refinement and improvement of initial mapping, allele/strand balance, and examination of spurious genes. Use of these sequence features appropriately in variant filtering could greatly improve validation rates, thereby saving time and costs in next-generation sequencing projects.
doi:10.1371/journal.pone.0038470
PMCID: PMC3371040  PMID: 22715385
13.  Integrative analysis of gene and miRNA expression profiles with transcription factor–miRNA feed-forward loops identifies regulators in human cancers 
Nucleic Acids Research  2012;40(17):e135.
We describe here a novel method for integrating gene and miRNA expression profiles in cancer using feed-forward loops (FFLs) consisting of transcription factors (TFs), miRNAs and their common target genes. The dChip-GemiNI (Gene and miRNA Network-based Integration) method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression between two biological conditions such as normal and cancer. GemiNI integrates not only gene and miRNA expression data but also computationally derived information about TF–target gene and miRNA–mRNA interactions. Literature validation shows that the integrated modeling of expression data and FFLs better identifies cancer-related TFs and miRNAs compared to existing approaches. We have utilized GemiNI for analyzing six data sets of solid cancers (liver, kidney, prostate, lung and germ cell) and found that top-ranked FFLs account for ∼20% of transcriptome changes between normal and cancer. We have identified common FFL regulators across multiple cancer types, such as known FFLs consisting of MYC and miR-15/miR-17 families, and novel FFLs consisting of ARNT, CREB1 and their miRNA partners. The results and analysis web server are available at http://www.canevolve.org/dChip-GemiNi.
doi:10.1093/nar/gks395
PMCID: PMC3458521  PMID: 22645320
14.  Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele 
PLoS ONE  2012;7(5):e37308.
Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.
doi:10.1371/journal.pone.0037308
PMCID: PMC3350485  PMID: 22606359
15.  Hsp90 Inhibition Suppresses Mutant EGFR-T790M Signaling and Overcomes Kinase Inhibitor Resistance 
Cancer Research  2008;68(14):5827-5838.
The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non–small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M– expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of lung cancers.
doi:10.1158/0008-5472.CAN-07-5428
PMCID: PMC3272303  PMID: 18632637
16.  Spectrum of Oncogenic Driver Mutations in Lung Adenocarcinomas from East Asian Never Smokers 
PLoS ONE  2011;6(11):e28204.
Purpose
We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.
Experimental Design
We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.
Results
152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.
Conclusion
The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.
doi:10.1371/journal.pone.0028204
PMCID: PMC3227646  PMID: 22140546
17.  Lung Adenocarcinoma From East Asian Never-Smokers Is a Disease Largely Defined by Targetable Oncogenic Mutant Kinases 
Journal of Clinical Oncology  2010;28(30):4616-4620.
Purpose
To determine the proportion of lung adenocarcinomas from East Asian never-smokers who harbor known oncogenic driver mutations.
Patients and Methods
In this surgical series, 52 resected lung adenocarcinomas from never-smokers (< 100 cigarettes in a lifetime) at a single institution (Fudan University, Shanghai, China) were analyzed concurrently for mutations in EGFR, KRAS, NRAS, HRAS, HER2, BRAF, ALK, PIK3CA, TP53 and LKB1.
Results
Forty-one tumors harbored EGFR mutations, three harbored EML4-ALK fusions, two harbored HER2 insertions, and one harbored a KRAS mutation. All mutations were mutually exclusive. Thus, 90% (47 of 52; 95% CI, 0.7896 to 0.9625) of lung adenocarcinomas from never-smokers were found to harbor well-known oncogenic mutations in just four genes. No BRAF, NRAS, HRAS, or LKB1 mutations were detected, while 15 had TP53 mutations. Four tumors contained PIK3CA mutations, always together with EGFR mutations.
Conclusion
To our knowledge, this study represents the first comprehensive and concurrent analysis of major recurrent oncogenic mutations found in a large cohort of lung adenocarcinomas from East Asian never-smokers. Since drugs are now available that target mutant EGFR, HER2, and ALK, respectively, this result indicates that prospective mutation testing in these patients should successfully assign a targeted therapy in the majority of cases.
doi:10.1200/JCO.2010.29.6038
PMCID: PMC2974342  PMID: 20855837
18.  Integrative genomic and proteomic analyses identify targets for Lkb1 deficient metastatic lung tumors 
Cancer cell  2010;17(6):547-559.
Summary
In mice, Lkb1 deletion and activation of KrasG12D results in lung tumors with a high penetrance of lymph node and distant metastases. We analyzed these primary and metastatic de novo lung cancers with integrated genomic and proteomic profiles and have identified gene and phosphoprotein signatures associated with Lkb1 loss and progression to invasive and metastatic lung tumors. These studies revealed that SRC is activated in Lkb1 deficient primary and metastatic lung tumors and that the combined inhibition of SRC, PI3K and MEK1/2 resulted in synergistic tumor regression. These studies demonstrate that integrated genomic and proteomic analyses can be used to identify signaling pathways that may be targeted for treatment.
doi:10.1016/j.ccr.2010.04.026
PMCID: PMC2901842  PMID: 20541700
19.  Neutrophil Elastase-Mediated Degradation of IRS-1 Accelerates Lung Tumor Growth 
Nature medicine  2010;16(2):219-223.
