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KRAS G12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRAS G12C -mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRAS G12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRAS G12C and Kras G12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer., Competing Interests: Declaration of interests A.J.A. has consulted for Anji Pharmaceuticals, Arrakis Therapeutics, AstraZeneca, Boehringer Ingelheim, Oncorus, Inc., Merck & Co., Inc., Mirati Therapeutics, Nimbus Therapeutics, Plexium, Revolution Medicines, Reactive Biosciences, Riva Therapeutics, Servier Pharmaceuticals, Syros Pharmaceuticals, T-knife Therapeutics. A.J.A. holds equity in Riva Therapeutics. A.J.A. has received research funding from Bristol Myers Squibb, Deerfield, Inc., Eli Lilly, Mirati Therapeutics, Novartis, Novo Ventures, Revolution Medicines, and Syros Pharmaceuticals. K.-K.W. has research funding and/or consulted for Janssen Pharmaceuticals, Pfizer, Bristol Myers Squibb, Zentalis Pharmaceuticals, Blueprint Medicines, Takeda Pharmaceuticals, Mirati Therapeutics, Novartis, Genentech, Merus, Bridgebio Pharma, Xilio Therapeutics, Allerion Therapeutics, Boehringer Ingelheim, Cogent Therapeutics, Revolution Medicines and AstraZeneca., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human lung adenosquamous cell carcinoma (LUAS), containing both adenomatous and squamous pathologies, exhibits strong cancer plasticity. We find that ALK rearrangement is detectable in 5.1-7.5% of human LUAS, and transgenic expression of EML4-ALK drives lung adenocarcinoma (LUAD) formation initially and squamous transition at late stage. We identify club cells as the main cell-of-origin for squamous transition. Through recapitulating lineage transition in organoid system, we identify JAK-STAT signaling, activated by EML4-ALK phase separation, significantly promotes squamous transition. Integrative study with scRNA-seq and immunostaining identify a plastic cell subpopulation in ALK-rearranged human LUAD showing squamous biomarker expression. Moreover, those relapsed ALK-rearranged LUAD show notable upregulation of squamous biomarkers. Consistently, mouse squamous tumors or LUAD with squamous signature display certain resistance to ALK inhibitor, which can be overcome by combined JAK1/2 inhibitor treatment. This study uncovers strong plasticity of ALK-rearranged tumors in orchestrating phenotypic transition and drug resistance and proposes a potentially effective therapeutic strategy., (© 2024 Qin et al.)

Lkb1 deficiency confers the Kras-mutant lung cancer with strong plasticity and the potential for adeno-to-squamous transdifferentiation (AST). However, it remains largely unknown how Lkb1 deficiency dynamically regulates AST. Using the classical AST mouse model (Kras LSL-G12D/+ ;Lkb1 flox/flox , KL), we here comprehensively analyze the temporal transcriptomic dynamics of lung tumors at different stages by dynamic network biomarker (DNB) and identify the tipping point at which the Wnt signaling is abruptly suppressed by the excessive accumulation of reactive oxygen species (ROS) through its downstream effector FOXO3A. Bidirectional genetic perturbation of the Wnt pathway using two different Ctnnb1 conditional knockout mouse strains confirms its essential role in the negative regulation of AST. Importantly, pharmacological activation of the Wnt pathway before but not after the tipping point inhibits squamous transdifferentiation, highlighting the irreversibility of AST after crossing the tipping point. Through comparative transcriptomic analyses of mouse and human tumors, we find that the lineage-specific transcription factors (TFs) of adenocarcinoma and squamous cell carcinoma form a "Yin-Yang" counteracting network. Interestingly, inactivation of the Wnt pathway preferentially suppresses the adenomatous lineage TF network and thus disrupts the "Yin-Yang" homeostasis to lean towards the squamous lineage, whereas ectopic expression of NKX2-1, an adenomatous lineage TF, significantly dampens such phenotypic transition accelerated by the Wnt pathway inactivation. The negative correlation between the Wnt pathway and AST is further observed in a large cohort of human lung adenosquamous carcinoma. Collectively, our study identifies the tipping point of AST and highlights an essential role of the ROS-Wnt axis in dynamically orchestrating the homeostasis between adeno- and squamous-specific TF networks at the AST tipping point., (© 2022. The Author(s).)

