0008-5472.CAN-11-3506.Full-Text.Pdf

Author Manuscript Published OnlineFirst on November 14, 2011; DOI: 10.1158/0008-5472.CAN-11-3506 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Novel transcriptional targets of the SRY-HMG box transcription factor SOX4 link

its expression to the development of small cell lung cancer

Sandra D. Castillo1, Ander Matheu2, Niccolo Mariani1, Julian Carretero3, Fernando Lopez-

Rios4, Robin Lovell-Badge2 and Montse Sanchez-Cespedes1*

Authors´Affiliations:1Genes and Cancer Group, Cancer Epigenetics and Biology Program-

PEBC), Bellvitge Biomedical Research Institute-IDIBELL, Barcelona, Spain. 2Division of Stem

Cell Biology and Developmental Genetics, MRC National Institute for Medical Research,

London, UK. 3Department of Physiology, Faculty of Medicine and Odontology, University of

Valencia, Spain. 4Hospital Universitario Madrid-Sanchinarro, Laboratorio Dianas Terapeuticas,

Madrid, Spain

Running title: Novel SOX4 targets and small cell lung cancer development

Keywords: SCLC, SOX4, SOX11, neuroendocrine lung tumors, oncogene

Disclosure of potential conflict of interest: No conflicts of interest were disclosed.

Word count: 5,428 Total number of figures and tables: 6

Accession number: Array data deposited in the Gene Expression Omnibus (GEO) under

accession number GSE31612.

*Corresponding Author: Montse Sanchez-Cespedes, Cancer Epigenetics and Biology

Program-PEBC (IDIBELL) 08908, Hospitalet de Llobregat, Barcelona, Spain; Tel:

+34932607132, Fax: +34932607219, Email: [email protected]

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 14, 2011; DOI: 10.1158/0008-5472.CAN-11-3506 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The HMG box transcription factor SOX4 involved in neuronal development is amplified and

overexpressed in a subset of lung cancers, suggesting that it may be a driver oncogene. In this

study, we sought to develop this hypothesis including by defining targets of SOX4 that may

mediate its involvement in lung cancer. Ablating SOX4 expression in SOX4-amplified lung

cancer cells revealed a gene expression signature that included genes involved in neuronal

development such as PCDHB, MYB, RBP1 and TEAD2. Direct recruitment of SOX4 to gene

promoters was associated with their upregulation upon ectopic overexpression of SOX4. We

confirmed upregulation of the SOX4 expression signature in a panel of primary lung tumors,

validating their specific response by a comparison employing embryonic fibroblasts from Sox4-

deficient mice. Interestingly, we found that small cell lung cancer (SCLC), a subtype of lung

cancer with neuroendocrine characteristics, was generally characterized by

high levels of SOX2, SOX4 and SOX11 along with the SOX4-specific gene expression

signature identified. Taken together, our findings identify a functional role for SOX genes in

SCLC, particularly for SOX4 and several novel targets defined in this study.

Introduction

As with other types of cancer, lung cancer is undergoing a therapeutic revolution

characterized by the identification of novel driver oncogenes and the generation of drugs that

inhibit their activity in a very specific manner. The success of erlotinib/gefitinib in EGFR-

mutant tumors and crizotinib in tumors carrying a translocation of the ALK oncogene are some

of the current paradigms (1). Since few tumors carry alterations of these genes, effort is required

to identify additional targetable oncogenes. We previously performed a wide-ranging DNA

copy number analysis of lung cancer cell lines and identified an amplicon on chromosome 6p22,

containing the SOX4 gene, which was expressed at very high levels and was the best candidate

for an oncogene in the amplicon (2).

The SOX4 gene belongs to the SOX family, which is divided into eight groups, A to H,

according to protein identity (3). Sox4, Sox11 and Sox12 form the Sox-C group, sharing a high

degree of identity in the high-mobility group domain, and in a group-specific transactivation

domain (4). Sox4 is predominantly expressed during embryonic development in the heart,

central nervous system, lung and thymus (5-7). SOX4 protein levels are increased in several

types of carcinoma (8-11), and knocking down SOX4 induces apoptosis and growth suppression

in cancer cells (12-14). Providing further evidence of the oncogenic potential of SOX4 we

reported that its over-expression in NIH3T3 cells increases the number of foci induced by the

mutant RHOA-Q63L (2). In addition, several independent studies have demonstrated that Sox4

(also known as ecotropic viral integration site 16, Evi16) is a frequent target of retroviral

insertional mutagenesis, leading to neoplasic transformation in murine hematopoietic cells (15-