Summary
Lung cancer is the leading cause of cancer death worldwide1. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness2-3. To determine the role of neutrophil elastase (NE or Elane) on tumor progression, we utilized the LSL-K-ras model of murine lung adenocarcinoma4 to generate LSL-K-ras/Elane−/− mice. Tumor burden was markedly reduced in LSL-K-ras/Elane−/− mice at all time points following induction of mutant K-ras expression. Kaplan-Meier life survival analysis demonstrated that while 100% of LSL-K-ras/Elane+/+ mice died, none of the mice lacking NE died. NE directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells where it degraded insulin receptor substrate-1 (IRS1). Co-immunoprecipitation studies showed that as NE degraded IRS1, there was increased interaction between PI3K and the potent mitogen platelet derived growth factor receptor (PDGFR) thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between NE and IRS1 in LSL-K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS1 as a key regulator of PI3K within malignant cells. Additionally, this is the first description of a secreted proteinase gaining access to a cell beyond its plasma membrane and altering intracellular signaling.
doi:10.1038/nm.2084
PMCID: PMC2821801  PMID: 20081861
20.  HLungDB: an integrated database of human lung cancer research 
Nucleic Acids Research  2009;38(Database issue):D665-D669.
The human lung cancer database (HLungDB) is a database with the integration of the lung cancer-related genes, proteins and miRNAs together with the corresponding clinical information. The main purpose of this platform is to establish a network of lung cancer-related molecules and to facilitate the mechanistic study of lung carcinogenesis. The entries describing the relationships between molecules and human lung cancer in the current release were extracted manually from literatures. Currently, we have collected 2585 genes and 212 miRNA with the experimental evidences involved in the different stages of lung carcinogenesis through text mining. Furthermore, we have incorporated the results from analysis of transcription factor-binding motifs, the promoters and the SNP sites for each gene. Since epigenetic alterations also play an important role in lung carcinogenesis, genes with epigenetic regulation were also included. We hope HLungDB will enrich our knowledge about lung cancer biology and eventually lead to the development of novel therapeutic strategies. HLungDB can be freely accessed at http://www.megabionet.org/bio/hlung.
doi:10.1093/nar/gkp945
PMCID: PMC2808962  PMID: 19900972
21.  Therapeutic anti-EGFR antibody 806 generates responses in murine de novo EGFR mutant–dependent lung carcinomas 
Journal of Clinical Investigation  2007;117(2):346-352.
Activating EGFR mutations occur in human non–small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%–30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.
doi:10.1172/JCI30446
PMCID: PMC1770949  PMID: 17256054
22.  Respiratory Failure Due to Differentiation Arrest and Expansion of Alveolar Cells following Lung-Specific Loss of the Transcription Factor C/EBPα in Mice¶  
Molecular and Cellular Biology  2006;26(3):1109-1123.
The leucine zipper family transcription factor CCAAT enhancer binding protein alpha (C/EBPα) inhibits proliferation and promotes differentiation in various cell types. In this study, we show, using a lung-specific conditional mouse model of C/EBPα deletion, that loss of C/EBPα in the respiratory epithelium leads to respiratory failure at birth due to an arrest in the type II alveolar cell differentiation program. This differentiation arrest results in the lack of type I alveolar cells and differentiated surfactant-secreting type II alveolar cells. In addition to showing a block in type II cell differentiation, the neonatal lungs display increased numbers of proliferating cells and decreased numbers of apoptotic cells, leading to epithelial expansion and loss of airspace. Consistent with the phenotype observed, genes associated with alveolar maturation, survival, and proliferation were differentially expressed. Taken together, these results identify C/EBPα as a master regulator of airway epithelial maturation and suggest that the loss of C/EBPα could also be an important event in the multistep process of lung tumorigenesis. Furthermore, this study indicates that exploring the C/EBPα pathway might have therapeutic benefits for patients with respiratory distress syndromes.
doi:10.1128/MCB.26.3.1109-1123.2006
PMCID: PMC1347037  PMID: 16428462
23.  Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma 
Nature Communications  2014;5:3261.
Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.
Non-small cell lung cancer manifests as adenocarcinoma and squamous cell carcinoma but tumours of mixed lineage are often found in humans. Here, the authors show that adenocarcinoma can transdifferentiate into squamous cell carcinomas in mice.
doi:10.1038/ncomms4261
PMCID: PMC3929783  PMID: 24531128

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