targeting creb pathway suppresses small cell lung cancer · oncogenes and tumor suppressors...

Oncogenes and Tumor Suppressors

Targeting CREB Pathway Suppresses Small CellLung CancerYifeng Xia1,2, Cheng Zhan2, Mingxiang Feng2, Mathias Leblanc3, Eugene Ke1,Narayana Yeddula1, and Inder M. Verma1

Abstract

Small cell lung cancer (SCLC) is the most deadly subtype oflung cancer due to its dismal prognosis. We have developed alentiviral vector-mediated SCLCmousemodel and have exploredthe role of both the NF-kB and CREB families of transcriptionfactors in this model. Surprisingly, induction of NF-kB activity,which promotes tumor progression in many cancer types includ-ing non–small cell lung carcinoma (NSCLC), is dispensable inSCLC. Instead, suppression of NF-kB activity in SCLC tumorsmoderately accelerated tumor development. Examination ofgene expression signatures of both mouse and human SCLCtumors revealed overall low NF-kB but high CREB activity.Blocking CREB activation by a dominant-negative form of PKA

(dnPKA) completely abolished the development of SCLC.Similarly, expression of dnPKA or treatment with PKA inhibitorH89 greatly reduced the growth of SCLC tumors in syngeneictransplantation models. Altogether, our results strongly suggestthat targeting CREB is a promising therapeutic strategy againstSCLC.

Implications: Activity of the transcription factor CREB is elevatedin SCLC tumors, which helps to maintain its neuroendocrinesignature and cell proliferation. Our results highlight the impor-tance of targeting the CREB pathway to develop new therapeuticsto combat SCLC. Mol Cancer Res; 16(5); 825–32. �2018 AACR.

IntroductionLung cancer represents nearly 20% of all cancer-related deaths

world-wide (1). Small cell lung cancer (SCLC) differs from othersubtypes of lung cancer (altogether called non–small cell lungcancer, NSCLC) by histopathology and general treatment strategyin the clinic. SCLC is also unique among all lung cancers for its cellof origin, spectrum of genetic mutations, and high potential tometastasize to distal organs. SCLC displays a neuroendocrine(NE) signature and is thus thought to be initiated from neuro-endocrine cells scattered underneath Clara cell and ciliated celllayer of bronchioles (2). Two major tumor suppressors, p53 andRb, are found extensively mutated in >90% of SCLC patients (3).Although it accounts only for�15%of all lung cancers inhumans,SCLC is the most malignant subtype with 5-year survival rate of

approved by the Institutional Animal Care and Use Committee(IACUC) of Salk Institute.

Clinical samplesHuman lung adenocarcinoma and SCLC samples were collect-

ed from surgeries performed in the Department of ThoracicSurgery, Zhongshan Hospital, Fudan University, with patientconsent forms. All procedures were carried out under InstitutionalReview Board (IRB)-approved protocols. Written informed con-sent was obtained from all patients and the study was conductedin accordance with recognized ethical guidelines.

Lentiviral production and mice infectionLentiviruses were produced as described previously (12). The

shRNA sequences used were: 50-GTACATGTGTAATAGCTCC-30

(p53); 50- AGCATATCTCCGACTAAATA-30 (Rb); 50- AAGATCAG-CATTCACAAATTA-30 (Pten). Viral titer was estimated byp24 ELISA with serial diluted viruses, and verified by infecting293-rtTA cells and examining Mycl1 expression by immunoblot-ting. For SCLC tumor model, 8-week-old R26-M2rtTA mice werepretreated with naphthalene (200 mg/kg, i.p.) 44 hours beforeviral infection, and then infected intra-tracheally with 2 � 105lentiviral particles using a protocol described before (7). Micewere switched to doxycycline diet (200 mg/kg; Bio-Serv) 1 weekafter the viral infection.

Histology, immunofluorescence staining, and immunoblottinganalysis

Mouse lung tumor samples collected at different time-pointswere fixed with 10% formalin, paraffin-embedded and sectionedfor hematoxylin and eosin staining (H&E) and immunofluores-cence staining. Elite ABC system (Vector Laboratories) wasappliedwhere staining signalwasweak. Immunoblotting analysiswas performed according to standard protocol. Antibodies werepurchased from Millipore (SPC, NCAM, both 1:2,000), Sigma(CGRP, 1:2,000), Abcam(NF-kBp65E379, 1:500), Cell SignalingTechnology (phos-CREB Ser133, Sox2, both 1:1,000), VectorLaboratories (Ki-67, 1:500; p53, 1:4,000), and Santa Cruz Bio-technology (L-Myc 1:1,000).