18). In the particular case of lung cancer, we previously reported the presence of a high level of

SOX4 amplification in a subset of lung primary tumors and cancer cell lines (2). The relevance

of SOX4 to lung cancer development has also been observed by others. A meta-analysis

examining the transcriptional profiles of human tumors found SOX4 to be one of 64 genes

uniquely up-regulated in cancer thereby making it part of a general gene expression signature of

cancer (19). Lung cancer was among the tumors with the greatest levels of SOX4 expression. In

addition, Sox4 was among the set of genes over-expressed in the lungs of c-myc transgenic

mice, and c-myc was also found to be one of the transcription factors with over-represented

Sox4 binding sites among the set of over-expressed genes (20).

SOX4 is involved in neural development and the maintenance of some stem cell types

(21, 22). Recent studies have reported SOX4 to be a direct TGF-beta target that activates SOX2

transcription while retaining the stem cell properties of glioma-initiating cells (21). In addition,

in normal hair follicles, Sox4 is expressed in the developing hair germ (23).

In spite of this, and although SOX4 was one of the first members of the SOX family to

be isolated and characterized, including the demonstration that it has separable DNA-binding

and transactivation domains, our present knowledge of the genes controlled by SOX4 activity is

scarce. This paper reports on the role of SOX4 in human lung carcinogenesis, focusing

especially on identifying novel targets of SOX4 transcriptional activity.

Materials and Methods

Cancer cell lines and primary tumors

Cancer cell lines were obtained from the American Type Culture Collection-ATCC

(Rockville, MD, USA) and grown under recommended conditions. DNA and RNA from

additional lung cancer cell lines used for real-time quantitative RT-PCR were kindly provided

by Luis M. Montuenga and Ruben Pio of the Centro de Investigación Medica Aplicada (CIMA),

University of Navarra, Spain, and Jun Yokota, National Cancer Center Research Institute,

Tokyo, Japan. Fresh frozen lung primary tumors were provided by the CNIO Tumour Bank

Network, CNIO, Spain, and were selected as previously described (24).

Expression plasmids and reporters

We stably transfected the H522 cell line with a Tet repressor (TetR) expression

construct, pCMB1b-TrS, kindly provided by MV de Wetering (Hubrecht Institute, The

Netherlands) using hygromycin selection. We examined TetR activity by transfecting the cells

with the pcDNA4/TO/Luc construct that expressed the firefly luciferase gene under the control

of a Tet operator and then measured the luciferase activity with the Dual Luciferase Assay kit

(Promega Madison, WI, USA). The pRL-Tk plasmid (Promega) that constitutively codified for

the Renilla luciferase was used to measure the transfection efficiency. Clones that stably

expressed the Tet repressor were transfected with pSUPERIOR-SOX4/S1 construct, kindly

provided by CA. Moskaluk (University of Virginia), which expressed a short hairpin against the

sequence 5´-AAGACGACCTGCTCGACCTGA-3´ of the SOX4 coding region under the

control of a Tet operator and selected using geneticin. These oligonucleotides specifically

inhibited the expression of SOX4 but not of other SOX family genes (13). Selected clones were

analyzed for SOX4 expression by quantitative RT-PCR and western blot before and after

doxycycline induction. For transient transfection, full-length human wt SOX4 and the mutant

SOX4 S395X carrying an HA tag were cloned as previously described (2).

Gene expression microarrays and real-time quantitative PCRs

mRNA was extracted using conventional methods, and 1μg of it was amplified from

each sample and used for gene expression microarray analysis. Universal Human Reference

RNA (P/N 740000; Stratagene, La Jolla, CA, USA), was used as reference for hybridization and

analysis. For labeling we used the commercial Two-Color Microarray-Based Gene Expression

Analysis kit (version 5.5). MMLV-RT retrotranscription of sample from a T7 promoter primer

was followed by a T7 RNA polymerase-catalyzed in vitro transcription reaction in the presence

of either Cy3-CTP or Cy5-CTP fluorophores. Cy3 labeling was used for the reference sample.