Tuberous sclerosis complex subunit 1 ( TSC1 ) and 2 ( TSC2 ) are frequently mutated in non-small cell lung cancer (NSCLC), however, their effects on antitumor immunity remained unexplored. A CRISPR screening in murine Kras G12D / Trp53 -/- (KP) model identified Tsc1 and Tsc2 as potent regulators of programmed cell death ligand 1 (Pd-l1) expression in vitro and sensitivity to anti-programmed cell death receptor 1 (PD-1) treatment in vivo. TSC1 or TSC2 knockout (KO) promoted the transcriptional and membrane expression of PD-L1 in cell lines. TSC2 -deficient tumors manifested an inflamed microenvironment in patient samples and The Cancer Genome Atlas dataset. In syngeneic murine models, KP- Tsc2 -KO tumors showed notable response to anti-PD-1 antibody treatment, but Tsc2 -wild-type tumors did not. Patients with TSC1 / TSC2 -mutant NSCLC receiving immune checkpoint blockade (ICB) had increased durable clinical benefit and survival. Collectively, TSC1 / TSC2 loss defines a distinct subtype of NSCLC characterized as inflamed tumor microenvironment and superior sensitivity to ICB.

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI., (© The Author(s) (2021). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Introduction: A comprehensive genomic analysis of paired primary tumors and their metastatic lesions may provide new insights into the biology of metastatic processes and therefore guide the development of novel strategies for intervention. To date, our knowledge of the genetic divergence and phylogenetic relationships among diverse metastatic lesions from cancer remains limited., Methods: We performed whole-exome sequencing in 84 tissue and blood samples from 26 patients with lung adenocarcinoma having liver metastases (LiM) or brain metastases (BrM) before any systemic therapy, with the goal to molecularly characterize the metastatic process. Mutational landscape and evolutionary patterns were compared between paired primary lesions (primary lesion of LiM or BrM) and metastases (metastatic site of LiM or BrM)., Results: We found that common driver mutations, including TP53 and EGFR, were highly consistent between paired primary and metastatic tumors. Although tumor mutational burden was comparable among groups, the LiM group had significantly higher mutational and copy number variational similarity than the BrM group between paired primary lesions and metastases (p = 0.019 and p = 0.035, respectively). Phylogenetic analysis further revealed that LiM-competent disseminations had a higher level of genetic similarity to their paired primary lesions and were genetically diverged from their primary tumors at a relatively later stage than those of BrM. These results suggest that LiM favorably followed the linear progression model, whereas BrM was more consistent with the parallel progression model., Conclusions: This study suggests that the mutational landscape and evolutionary pattern was distinctly different between the LiM and BrM of lung adenocarcinoma., (Copyright © 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 ( Npm1 ) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo . Furthermore, KRAS -mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. SIGNIFICANCE: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC., (©2020 American Association for Cancer Research.)

Alterations of gut microbiota have been implicated in multiple diseases including cancer. However, the gut microbiota spectrum in lung cancer remains largely unknown. Here we profiled the gut microbiota composition in a discovery cohort containing 42 early-stage lung cancer patients and 65 healthy individuals through the 16S ribosomal RNA (rRNA) gene sequencing analysis. We found that lung cancer patients displayed a significant shift of microbiota composition in contrast to the healthy populations. To identify the optimal microbiota signature for noninvasive diagnosis purpose, we took advantage of Support-Vector Machine (SVM) and found that the predictive model with 13 operational taxonomic unit (OTU)-based biomarkers achieved a high accuracy in lung cancer prediction (area under curve, AUC = 97.6%). This signature performed reasonably well in the validation cohort (AUC = 76.4%), which contained 34 lung cancer patients and 40 healthy individuals. To facilitate potential clinical practice, we further constructed a 'patient discrimination index' (PDI), which largely retained the prediction efficiency in both the discovery cohort (AUC = 92.4%) and the validation cohort (AUC = 67.7%). Together, our study uncovered the microbiota spectrum of lung cancer patients and established the specific gut microbial signature for the potential prediction of the early-stage lung cancer.

Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS -mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responses-such as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D / Trp53 -/- LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a deficiency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identified Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a deficiency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD. See related commentary by Menzel and Black, p. 179 . This article is highlighted in the In This Issue feature, p. 161 ., (©2019 American Association for Cancer Research.)

Background: Lung cancer is a very heterogeneous disease that can be pathologically classified into different subtypes including small-cell lung carcinoma (SCLC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and large-cell carcinoma (LCC). Although much progress has been made towards the oncogenic mechanism of each subtype, transcriptional circuits mediating the upstream signaling pathways and downstream functional consequences remain to be systematically studied., Results: Here we trained a one-class support vector machine (OC-SVM) model to establish a general transcription factor (TF) regulatory network containing 325 TFs and 18724 target genes. We then applied this network to lung cancer subtypes and identified those deregulated TFs and downstream targets. We found that the TP63/SOX2/DMRT3 module was specific to LUSC, corresponding to squamous epithelial differentiation and/or survival. Moreover, the LEF1/MSC module was specifically activated in LUAD and likely to confer epithelial-to-mesenchymal transition, known important for cancer malignant progression and metastasis. The proneural factor, ASCL1, was specifically up-regulated in SCLC which is known to have a neuroendocrine phenotype. Also, ID2 was differentially regulated between SCLC and LUSC, with its up-regulation in SCLC linking to energy supply for fast mitosis and its down-regulation in LUSC linking to the attenuation of immune response. We further described the landscape of TF regulation among the three major subtypes of lung cancer, highlighting their functional commonalities and specificities., Conclusions: Our approach uncovered the landscape of transcriptional deregulation in lung cancer, and provided a useful resource of TF regulatory network for future studies.

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a Kras G12D/ + mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx , Ptip , Acp5 , Acacb , and Clu , whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the Kras G12D/ + mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx -knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis., Competing Interests: The authors declare no conflict of interest.

The non-receptor tyrosine kinase BMX has been reported in several solid tumors. However, the alternative splicing of BMX and its clinical relevance in lung cancer remain to be elucidated. Exon1.0 array was used to identify a novel alternative splicing of BMX, BMXΔN, which was confirmed by rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. BMXΔN, resulting from exon skipping with excluding exon 1 to exon 8 of BMX gene, was found in 12% human lung adenocarcinoma specimens. BMXΔN is not found in paired pathologically normal lungs and positively correlated with EGFR mutation in lung adenocarcinomas. Moreover, BMXΔN increases cell proliferation, neoplastic transformation, and migratory property of human non-small cell lung cancer cells. The function of BMXΔN in lung cancer might be presumably due to enhanced ERK signaling.

Lung squamous cell carcinoma (SCC) is one of the major subtypes of lung cancer. Our current knowledge of oncogenic drivers in this specific subtype of lung cancer is largely limited compared with lung adenocarcinoma (ADC). Through exon array analyses, molecular analyses and functional studies, we here identify the TRA2B-DNAH5 fusion as a novel oncogenic driver in lung SCC. We found that this gene fusion occurs exclusively in lung SCC (3.1%, 5/163), but not in lung ADC (0/119). Through mechanistic studies, we further revealed that this TRA2B-DNAH5 fusion promotes lung SCC malignant progression through regulating a SIRT6-ERK1/2-MMP1 signaling axis. We show that inhibition of ERK1/2 activation using selumetinib efficiently inhibits the growth of lung SCC with TRA2B-DNAH5 fusion expression. These findings improve our current knowledge of oncogenic drivers in lung SCC and provide a potential therapeutic strategy for lung SCC patients with TRA2B-DNAH5 fusion.