Primary cell culture and syngeneic transplant modelPrimary tumors isolated from SCLC lentiviral model were

dissociated with trypsin and cultured inmodifiedHITESmediumDMEM/F12 (1:1) supplemented with Glutamax, 4 mg/mLHydro-cortisone (Sigma), 5 ng/mL murine EGF (Invitrogen), insulin–transferrin–selenium mix/solution (Invitrogen) and 10% FBS.Culture dishes were precoated with poly 2-hydroxyethyl meth-acrylate (poly-HEMA; Sigma) to prevent cell adhesion and dif-ferentiation. Mycoplasma testing was performed using Commer-cial Kit (Lonza). SCLC cells (106) infected with different expres-sion vectors were transplanted subcutaneously into flank regionsof FVB hosts. Tumors were collected 4 weeks later, weighed andfixed for histopathologic analysis. PKA inhibitor treatment (H89,10 mg/kg in 75% DMSO/PBS, i.p.) was carried out 2 weeks afterthe transplantation for another 2 weeks.

Quantitative RT-PCRTotal RNA isolated from homogenized tumor samples or cell

lines was reverse transcribed using qScript cDNA SuperMix(Quantabio). Quantitative PCR was performed in triplicate using7900HT Fast Real-Time PCR system with SYBR green method

(Applied Biosystems). Results were analyzed for the relativeexpression of mRNAs normalized against GAPDH. A completelist of primers used in the study is shown in SupplementaryTable S2.

Electrophoretic mobility shift assayElectrophoretic mobility shift assay (EMSA) was carried out

using LightShiftChemiluminescent EMSAKit fromThermoFisherScientific and their standard protocol. Consensus DNA bindingsequences were listed as following. NF-kB 50-AGT TGAGGGGACTTT CCC AGG C-30; CREB 50-AGA GAT TGC CTG ACG TCA GAGAGC TAG-30; OCT1 50-TGT CGA ATG CAA ATC ACT AGA A-30.

RNAseq and GSEA analysisTotal RNA isolated from homogenized primary tissue samples

(eight normal lungs, six SCLC-luci tumors, and six SCLC-IkBaMtumors) was used to make stranded mRNA library. Single-endRNAseq was carried out using standard protocol on IlluminaHiSeq 2500 sequencer. Reads were then mapped to mouse(mm9) genome sequence using STAR. Differential gene expres-sion was analyzed using Cuffdiff. Human SCLC RNAseq raw dataof 19 normal lungs, 21 primary tumors, and 21 cell linesshared from Dr. Seshagiri group (13) were reanalyzed using asimilar method as described above. FPKM values of differentiallyexpressed genes, NF-kB genes, and CREB genes from multiplesamples were clustered and heatmaps were drawn with Cluster/TreeView softwares.

Statistical analysisStatistical significance of the differences of gene expression or

tumor size was evaluated using Student unpaired two-tailed t test.The Kaplan–Meier curves were analyzed by log-rank test.

Accession numbersRaw data of microarray analysis have been deposited to NCBI

Gene Expression Omnibus (GEO) with accession numberGSE89857.

ResultsA lentiviral mouse model of SCLC

Currently available mouse models of SCLC are all based onconditional knockout in lung cells of different tumor suppressorsincluding p53, Rb, p130, and Pten (6, 14–16). To faithfullyrecapitulate the human disease, we have successfully expandedour lentiviral vector-mediated cancer model to study SCLC inwild-type mice (Fig. 1A). Our result is consistent with earlierreports that loss of both p53 and Rb are indispensable to induceneuroendocrine tumors in lung whereas loss of Pten or over-expression ofMycl1 further accelerates tumor progression (6, 15).Interestingly, endogenous Mycl1 mRNA upregulation and Ptendownregulation are readily detected in tumors induced withshp53-shRb vector (Supplementary Fig. S1A and S1B). Consis-tently, if Mycl1 expression was lost upon the withdrawal ofdoxycycline,massive tumor shrinkagewas observed (Supplemen-tary Fig. S1C). All these observations indicated the requirementof enhanced Mycl1 expression and Pten suppression in SCLCdevelopment. Infected with a four-in-one vector (Mycl1-shp53-shRb-shPten), mice typically developed advanced SCLC within4months andhad amedium survival timeof 147days (comparedto 475 days with the shp53-shRb vector; Fig. 1B).