Hybridization was performed on the Whole Human Genome Microarray (4x44K), scanned with

a G2505B DNA microarray scanner and quantified using Agilent Feature Extraction Software

(version 9.5) (Agilent, Santa Clara, CA, USA). Fluorescence intensity from each array element

was subtracted from the local background and data normalized as previously described (24). We

further selected transcripts that fulfilled the following criteria: i) repressed at least 2 times in the

H522Tr-shSOX4-1 at 48 and 96h after induction of shSOX4 relative to the level at 0h, ii) no

changes in gene expression in the parental H522 or in H522TRshSOX4. mRNA and genomic

DNA were measured by real-time quantitative PCR. DNase-treated RNA was reverse-

transcribed. cDNA and genomic DNA were amplified using an ABI Prism 7900 Sequence

detector (Applied Biosystems, CA, USA), and levels of genes were measured by SYBR green

real-time PCR. Reactions were performed in triplicate. As controls we used the human GAPDH,

B-ACTIN and TATA box binding protein (TBP) to correct for inter-individual/tumor variations.

The primer sequences used are included in Supplementary Table 2.

Antibodies,western blots and immunostaining

For western blotting, cells were scraped from the dishes into the lysis buffer. A total of

25µg of total protein was separated by SDS–PAGE and blotted with rabbit anti-SOX4 (A574)

1:5000 (CS-129-100, Diagenode, Liège, Belgium), mouse anti-SOX2 1:2500 (R&D Systems);

rabbit anti-NSE 1:1000 (Abcam) or mouse anti-GAPDH. The secondary goat anti-mouse-

IgG:HRP and goat anti-rabbit:HRP antibodies (DAKO, Glostrup, Denmark) were added at

1:5000. We performed immunohistochemistry of SOX9 (1:750 dilution; Chemicon, Temecula,

CA) in the autostainer Low FLEX (DAKO), using previously described protocols (25). Sections

were counterstained with hematoxylin and evaluated by two independent researchers.

Chromatin immunoprecipitation assays

Cells were fixed in 1% formaldehyde for 10 min at 37ºC. Cross-linking was quenched

by adding 125mM glycine. Cells were then washed with cold PBS, harvested and resuspended

in SDS lysis buffer containing a protease inhibitor cocktail. Chromatin was sheared by

sonication (average length 0.25-1Kb) and incubated with 60µl protein A/G agarose/salmon

sperm DNA (50% slurry) (Millipore, Billerica, MA, USA) with gentle agitation for 30min. The

supernatant was then immunoprecipitated with anti-SOX4 antibody 1:500 or its matched non-

immune crude serum 1:500 (IgG) (Diagenode) at 4ºC overnight. Protein A/G agarose (60µl of a

50% slurry) was then added and incubated for 1h. Pellets were washed and protein-DNA cross-

links were reversed by overnight incubation at 65°C with proteinase K. DNA was purified

following a conventional phenol–chloroform protocol and eluted in 50µl water. At least two

independent ChIP experiments were performed. Real-time quantitative PCR was performed

using SYBR Green Master Mix on an ABI Prism 7900 (Applied Biosystems, CA, USA). The

primer sequences used are included in Supplementary Table 2.

Mouse embryonic fibroblast processing and RNA extraction from mouse tissues

Mice were housed in the pathogen-free barrier area of the National Institute for Medical

Research, London. Tissues from young (1-2months) and old (1.5-2years) C57BL6 mice were

homogenized at high speed and total RNA extracted with trizol. Mouse embryo fibroblasts

(MEFs) were isolated and cultured as previously described (26) from Sox4+/+, Sox4+/- and Sox4-/-

E13.5 mouse embryos. Mice were genotyped by PCR as described (26).

Statistical and bioinformatic analysis

Statistical analyses were performed with SPSS software. Differences between groups

were analyzed by the unpaired t test or Fisher's exact test, as appropriate. Differences in gene

expression between lung tumors with and without high levels of SOX4 were determined by the

Mann-Whitney U test. Correlations between SOX genes were estimated by Spearman’s rank

correlation. Values of p<0.05 were considered statistically significant. For the Gene Set

Enrichment Analysis (GSEA), raw data from the Gene Expression Omnibus (GEO) database

repository (28) were normalized using the Robust Multiarray Average (RMA) algorithm

available in Bioconductor’s affy package. We used the limma package to obtain limma-

moderated t statistics to build a ranked list of expression. Gene set enrichment analysis (GSEA)

was applied to this ranked list. After testing for normality of the data with the Kolmogorov-

Smirnoff test, our gene sets were considered significantly enriched between classes under

comparison for values of FDR<0.25, a well-established cut-off for the identification of

biologically relevant gene sets (29).