LKB1 regulates both cell growth and energy metabolism. It remains unclear how LKB1 inactivation coordinates tumor progression with metabolic adaptation in non-small cell lung cancer (NSCLC). Here in Kras(G12D);Lkb1(lox/lox) (KL) mouse model, we reveal differential reactive oxygen species (ROS) levels in lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). ROS can modulate ADC-to-SCC transdifferentiation (AST). Further, pentose phosphate pathway deregulation and impaired fatty acid oxidation collectively contribute to the redox imbalance and functionally affect AST. Similar tumor and redox heterogeneity also exist in human NSCLC with LKB1 inactivation. In preclinical trials toward metabolic stress, certain KL ADC can develop drug resistance through squamous transdifferentiation. This study uncovers critical redox control of tumor plasticity that may affect therapeutic response in NSCLC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Whether the Hippo pathway contributes to cell lineage transition under pathological conditions, especially tumorigenesis, remains largely unknown. Here we show that YAP, the major effector of the Hippo pathway, displays a distinct activation pattern in lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC); YAP is initially activated by LKB1 loss in lung ADC, which upregulates ZEB2 expression and represses DNp63 transcription in a default manner. During transdifferentiation, YAP is inactivated, which in turn relieves ZEB2-mediated default repression of DNp63 and triggers squamous differentiation reprogramming. Disruption of the YAP barrier for phenotypic transition significantly accelerates squamous transdifferentiation, whereas constitutive YAP activation conversely inhibits this transition. More importantly, ectopic DNp63 expression rescues the inhibitory effect of YAP on squamous transdifferentiation. These findings have established YAP as an essential barrier for lung cancer cell fate conversion and provided a mechanism for regulating cancer plasticity, which might hold important implication for YAP-targeted therapies.

Metastasis is the major cause for high mortality of lung cancer with the underlying mechanisms poorly understood. The scaffolding protein neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) has been identified as a pro-metastasis gene in several types of cancers including melanoma and breast cancer. However, the exact role and related mechanism of NEDD9 in regulating lung cancer metastasis still remain largely unknown. Here, we demonstrate that NEDD9 knockdown significantly inhibits migration, invasion and metastasis of lung cancer cells in vitro and in vivo. The pro-metastasis role of Nedd9 in lung cancer is further supported by studies in mice models of spontaneous cancer metastasis. Moreover, we find that NEDD9 promotes lung cancer cell migration and invasion through the induction of epithelial-mesenchymal transition (EMT) potentially via focal adhesion kinase activation. More importantly, NEDD9 expression inversely correlates with E-cadherin expression in human lung cancer specimens, consistent with the findings from in vitro studies. Taken together, this study highlights that NEDD9 is an important mediator promotes lung cancer metastasis via EMT., (© 2013 UICC.)

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.

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.

Background: AAA+ nuclear coregulator cancer associated (ANCCA) is found to be overexpressed in various cancer types and could play a role in common and fundamental cellular processes. A recent study suggested that ANCCA was a likely driver whose expression explained the behavior of differentially expressed proliferation-related genes in lung adenocarcinoma. However, protein expression of ANCCA in lung adenocarcinoma and its association with clinicopathologic parameters and commonly reported driver mutations remains unexplored., Methods: ANCCA expression was evaluated by immunohistochemistry in 143 surgically resected lung adenocarcinomas and was correlated with clinicopathologic and molecular variables including adenocarcinoma histologic subtypes, tumor, node, metastasis status, relapse-free survival, overall survival, EGFR mutations, KRAS mutations, HER2 mutations and ALK fusions., Results: Positive ANCCA expression was significantly associated with male sex, smokers, poorly differentiated tumors, nonlepidic predominant subtype, more advanced T stage, lymph nodal metastasis and late disease stage. Cox multivariate analysis revealed that ANCCA-positive expression was an independent predictor of worse relapse-free survival [hazard ratio (HR) 1.736, 95 % confidence interval (CI) 1.075-2.804; P = .024) and overall survival (HR 7.758, 95 % CI 2.955-20.370; P < .001). The addition of ANCCA protein expression to the prognostic model using pathologic stage markedly improved the prognostic accuracy; the concordance index increased from .692 to .788, and the Akaike information criterion decreased from 354.20 to 336.11., Conclusions: We have identified ANCCA protein expression as a novel independent poor prognostic indicator in lung adenocarcinoma. Prospective studies are warranted to validate its potential prognostic value in combination with the current staging system.