Xia et al.

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Figure 1.

Lentiviral vector-mediated SCLC mouse model highly recapitulates human tumors. A, Schematic of transducing lentiviral vector. Mycl1 is controlled by a TREpromoter (top) and activated in Rosa26-M2rtTA mice upon doxycycline chow or directly by a CMV promoter (bottom). shPten is also driven by a CMVpromoter in a miR-E backbone immediately after Mycl1, whereas other shRNAs are driven by U6 or H1 promoters. B, Kaplan–Meier survival curves showingeffects of lentiviral vector carrying different payloads.Median survival timeof the groupswas 475days (GFP-shp53-shRb, n¼9), 323days (Mycl1-shp53-shRb,n¼ 11),188 days (Mycl1-shp53-shRb(mU6), n ¼ 6, shRb(mU6) has better Rb knockdown by mouse U6 promoter), 250 days (Myc-shp53-shRb, n ¼ 7), 201 days(Cherry-3shRNA, n ¼ 8), and 147 days (Mycl1-3shRNA, n ¼ 5), respectively. C, Histopathologic analysis of SCLC tumors induced by TRE-Mycl1-shp53-shRb vector,showing tumor initiating from bronchus (i), in low magnification view (ii), forming nested pattern (iii), in high magnification view (iv), forming massive necrosis (v),spreading along vasculature (vi), invading to big bronchus (vii). Tumors stained negative for SPC and positive for CGRP, Ki67, NCAM, Sox2. Scale bars, 100 mm.D,Viewofwhole lung, liver and kidney, showing primary lung tumor (white circle),metastases tomediastinal lymph node (black circle), liver (right, yellow circle), andkidney (right inset, green circle). Scale bars, 5mm. E,Histopathologic analysis ofmetastases to liver, muscle, kidney, andmediastinal lymph node. Scale bars, 100 mm.

Targeting CREB in SCLC

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Histopathology of tumors induced by these vectors hadfeatures of neuroendocrine tumors: small cell-size, minimal cyto-plasm, salt and pepper chromatin, high mitotic index, massivenecrosis, positive for NCAM and CGRP, and negative for SPCstaining (Fig. 1C). These tumors were highly invasive within thelungs and metastasized to other organs (frequently to draininglymph nodes and liver, and rarely to muscle, kidney; Fig. 1Dand E). Furthermore, cell lines derived from these SCLC tumorswere easily transplantable in syngeneic hosts and grafts boreindistinguishable pathology as compared to the primary tumors(Supplementary Fig. S1D).

Inhibition of NF-kB pathway accelerates SCLC progression inmouse

It has been shown that blocking the NF-kB pathway can limittumor progression and prolong survival in many cancer typesincluding lung adenocarcinoma harboring Kras mutation (9).We next asked whether this also happens in our SCLCmodel. Wetherefore generated a lentiviral vector containing the dominantnegative form of IkBa (IkBaM) which cannot be phosphorylatedor ubiquitinylated, and hence is resistant to proteosomal degra-dation thereby blocking NF-kB activation in cells (Fig. 2A).Surprisingly, inhibition of NF-kB moderately accelerated SCLCprogression and resulted in a median survival time of 288 days ascompared to 328 days in control group (Fig. 2B).