Results

Differential gene expression upon SOX4 depletion in lung cancer cells

The NCI-H522 lung cancer cell line (henceforth referred to as H522) is known to

feature SOX4 gene amplification (>20 copies/nuclei) and SOX4 over-expression (2). To identify

transcriptional targets regulated by SOX4 we generated H522-derived cells that down-regulate

SOX4 in an inducible manner with a shSOX4 that was reported to specifically deplete SOX4

expression (13). Several clones were found to down-regulate SOX4 in an inducible-dependent

manner. A stable clone, H522Tr-shSOX4-1, which down-regulates SOX4 expression by 90%,

48h after the addition of doxycycline, was chosen for further analysis (Fig. 1A).

To determine the gene expression profile characteristic of SOX4-depleted expression

we compared the global gene expression of the parental H522 cells with that of H522Tr-

shSOX4-1 cells 0, 48 and 96h after inducing shSOX4 with doxycycline. We found 123 genes

with a more than two-fold change of expression (85 down-regulated and 38 up-regulated) in the

H522Tr-shSOX4-1 cells after shSOX4-inducible expression relative to the H522-parental and to

the H522Tr-shSOX4-1 cells before adding doxycycline (at 0 h) (Supplementary Table S1). We

then measured the expression of these transcripts by quantitative RT-QPCR in the H522Tr-

shSOX4-1 cells to validate the microarray observations. Half of the 50 down-regulated genes

chosen for validation were confirmed and selected for further analysis. The TEAD2 and TUBB3

genes were previously identified as Sox4 transcriptional targets in neural progenitors (30-33).

All the transcripts, including the TEAD2 and TUBB3 controls, showed an approximately 50%

drop in expression after interfering SOX4 expression (Fig. 1B), validating the robustness of our

microarray assay. It is of note that many of the transcripts found to be down-regulated after

inducing shSOX4 (e.g., EGR3, MLLT11, NBEA, PCDHBs, RBP1, TMEFF1, TUBB3, VASH2)

are highly specific to neural-derived tissues, including brain, spinal cord and retina (GSE7905

from GEO database).

Next, we aimed to determine whether these observations could be extended to other

lung cancer cell lines. To this end we transfected the H1299 cells, which have low levels of

endogenous SOX4, with a construct carrying wild type SOX4 or a mutant form (S395X). The

latter is devoid of transactivation activity due to a mutation that eliminates the serine-rich C-

terminal domain of SOX4 (2) (Fig. 1C). As shown in Fig. 1B, ectopic expression of the wild

type, but not the mutant SOX4 form, leads to a significant, >2-fold up-regulation of most

targets. In particular, EGR3 and RBP-1 were strongly up-regulated (>16 times) relative to the

control and to the SOX4-mutant.

It has previously been described that SOX4 regulates the expression of SOX2 in

glioma-initiating cells (21). Although SOX2 was not down-regulated in our microarray analysis

after depletion of SOX4, we specifically tested for SOX2 levels in a western blot. No change in

protein expression levels of SOX2 was observed after ectopic expression of SOX4 in the H1299

cells or after abrogation of SOX4 in the H522Tr-shSOX4-1 cells (Fig. 1C-D).

Analysis of SOX4 recruitment to the gene promoters

We developed a quantitative ChIP assay to investigate which of the genes down-

regulated after SOX4 depletion were direct transcriptional targets of SOX4. The SOX4 HMG-

box binds preferentially to the 4-bp DNA-motif 5´-ACAA-3´ sequence (34) and we searched for

putative SOX4 binding sequences in the promoters of these genes. We used the MatInspector

program (Genomatix) (35) and designed primers flanking the regions enriched in these

sequences (Supplementary Figure S1). To determine the specificity of SOX4 occupancy in the

selected regions we also screened distant regions (>5,000bp from the SOX4 binding sequences)

that were considered negative controls. We immunoprecipitated the H522Tr-shSOX4-1

chromatin with the rabbit polyclonal anti-SOX4 antibody and its matched crude serum as IgG

control (Fig. 2A) followed by quantitative PCR. We confirmed the recruitment of SOX4 to the

known targets TEAD2 and TUBB3 (30-33) (Fig. 2B). We also determined that the MYB and

VASH2 promoters were significantly enriched (>4-fold) in the anti-SOX4 immunoprecipitated

chromatin from the cells prior to doxycycline induction, compared with cells after depletion of

SOX4 expression and IgG immunoprecipitates (Fig. 2C). In all cases tested, the enrichment was

specific to the indicated SOX4 binding regions, and was negative in the control regions. This

indicates that SOX4 binds to the MYB and VASH2 promoters. The protocadherin genes,

PCDHA, PCDHB and PCDHG, are tandemly arranged in clusters on the same chromosome. To

determine the possible direct involvement of SOX4 in the transactivation of the different

PCDHB genes we first screened for binding of SOX4 in regions upstream of individual genes

but found no SOX4 recruitment. While performing the screening, the PCDHB cluster was found

to be transcriptionally regulated by a control region downstream of the PCDHG cluster (36).

