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Nuclear Factor I/B is an Oncogene in Small Cell Lung Cancer (2011)

Nuclear Factor I/B is an Oncogene in Small Cell Lung Cancer (2011)

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Published by James Clement
Small cell lung cancer (SCLC) is an aggressive cancer
often diagnosed after it has metastasized. Despite the
need to better understand this disease, SCLC remains
poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude
focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.
Small cell lung cancer (SCLC) is an aggressive cancer
often diagnosed after it has metastasized. Despite the
need to better understand this disease, SCLC remains
poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude
focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

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Published by: James Clement on Jul 20, 2011
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 10.1101/gad.2046711Access the most recent version at doi: 2011 25: 1470-1475
Genes Dev.
Alison L. Dooley, Monte M. Winslow, Derek Y. Chiang, et al.
Nuclear factor I/B is an oncogene in small cell lung cancer
MaterialSupplemental
References
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 Cold Spring Harbor Laboratory Presson July 20, 2011 - Published by genesdev.cshlp.orgDownloaded from 
 
RESEARCH COMMUNICATION
Nuclear factor I/B is anoncogene in small celllung cancer
Alison L. Dooley,
1
Monte M. Winslow,
1
Derek Y. Chiang,
2,3,6
Shantanu Banerji,
2,3
Nicolas Stransky,
2
Talya L. Dayton,
1
Eric L. Snyder,
1
Stephanie Senna,
1
Charles A. Whittaker,
1
Roderick T. Bronson,
4
Denise Crowley,
1
Jordi Barretina,
2,3
Levi Garraway,
2,3
Matthew Meyerson,
2,3
and Tyler Jacks
1,5,7
1
David H. Koch Institute for Integrative Cancer Research,Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139, USA;
2
The Broad Institute,Cancer Program, Cambridge, Massachusetts 02142, USA;
3
Department of Medical Oncology, Center for Cancer GenomeDiscovery, Dana-Farber Cancer Institute, Boston,Massachusetts 02115, USA;
4
Department of Pathology, TuftsUniversity School of Medicine and Veterinary Medicine, NorthGrafton, Massachusetts 01536, USA;
5
Howard Hughes MedicalInstitute, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139, USA
Small cell lung cancer (SCLC) is an aggressive canceroften diagnosed after it has metastasized. Despite theneed to better understand this disease, SCLC remainspoorly characterized at the molecular and genomic levels.Using a genetically engineered mouse model of SCLCdriven by conditional deletion of
Trp53
and
Rb1
in thelung, we identified several frequent, high-magnitudefocal DNA copy number alterations in SCLC. We un-covered amplification of a novel, oncogenic transcriptionfactor, Nuclear factor I/B (
 Nfib
), in the mouse SCLCmodel and in human SCLC. Functional studies indicatethat
NFIB
regulates cell viability and proliferation duringtransformation.
Supplemental material is available for this article.Received March 1, 2011; revised version accepted May 31,2011.
Small cell lung cancer (SCLC) is a highly lethal form of cancer that comprises
;
20% of all lung cancer cases(Wistuba et al. 2001; Meuwissen and Berns 2005). Un-fortunately, SCLC is frequently diagnosed only aftermetastatic spread of the disease, and at present only 5%ofpatientssurvivebeyond5yearsafterdiagnosis(Wordenand Kalemkerian 2000; Cooper and Spiro 2006). Someinsight has been gained as to the underlying mechanismsof this aggressive disease, including the identification of loss-of-function mutations in the tumor suppressor genes