Histopathologic analysis of these IkBaM tumors did not revealany differences in neuroendocrine markers (CGRP, NCAM) andmitotic index, compared to control tumors (luciferaseimaging; Fig. 2C). Further, control tumors did not reveal signif-icant differences in terms of differentially expressed genes (SCLCvs. normal lung) or the 36 SCLC signature genes defined by theBerns group (Supplementary Fig. S2A and S2B; ref. 17). Interest-ingly, SCLC samples had substantially lower NF-kB induciblegenes expression compared to normal lung tissues, even whenIkBaM was not present in the vector (luci SCLC tumors, Fig. 2D;also see human SCLC tumors in Supplementary Fig. S3A). Toexamine the actual NF-kB activity in SCLC tumor cells, we carriedout EMSA using consensus probes for NF-kB and other commontranscription factors. Surprisingly, we found that NF-kB bindingactivity was much lower in SCLC cells compared with lungadenocarcinoma cells induced by Kras-shp53 vector; however,SCLC cells had higher CREB binding activity (Fig. 2E; alsosee Fig. 2D and Supplementary Fig. S3B). This observation wasconsistent with many other reports that NF-kB and CREB path-ways counteract each other at multiple levels (18). Besides, GSEAanalysis showed that compared to control tumors, SCLC-IkBaMtumors were enriched for neuronal genes in line with the neuro-endocrine nature of the tumors (Fig. 2F). We further examinedNF-kB and CREB pathway activities in human lung cancersamples by IHC. As shown in Fig. 2G and SupplementaryTable S1, SCLC tissues indeed had low p65 nuclear staining buthigh phospho-CREB staining, which further supported the geneexpression data described earlier. Thus, the data presented heresuggests that increase in tumor progression from downregulationof NF-kB ultimately leads to high CREB activity which is requiredfor maintaining neuronal signature and for promoting cell pro-liferation in SCLC tumors.

Wehave previously shown that knockout of IKK2 in tumor cellsblocks NF-kB activation in a Kras-induced lung adenocarcinomamodel, leading to significantly impaired cell proliferation (7).This dichotomy in the role of the NF-kB family in SCLC and

NSCLC supports the notion that mutations commonly found inSCLC and lung adenocarcinoma rely on different signal pathwaysfor cell proliferation. To determine the generality of this hypoth-esis, we transformed mouse embryonic fibroblasts (MEF) withsimilar vectors that induced in our NSCLC and SCLC mousemodels. Consistent with in vivo data, IkBaM potently blockedtransformation (or cell proliferation) induced by Kras-shp53 butnot Mycl1-shp53-shRb (Supplementary Fig. S4A). More interest-ingly, Kras-shp53 vector induced enhanced NF-kB DNA bindingactivity whereas Mycl1-shp53-shRb vector induced higher CREBactivity even in these MEF cells (Supplementary Fig. S4B).

Role of CREB in maintaining neuroendocrine signature andhigh proliferation in SCLC

SCLC initiates from lung neuroendocrine cells (19) as tumorsexpress neuronalmarkers (Fig. 1C). A number of cells of neuronalorigin rely on CREB pathway for cell proliferation even thoughNF-kB and CREB pathwaysmay counteract each other at multiplelevels (20–22). To test the role of either signaling pathways ininduction of SCLC, we used lentiviral vectors expressing eitherIkBaM (to inhibit NF-kB activity) or dnPKA (to inhibit CREBpathway) in addition to three shRNAs against tumor suppressors(p53,Rb, and Pten; Fig. 3A).Mice transducedwith luciferase vector(as control) had median survival of 291 days. Mice expressingIkBaM had significantly accelerated tumor progression (mediansurvival of 186 days), while expressing IKK2SE (constitutivelyactive form of IKK2 leading to high basal NF-kB activity) delayedtumor progression (median survival of 396 days). Interestingly,expression of a dominant negative form of PKA (dnPKA) severelyimpaired tumor development and prolonged mouse survival(over 500 days; Fig. 3B).

To demonstrate that NF-kB and CREB pathways in SCLC cellsare antagonistic to each other, we infected them with 5xkB or3xCREB luciferase reporter constructs. In these SCLC cells, TNFatreatment activated the 5xkB reporter while inhibited the 3xCREBreporter. In contrast, forskoline treatment activated the 3xCREBreporter while inhibited the 5xkB reporter (Fig. 3C). Furthermore,TNFa treatment remarkably downregulated neuroendocrinesignature genes, including Calca, Syp, and Chga, in SCLC cells(Fig. 3D).

Targeting CREB pathway suppresses SCLC growth in vivoBasedon these observations,wehypothesized that blocking the

CREB pathway instead of the NF-kB pathway could benefit SCLCpatients. Therefore, we tested this idea by examining the in vivogrowth of a SCLC cell line derived from the 3-shRNA vector in asyngeneic transplant model. Upon induction of dnPKA, tumorswere substantially reduced in size (Fig. 4A). Apparently, thistumor reduction was due to the impaired activity of CREBpathway (see reduced CREB phosphorylation as compared tocontrol) and cell proliferation (Fig. 4B). As there are no specificPKA inhibitors available for in vivo study, we treatedmice carryingSCLC grafts with the broad spectrum PKA inhibitor H89 for 2weeks, and the compound reduced tumor size about 40% onaverage (Fig. 4C). All these results strongly suggest the possibilityof targeting SCLC by inhibiting the CREB pathway.