Therefore, we searched and found SOX4 binding sequences within this control region. Next, we

tested and confirmed SOX4 recruitment to this regulatory region (Fig. 2C), although it remains

to be understood how the binding of SOX4 to this region affects the transcriptional activation of

the PCDHB cluster. Thus, we found evidence that the PCDHB cluster, as well as the MYB and

VASH2 genes, are direct SOX4 transcriptional targets. Other genes that are strongly up-

regulated by SOX4 were not found to recruit SOX4 to their promoters (EGR3, GPC2, RBP1), at

least in the regions that we selected for the analysis.

Expression of SOX4 targets is increased in lung tumors with SOX4 gene over-expression

and is decreased in Sox4-/- mouse embryonic fibroblasts

We extended our analysis to lung primary tumors selected on the basis of high (SOX4-

H) and low (SOX4-L) levels of SOX4 gene expression. We compared the differences in the

expression of the transcripts between the two groups. As depicted in Fig. 3A, most of the

transcripts, especially CCNG2, DLG3, VASH2, PCDHB9, -11 and -14, were expressed at

significantly higher levels in the SOX4-H group than in the SOX4-L group. We also used Gene

Set Enrichment Analysis (GSEA) to compare our SOX4 specific gene expression signature,

composed of the genes most significantly down-regulated in H522TR-shSOX4 with the dataset

containing the gene expression profile of the SOX4-H and SOX4-L lung primary tumors (37)

(GEO Series accession number GSE8569). The GSEA showed that the set of down-regulated

genes after inducing the expression of the shSOX4 were up-regulated in the SOX4-H tumors

(Fig. 3B). Collectively, these observations are consistent with these being transcriptional targets

of SOX4 and having a role in lung carcinogenesis.

Sox4 is key to embryogenesis and, while Sox4+/- embryos are viable, Sox4-/- embryos die

by day 14 of embryonic development (27). Taking advantage of the availability of mouse

fibroblasts (MEFs) from Sox4-/- and Sox4+/+ embryos, we characterized the expression levels of

the newly identified SOX4 targets in this material (Fig. 3C). Compared with mouse lung tissues,

some of the targets were expressed at very low levels in the Sox4+/+ MEFs (i.e. Dlg3, Dock4,

Egr3, Mta2, Myb, Rbp1 and most Pcdhb-transcripts) and were then excluded from the analysis.

Our results showed that the Gpc2, Rnf122, Tead2, Tubb3 and Vash2 transcripts exhibited

significant down-regulation in the Sox4-/- MEFs, relative to their wild type counterparts

(Fig.3D).

Ageing is associated with decreased expression of Sox4 and its targets in lung tissue

Ageing tissues experience changes in the expression levels of some tumor suppressors

and oncogenes that have been associated with a diminished potential for self-renewal of

multipotent progenitors. In particular, an increase in the levels of p16/INK4a has been reported

in several tissues including the neural system and in beta cells from the pancreas of ageing mice

(38, 39). This background, coupled with the reported connection of Sox4 with the maintenance

of specific stem cell properties (23), prompted us to test whether the levels of Sox4 in the lung

tissue vary as mice age. We used real-time quantitative RT-PCR to compare the gene expression

levels of Sox4 in different tissues (lung, spleen, liver, kidney and heart) of young (1-2month)

and old (1.5-2 years) C57BL6 mice, from four and five different individuals, respectively. As a

positive control we included p16INK4a. We found that Sox4 levels were significantly reduced

(>70%) in the lungs of old mice compared with young individuals (Fig. 4A). We also tested the

gene expression levels in many of the newly identified targets of Sox4. Again we discarded

some of the transcripts because they were expressed at very low levels in normal lung (e.g.

Pcdhb2, Pcdhb5, Pcdhb7, Tubb3 and Vash2). Among those depicting measurable levels of gene

expression, ten (Ccng2, Dlg3, Dock4, Gcp2, Mta2, Nbea, Rbp1, Rnf122, Tead2 and Tmeff1)

exhibited significantly higher levels in the lungs of young mice than in their older counterparts

(Fig. 4B).