Trp53
(Yokotaetal.1987;Takahashietal.1989,1991)and
RB1
(Harbour et al. 1988; Yokota et al. 1988), which areobserved in 75% and 90% of SCLC cases, respectively(Wistuba et al. 2001; Meuwissen and Berns 2005). Inaddition, MYC family members (C-MYC, L-MYC, andN-MYC) are frequently amplified in SCLC (Nau et al.1985; Meuwissen and Berns 2005). However, very little isknown about other functionally relevant alterations inSCLC, and a more complete understanding of the diseaseis required to allow the development of new targetedtreatments.Whole-genome profiling has been used to gain infor-mation about copy number alterations, point mutations,and translocations in tumors (Campbell et al. 2008; Leyetal.2008;Mardisetal.2009).Onerecentexaminationof 33 primary SCLC tumors and 13 SCLC cell lines identi-fied MYC family amplifications in 82% of tumors and62% of cell lines (Voortman et al. 2010). Another studyidentified 22,000 point mutations in a SCLC cell line, themajority of which were G–T transversions, a hallmark of carcinogens present in tobacco smoke (Toyooka et al.2003; Lewis and Parry 2004; Pleasance et al. 2010). Inother cancer types, comparative studies using mousemodels have aided in narrowing lists of candidate genes(Kim et al. 2006; Zender et al. 2006, 2008). Thus, weanalyzedthegenomicalterationsthatoccurduringtumorprogression in a mouse model of SCLC to identifyoncogenes in this cancer type.
Results and Discussion
Genetically engineered mouse model of metastatic SCLC
Bernsandcolleagues(Jonkersetal.2001;Vooijsetal.2002;Meuwissen et al. 2003; Sage et al. 2003) have developeda mouse model of SCLC (mSCLC) that involves theinactivation of the
Trp53
and
Rb1
tumor suppressor genesusing conditional (‘‘floxed’’) alleles in
p53
 fl/fl 
;Rb
 fl/fl 
mice(Supplemental Fig. S1). Inhalation of adenovirus contain-ing Cre recombinase results in infection of lung epithelialcells that develop into tumors resembling human SCLChistopathologically (Supplemental Fig. S1; Meuwissenet al. 2003; DuPage et al. 2009). These mice have a me-dian survival time of 350 d, during which the tumorsbecome malignant and metastatic (Supplemental Fig. S1;Meuwissen et al. 2003). Similar to human SCLC, themSCLC metastasized to the thoracic lymph nodes, liver,adrenalglands,andbone(SupplementalFig.S1;Meuwissenet al. 2003). Thus, this model provided a platform withwhich to identify genetic alterations that occur duringtumor progression.
Identification of Nuclear factor I/B (Nfib)amplifications
To determine the genetic alterations that occur inmSCLC, primary tumors and metastases were dissectedand used for histology, DNA and RNA isolation, and thederivationofcelllines(SupplementalFig.S1).Eachtumorwas verified histopathologically to be SCLC, and tumorpurity wasassessed byPCR for the recombined
Trp53
and
[
Keywords
: small cell lung cancer; mouse model; Nuclear factor I/B]
6
Present address: Lineberger Comprehensive Cancer Center, 450 WestDrive, CB #7295, Chapel Hill, North Carolina 27514, USA.
7
Corresponding author
.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.2046711.
1470 GENES
&
DEVELOPMENT 25:1470–1475
Ó
2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org
 Cold Spring Harbor Laboratory Presson July 20, 2011 - Published by genesdev.cshlp.orgDownloaded from 
 