DiscussionOver quarter of a million people are diagnosed annually with

SCLC, one of the most deadly cancers with very poor prognosis.

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Figure 2.

SCLC has low NF-kB and high CREB activity. A, Diagram of lentiviral vector design. Luciferase or IkBaM gene is coexpressed with Mycl1 by 2A ribosomalskipping mechanism. B, Kaplan–Meier curve showing mice survival. Median survival time of the two groups was 328 days (luci, n¼ 17), and 288 days (IkBaM, n¼ 12)respectively, P ¼ 0.0029. C, Histopathologic analysis of IkBaM tumors showed no difference compared to control tumors in Fig. 1. D, Heatmap of theexpression values of 146 NF-kB target genes (left) and 245 CREB target genes (right) in normal lung, luci tumor, and IkBaM tumor. E, EMSA showing SCLC tumorshad lower NF-kB and higher CREB binding activity than lung ADC. F, GSEA analysis of gene sets enriched in IkBaM tumor vs. control luci tumors. G, Human SCLCand lung adenocarcinoma samples were stained for Ki67, p65, and phos-CREB (left). Total 10 SCLC and 10 adenocarcinoma samples were analyzed and theresults were summarized in Supplementary Table S1. Mouse SCLC and lung adenocarcinoma (KrasG12D-shp53) samples were stained for phos-CREB (right).Insets were in higher magnification showing nuclear staining. Scale bars, 100 mm.






Targeted therapies have been rapidly developed in the last twodecades against NSCLC with EGFR and ALKmutations; however,little progress has been made for SCLC. This, in part, is due to thefact that thoracic surgery is rarely performed in SCLC patients andas a result tumor sample accessibility is very limited. Previousgenetic mouse models of SCLC by p53 and Rb knockout recapi-tulates human disease but the latency is extremely long. Althoughsome improvement has been made by further knocking out p130or Pten, time consuming, extensive mouse breeding is required(14–16). We leveraged our lentiviral delivery strategy to induceSCLC in wild-type mice with a single vector carrying all thenecessary mutations (Fig. 1A). The vector can be further modifiedto include new combination of mutations for studying their rolesin tumor initiation and development, or luciferase/GFP reportergenes for in vivo tracking tumor response upon treatment inpreclinical study settings. Thus, this model system offers theflexibility for both basic and preclinical studies.

The role of NF-kB in cancer initiation, development, andmetastasis has been extensively studied, and in general pro-motes tumoriogenesis. Interestingly, NF-kB has been shown to

also act as a tumor-suppressor in both a diethylnitrosamine(DEN)-induced hepatocellular carcinoma (HCC) model and a7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin squamous cell carcino-ma (SCC) model (23, 24). These two seemingly dissimilartumor models share one common feature: NF-kB and JNK aretwo major signaling pathways downstream of TNFR1, whichusually counter-regulate each other. Upon chemical treatmentand NF-kB inhibition in hepatocytes or keratinocytes, JNKsignaling is unleashed, which leads to excessive oxidative stress,DNA damage, and eventually induces tumorigenesis. In thescenario of SCLC, Mycl1 gene is usually amplified leading tooverexpression, which may lead to activation of PKA pathwayby upregulation of Prkacb expression (25). Indeed, we observedhigh Prkacb mRNA in mouse SCLC but not adenocarcinomatumor samples (data not shown). Intriguingly, PKA and CREBactivity has also been reported in NSCLC, which may contributeto cancer metastasis and drug resistance; however, the mech-anism of PKA activation in NSCLC could be very different fromSCLC (26, 27).

Figure 3.