Analysis and characterization of gene expression levels among the SOX family of genes in

human lung cancer

We previously reported that SOX4 expression levels were significantly higher in small

cell lung cancer (SCLC) than in non-small cell lung cancer (NSCLC) histopathology, indicating

differences in their underlying biology (2). A functional, but non-overlapping relationship has

been reported between SOX4 and other members of the SOX family of transcription factors (6,

30, 32, 33). Thus, we searched for a possible preferential expression of different SOX-family

members (i.e., SOX2, SOX4, SOX9, SOX11 and SOX12) in the distinct and most common lung

cancer histopathologies. First, we tested a panel of lung cancer cell lines, using quantitative real-

time RT-PCR, and found that the expression of SOX2, SOX4 and SOX11 transcripts was

significantly higher in SCLC than in NSCLC (Fig. 5A and Supplementary Fig. S2). This

occurred in the absence of gene amplification (data not shown). The differences were

particularly striking in the case of SOX11, in which nine out of 18 SCLCs had very high levels

of SOX11 (P<0.005) (Fig. 5A). For unknown reasons, commercially available lung cancer cell

lines are under-represented in the squamous cell carcinoma type, which affects the comparative

assessment of differences between the two main NSCLC histopathologies: adenocarcinomas

(ACs) and squamous cell carcinomas (SCCs). To overcome this hurdle we also tested in

primary lung tumors: 40 SCCs and 30 ACs. We found that levels of SOX2 and SOX9 were

significantly higher in SCCs while high levels of SOX4 predominated in the AC type

(Supplementary Fig. S3A). It is important to note that SOX2 is located on chromosome 3q, a

region that is frequently amplified in SCC and also includes the PIK3CA oncogene (24, 40). In

our samples, over-expression of SOX2 was concordant with the presence of gene amplification,

as we previously observed (24). In the case of SOX9, taking advantage of available antibodies,

we performed immunostaining and confirmed the high protein levels of SOX9 and its

association with the SCC histopathology (Fig. 5B). Although more commonly over-expressed in

SCC, strong SOX9 immunostaining was also observed in some ACs. No differences in the

levels of SOX12 were observed among the different types of lung cancer.

Finally, we determined the correlations between the levels of the different SOX family

genes. In lung cancer cell lines, there were significant direct correlations between all pairs of

SOX transcripts, except SOX9 (Supplementary Fig. S3B). The most significant direct

correlation involved SOX4 and SOX2 expression levels, which occurred in both NSCLC and

SCLC cell lines and suggests a functional correlation. The exception was in the lung primaries

of the SCC type, which probably reflects the fact that SOX2 over-expression in this type of lung

cancer is mainly due to gene amplification.

The gene expression signature of SOX4 relates to that of small cell lung cancer

Taking into account the following observations: i) the high level of expression of

several SOX-family genes in the SCLC type; ii) the neural-derived origin of SCLC and iii) the

neural-tissue specificity of many of the SOX4 targets (Supplementary Table S1), we decided to

examine whether there was a relationship between the gene expression signature of SOX4 and

that of SCLC. First, we compared the expression levels of the various SOX4 targets identified

here with a dataset containing the gene expression profile of a panel of 68 cells lines (48 and 20

of the NSCLC and SCLC types, respectively) extracted from the GEO database (GSE4824). As

depicted in figure 6A, most of the SOX4 targets exhibited higher expression levels in the SCLC

than in the NSCLC type. Two of the lung adenocarcinoma cell lines, H2009 and H2887, had a

profile for the SOX4 targets with similarities to that of SCLC. One of these cell lines, H2009,

had simultaneous mutations at KRAS and RB (http://www.sanger.ac.uk/). While the former

mutation is characteristic of lung adenocarcinomas, the latter is almost exclusively found in

SCLCs. This is puzzling as it suggests that this cell line has a neuroendocrine origin.

Next, we used the same dataset to obtain the gene expression profile of the SCLC cell

lines (Fig. 6B) and to perform GSEA. Our results show significant similarities between the

genes down-regulated after inducing the expression of shSOX4 in the H522Tr-shSOX4-1 and

the SCLC gene expression signature (Fig. 6C). Taken together these observations suggest that

SOX4 is a key element required for the development of small cell lung cancer.