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.

Purpose: Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA., Experimental Design: Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants., Results: Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20)., Conclusions: Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes., (©2012 AACR.)

High glycolysis, well known as "Warburg effect," is frequently observed in a variety of cancers. Whether the deregulation of miRNAs contributes to the Warburg effect remains largely unknown. Because miRNA regulates gene expression at both mRNA and protein levels, we constructed a gene functional association network, which allows us to detect the gene activity instead of gene expression, to integratively analyze the microarray data for gene expression and miRNA expression profiling and identify glycolysis-related gene-miRNA pairs deregulated in cancer. Hexokinase 2 (HK2), coding for the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143. Interestingly, miR-143 expression was inversely associated with HK2 protein level but not mRNA level in human lung cancer samples. miR-143, down-regulated by mammalian target of rapamycin activation, reduces glucose metabolism and inhibits cancer cell proliferation and tumor formation through targeting HK2. Collectively, we have not only established a novel methodology for gene-miRNA pair prediction but also identified miR-143 as an essential regulator of cancer glycolysis via targeting HK2.

Introduction: The epidermal growth factor receptor (EGFR) gene status including tyrosine kinase domain somatic mutations, increased copy number, and protein overexpression are reported to be associated with response to EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer. The purpose of this study was to elucidate the prevalence of activated EGFR gene and the association between mutation, copy number, and protein overexpression., Patients and Methods: In a cohort of consecutive patients with lung adenocarcinoma, polymerase chain reaction and direct sequencing (n = 89) were conducted through exons 18 to 21. Fluorescence in situ hybridization (FISH) (n = 89) and single-nucleotide polymorphism (SNP) array 6.0 (n = 77) were used to detect the gene copy number. The protein expression of EGFR was detected by standard immunohistochemistry (IHC) (n = 89)., Results: Fifty-nine (66.3%) patients harbored somatic mutations of EGFR in tyrosine kinase domain, 55.1% were positive by IHC and 44.9% were positive by FISH, and 66.2% showed gain of copy number according to SNP array 6.0. EGFR somatic mutations are more common in women, never smokers, and tumors with better differentiation. Increased copy number detected by both FISH and SNP array 6.0 analysis is significantly correlated with mutations of EGFR., Conclusions: The EGFR somatic mutation rate is significantly higher in Chinese patients with lung adenocarcinoma than western countries. Nevertheless, we found comparable FISH and IHC-positive rates between different ethnics. Considering that FISH may be affected by tumor heterogeneity and other factors, SNP array 6.0 analysis is a good alternative method to detect EGFR copy number variations.

LKB1 loss-of-function mutations, observed in ∼30% of human lung adenocarcinomas, contribute significantly to lung cancer malignancy progression. We show that lysyl oxidase (LOX), negatively regulated by LKB1 through mTOR-HIF-1α signaling axis, mediates lung cancer progression. Inhibition of LOX activity dramatically alleviates lung cancer malignancy progression. Up-regulated LOX expression triggers excess collagen deposition in Lkb1-deficient lung tumors, and thereafter results in enhanced cancer cell proliferation and invasiveness through activation of β1 integrin signaling. High LOX level and activity correlate with poor prognosis and metastasis. Our findings provide evidence of how LKB1 loss of function promotes lung cancer malignancy through remodeling of extracellular matrix microenvironment, and identify LOX as a potential target for disease treatment in lung cancer patients.

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.

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.