SCLC relies on high CREB and low NF-kB to maintain neuroendocrine signature and high proliferation. A, and B, Diagram of lentiviral vector designing.Kaplan–Meier curve showingmice survival. Median survival time of the four groupswas 396 days (IKK2SE, n¼ 8), 186 days (IkBaM, n¼ 5), undefined (dnPKA, n¼ 11),and 291 days (luci, n ¼ 7) respectively. P ¼ 0.0354 (IkBaM vs. luci), P ¼ 0.0142 (IKK2SE vs. luci), P < 0.0001 (dnPKA vs. luci). C, primary SCLC cells derivedfrom shp53-shRb-shPTEN model were infected with 5xkB reporter or 3xCREB reporter, and treated with TNFa (10 ng/mL) or forskolin (5 mmol/L) for 8 hours,and luciferase activity was measured. D, Same SCLC cells in C were treated with TNFa (10 ng/mL) for 24 hours, and the expression of five neuroendocrinesignature genes were analyzed with quantitative RT-PCR.


Xia et al.




Our current work indicates that, in contrast to many othertypes of cancer, SCLC does not benefit from high NF-kBactivity with regard to tumor cell proliferation. Thus, NF-kBinhibition may not directly benefit patients with SCLC ingeneral, although we cannot exclude the possibility that NF-kBinhibitor could work well in certain scenarios, for example, toovercome chemo-resistance, which usually is linked to NF-kBactivation. However, our data suggests that manipulating thePKA–CREB pathway should be further pursued for the sakeof developing new therapeutic strategies against this deadlydisease. The big hurdle for this therapy, however, is that mostof the current available PKA inhibitors are ATP competitiveantagonists and not very specific. Furthermore, the PKA–CREBpathway is responsible for many other important biophysio-logical activities, especially for neuronal system. Interestingly,Jahchan and colleagues have recently reported that tricyclicantidepressants could effectively suppress SCLC tumorgrowth both in vitro and in animals through blockingautocrine survival signals involving neurotransmitters andtheir G protein–coupled receptors (GPCR). These GPCRs usu-ally activate PKA pathway via Gas, adenyl cyclase, and cAMPand indeed they observed that tricyclic antidepressants couldeffectively reduce PKA–CREB activity in SCLC cells and thus

attenuate tumor growth in animals (28). Altogether, ourresults support the notion that targeting the PKA–CREB path-way, is a novel strategy in fighting against SCLC—the mostmalignant form of human lung cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Xia, I.M. VermaDevelopment of methodology: Y. XiaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Xia, C. Zhan, M. Feng, N. YeddulaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Xia, C. Zhan, M. Feng, M. Leblanc, E. Ke,N. Yeddula, I.M. VermaWriting, review, and/or revision of the manuscript: Y. Xia, M. Leblanc,I.M. VermaStudy supervision: I.M. Verma

AcknowledgmentsThis work was supported in part by grants from the NIH

(5R01CA195613-20 to I.M. Verma), the Cancer Center Core Grant (P30CA014195-38), the H.N. and Frances C. Berger Foundation, and the LeonaM. and Harry B. Helmsley Charitable Trust Grant No. 2017-PG-MED001.

Figure 4.

Targeting CREB pathway suppresses SCLC growth in vivo. A, SCLC cells were infected with GFP (Ctrl) or dnPKA vector and transplanted into syngeneic hosts.Tumors were harvested after 4 weeks and weighed. B, Immunohistochemistry staining of the tumors from (A). Scale bars, 100 mm. Quantification of positivestaining cells were shown on the right. C, SCLC cells were transplanted into syngeneic hosts. H89 treatment was started 2 weeks after the transplantationfor another 2 weeks, and then tumors were harvested and weighed.






This work was also supported by The National Natural Science Foundationof China (No. 81672268 to Y. Xia).

The authors appreciate the generosity of Dr. S. Seshagiri for sharing humanSCLC RNAseq data (accession no.: EGAS00001000334). We also thank M.Schmitt, G. Estepa, and B. Coyne for technical and administrative support. I.M.Verma is an American Cancer Society Professor of Molecular Biology, and holdsthe Irwin and Joan Jacobs Chair in Exemplary Life Science.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 9, 2017; revised October 25, 2017; accepted January 31,2018; published first March 9, 2018.

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http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-17-0576http://mcr.aacrjournals.org/content/suppl/2018/03/09/1541-7786.MCR-17-0576.DC1http://mcr.aacrjournals.org/content/16/5/825.full#ref-list-1http://mcr.aacrjournals.org/content/16/5/825.full#related-urlshttp://mcr.aacrjournals.org/cgi/alertsmailto:[email protected]://mcr.aacrjournals.org/content/16/5/825http://mcr.aacrjournals.org/

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