Discussion

We report the changes in global gene expression after depletion of SOX4 in a lung

cancer cell line that has SOX4 over-expression due to gene amplification. Taking this approach

we identified novel and high confidence direct and indirect targets of SOX4 activity. We

validated our findings in various models, attesting to the robustness of our approach. Chromatin

immunoprecipitation of SOX4 indicated that some of these are direct targets, activated by the

position of SOX4 in their promoters, while others are indirectly activated by SOX4. Apart from

TEAD2 and TUBB3, previously described as SOX4 direct targets in neural progenitors (30-33),

we identified novel genes that recruit SOX4 to their promoters, including the MYB and VASH2

genes. Some human protocadherin (PCDH) genes are clustered together on chromosome 5 and

classified into three subfamilies, PCDHA, PCDHB and PCDHG. Of special interest are the

PCDHB genes, many of which are down-regulated after SOX4 depletion. The PCDH proteins

are located, at least in part, at synapses and several mouse models lacking Pcdhs have a wide

variety of neural malformations, suggesting that the Pcdhs regulate neural formation. Each

clustered PCDH isoform has its own promoter but it has recently been reported that genes of the

Pcdh-β family are controlled by a region located downstream of the Pcdh-γ cluster. This region

activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases

their expression levels (36). We found that this control region, containing many SOX4 binding

sites, strongly recruits SOX4, implying that it is an important regulator of the PCDHB gene

family. Although the exact mechanisms that underlie this regulation remain to be elucidated, the

formation of a looped architecture is a possible explanation that has been reported for other

transcription factors. Apart from the PCDHBs, other genes up-regulated by SOX4, including

direct and indirect targets (e.g., EGR3, DLG3, DOCK4, MLLT11 and TMEFF1), are also

predominantly expressed in neural-derived tissues (i.e., brain, spinal cord and retina). These

observations are in agreement with the reported connection between SOX4 and neural

development and developmental potential (22, 23, 30). Others have searched for SOX4 targets

in human hepatocellular and prostate cancer cells (34, 41). The overall overlap of targets is low,

possibly reflecting that SOX4 controls specific subset of genes in different cellular contexts.

Although it was reported that SOX2 constitutes a direct target of SOX4 in glioma cells (21), we

could not confirm this in lung cancer-derived cells.

Sox4+/- mice are viable but Sox4 homozygous null mutants are embryonic lethal due to

cardiac defects and other abnormalities (27). This indicates an involvement of Sox4 in

embryonic development, which is consistent with the observed fluctuations of Sox4 levels

during embryogenesis; these are significantly reduced in later stages (42). In addition to

embryogenesis, we have shown that levels of Sox4 and of most Sox4 targets are reduced in the

lungs of older mice compared with those of younger ones, thereby implying a role for Sox4 in

lung ageing. In parallel, the levels of the Cdkn2a tumor suppressor were significantly increased

in aged lungs. This agrees with the previously reported increase in the expression of Cdkn2a in

the neural system and in beta cells from the pancreas of ageing mice (38, 39) and suggests that

genes involved in cancer development also have a role during ageing, possibly related to the

diminished self-renewal potential of multipotent progenitors. In this regard, a crucial role for

Sox4 in cell fate decisions and maintenance of stem cell properties has been described (21, 23).

A variety of Sox family genes are expressed in the developing lung, including Sox2,

Sox4, Sox9 and Sox11, and become repressed as embryonic development progresses (33, 42,

43), indicating that this family of genes actively control lung embryogenesis. On the other hand,

lung cancer comprises several histopathological lineages that arise from various genetically

distinct cell types (43). In this study, we found strong differences in the pattern of expression of

SOX-related genes (SOX2, SOX4, SOX9 and SOX11) among the lung cancer types. In non-small

cell lung cancer (NSCLC), increased levels of SOX4 preferentially occurred in adenocarcinomas

while SOX2 and SOX9 were more commonly up-regulated in the squamous cell carcinoma

(SCC) type. It is important to note that SOX2 is located on chromosome 3q26 and is amplified

and overexpressed in lung SCCs (24, 40). Moreover, shRNA targeting SOX2 decreased colony

formation in SOX2-amplified cells lines (40), supporting the causative effect of SOX2

amplification in lung cancer development. However, since the 3q26 amplicon includes another

bona fide oncogene, PIK3CA (24), it is not possible to determine which is the target for gene

amplification in each case. Interestingly, SOX2, SOX4 and SOX11 exhibited very strong

expression levels in most small cell lung cancers (SCLCs), implying specific roles for the SOX

family in this lung cancer type. This occurs in the absence of gene amplification and indicates

that there are alternative mechanisms for SOX-gene up-regulation in this lung tumor type. Sox4

and Sox2 are known to be TGFβ target genes (21) and it cannot be ruled out that the high levels

of SOX4 and SOX2 in SCLC in the absence of an associated gene alteration may be due to an

active TGFβ pathway. While the high expression levels of SOX4 in SCLC had been described

by us and others (2, 44), the strong and unique association between high levels of SOX11 and

the SCLC type is a new finding. Here, we also report that the SOX4-associated expression

profile has significant similarities with the SCLC gene expression signature. Collectively, these

observations strongly implicate SOX4 and other SOX family genes in the development of the