Rb1
alleles (Supplemental Fig. S1; data not shown). Theanalysis of DNA copy number alterations in murinetumor models has previously aided in the identificationof functionally important genes inseveral human cancers(Kim et al. 2006; Zender et al. 2006, 2008). Thus, weanalyzed mSCLC tumors and metastases using next-generation sequencing-based DNA copy number analysis(Chiang et al. 2009). These data show that while themajority of the genome was surprisingly unaltered, sev-eral high-level focal amplifications and deletions wereobservedintumorspecimens(Fig.1A;SupplementalFigs.S2–S4; Supplemental Table 1). In particular, we identifiedtworecurrentfocalamplificationscenteredaround82Mband 122 Mb on mouse chromosome 4 and a heterozy-gous deletion spanning from
;
148.5 Mb to the end of chromosome 4 (Fig. 1B). Although one of the focal am-plifications on chromosome 4 contained a known proto-oncogene involved in SCLC, L-myc (
Mycl1
) (Nau et al.1985), the other focal amplification contained no genespreviously implicated in this disease (Fig. 1C; Supple-mental Fig. S3). To identify the relevant targets withinthe amplified region, the amplification breakpoints weremapped using statistical change point analysis of thenormalized copy number ratios (Chiang et al. 2009).Nuclear factor I/B (
Nfib
) was the only gene within thisregion amplified in each of the samples (Fig. 1C). Further-more,
Nfib
is located at the apex of the amplified peak intumors and tumor-derived cell lines (Supplemental Fig.S2). Thus,
Nfib
represents a newly identified amplifiedgene in SCLC.
Nfib
is a CCAAT-box-binding transcription factor thatregulates the expression of lung differentiation genes(Santoro et al. 1988; Steele-Perkins et al. 2005).
Nfib
knockout mice have lung hypoproliferation and differ-entiation defects, in addition to brain defects, and dieshortly after birth (Gru¨ nder et al. 2002). The chromo-somal region containing
Nfib
has been reported to befrequently amplified in a mouse model of prostate cancer(Zhou et al. 2006) and in patients with triple-negativebreastcancer(Hanetal.2008).Basedontheidentificationof 
Nfib
as an amplified gene in SCLC and its potentialimportance in other prevalent tumor types, we chose toexamine
Nfib
further.The DNA copy number of 
Nfib
and the expression of 
Nfib
mRNA was determined using real-time PCR ina panel of 28 mSCLC-derived cell lines (Fig. 2A,B). Outof 28 cell lines, 16 had
Nfib
and six had
L-myc
amplifi-cations (Fig. 2A; Supplemental Fig. S5). Notably, fourmSCLC cell lines had amplified both
Nfib
and
L-myc
(Supplemental Fig. S5). mSCLC cell lines with increased
Nfib
copy number also expressed high levels of 
Nfib
(Fig.2B). Interestingly, two cell lines with normal
Nfib
copynumber expressed high levels of 
Nfib
mRNA, suggestingthat mechanisms other than genomic alteration mayincrease Nfib levels in SCLC (Fig. 2B).To confirm
Nfib
amplification in mouse tumors, weperformed fluorescence in situ hybridization (FISH). FISHconfirmed the amplification of 
Nfib
in the lymph nodemetastasis analyzed in Figure 1 (Fig. 2C,D). Interestingly,in a primary tumor, we found that
Nfib
amplificationclearly correlated with a region of increased Nfib expres-sion(Fig.2E,F).Inanormallung,Nfibproteinwaslocalizedappropriatelytothenucleusofalveolar typeIIcells(Steele-Perkins et al. 2005) and was also nuclear in mSCLC.Additionally, Nfib protein was detected in a subset of lungneuroendocrine cells (Supplemental Fig. S6). Consistentwith data from human tumor samples (Bhattacharjee et al.2001), Nfib was not detected in lung adenomas that oc-casionally arise in this mouse model (Supplemental Fig.S7). Furthermore, we observed that both lymph node andliver metastases very frequently expressed high levelsof Nfib (Supplemental Fig. S7). These data confirm theamplification and increased expression of 
Nfib
in mSCLCtumors and local and distant metastases.
NFIB amplifications in human SCLC
We next examined whether
NFIB
is amplified and/orexpressed in human SCLC. Copy number analysisrevealed a broad region of amplification on chromosome9p23 encompassing 210 genes. GISTIC analysis identifiedan
;
200-kb minimal region of amplification containingonlyonegene,
NFIB
(Beroukhimetal.2007).Intotal,16of 46 human SCLC cell lines had
NFIB
copy number gains(Fig. 3A). Interestingly, 11 of the cell lines with
NFIB
amplification alsohad
L-MYC
amplification,and15outof the 16 cell lines with amplification of 
NFIB
displayedadditional amplification of one of the
MYC
family mem-bers (Supplemental Fig. S5; data not shown). Increased
NFIB
copy number was confirmed by real-time PCR(Supplemental Fig. S8). Additionally,
NFIB
amplificationwas detected by FISH in 15% of primary human tumorsamples(Fig.3B).WenextaddressedwhetherNFIBprotein
Figure 1.
Nfib is amplified in mSCLC tumors. (
 A
) Log
2
ratio of tumor to somatic DNA copy number across the whole genome of a mSCLC lymph node metastasis cell line. The X chromosome hasa copy number ratio of 2 due to the male reference genome, whilethe sample was derived from a female mouse. (
B
) DNA copy numberratio of chromosome 4 of the same sample as in
A
. Interestingly, theregion between the two focal amplifications is near diploid andcontains the tumor suppressor gene
Cdkn2a
. Despite the well-known role of 
Cdkn2a
in regulating p53 and Rb, which are alreadydeleted in tumors, the fact that the copy number of this gene is keptlow is consistent with this locus regulating other Rb familymembers (Schaffer et al. 2010). Copy number data are plotted asthe tumor to somatic copy number ratio. (
C
) Integrated genomeviewer (IGV) plot of the DNA copy number of position 79.5–84.5 Mbon chromosome 4. The bar indicates the log
2
copy number ratio of tumor to somatic reference sample. The dotted line indicates theboundaries of the minimally conserved region. (T) Primary lungtumor; (Liv) liver metastasis; (LN) lymph node metastasis; (graylabels) tumor samples; (black labels) cell lines.
Nuclear factor I/B is an oncogene in SCLCGENES
&
DEVELOPMENT 1471
 Cold Spring Harbor Laboratory Presson July 20, 2011 - Published by genesdev.cshlp.orgDownloaded from 

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