SCLC type. The SOX4 and SOX11 proteins are members of the Sox-C group, are molecularly

very similar (4) and both are critical to the induction of the expression of neuronal traits and the

maintenance of neural stem cells (21, 30). Taken into account this background and the fact that

many of the SOX4 targets are related to neural tissues, the high levels of SOX4 and SOX11 in

SCLC may also indicate specific cells of origin. In fact, it has been suspected for a long time

that the SCLC type arises from neuroendocrine cells, commonly found in clusters known as

neuroendocrine bodies. Recent observations, using mouse models for targeted Trp53 and Rb1

inactivation in distinct cell types of the adult lung, support the neuroendocrine origin of SCLC

(45). It is likely that along with their role in the stemness of the neuroendocrine cells of the

lung, SOX4 and possibly SOX11, are required for the development of SCLC.

In conclusion, through the abrogation of SOX4 expression in lung cancer cells carrying

SOX4 amplification and over-expression we have identified novel transcriptional targets for

SOX4. We demonstrate that these are up-regulated in lung tumors with SOX4 over-expression,

indicating involvement in lung cancer development. We have also found that SOX4 and other

SOX family genes are strongly up-regulated in the SCLC type. Given the neural-related nature

of many of the SOX4 targets identified here and the similarity between the gene expression

signatures of SOX4 and SCLC we propose that SOX4 is involved in the development of lung

tumors with neuroendocrine characteristics, especially of the SCLC type.

Acknowledgments

We acknowledge the technical assistance of Albert Coll (PEBC) and of Orlando

Dominguez at the Genomics Unit (CNIO).

Grant Support

SD Castillo was supported for fellowships from the CAM, AGAUR and EMBO short-

term fellowship-ASTF-125-2009 and A Matheu by a postdoctoral fellowship from the HFSP.

Work of R Lovell-Badge is funded by UK-MRC (U117512772) and of M Sanchez-Cespedes by

MICINN (SAF2008-02698), RTICC (RD06/0020/0062) and EU-Framework Programme

(HEALTH-F2-2010-258677).

Author contribution

SD Castillo, A Matheu, R Lovell-Badge and M Sanchez Cespedes conceived and

designed the experiments. S D. Castillo, A Matheu and N Mariani performed the experiments. F

Lopez-Rios contributed to the histopathological and immunohistochemical analysis and

evaluation of the data. J Carretero did the bioinformatic analysis. S D. Castillo and MSanchez

Cespedes analyzed the data and wrote the paper.

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Figure legends

Figure 1. Changes in gene expression after depletion of SOX4 in the H522Tr-shSOX4-1 cells.

(A) Western blot of SOX4 of the H522 parental, H522Tr-shSOX4-1 derived cells with (+) or

without (-) doxycycline (2.5ng/µl) after 48h. (B) Representative examples of real-time

quantitative RT-PCR of the indicated transcripts relative to GAPDH. The values were also

relative to the un-induced control, for the H522Tr-shSOX4-1 (dox+) and to the parental H1299

(pCEFL-mock) for the H1299 pCEFL-SOX4-wt and H1299 pCEFL-SOX4-S395X. Bars, mean

±SD from triplicates, from at least two independent experiments. (C) Western blot of SOX4 and

SOX2 of the H1299, transfected with pCEFL-mock, pCEFL-SOX4 and pCEFL-SOX4-S395X.

GAPDH is also shown for protein loading comparison. (D) Western blot of SOX4 and SOX2 of

the H522Tr-shSOX4-1.

GAPDH is also shown for protein loading comparison.

Figure 2. Chromatin immunoprecipitation identifies novel SOX4 transcriptional targets.

(A) On the left, western blot of SOX4, depicting its proper depletion after shSOX4 induction by

doxycycline treatment and GAPDH as loading control. On the right, western blot of precipitated

SOX4, showing the presence and absence of SOX4 in the precipitates from the H522Tr-

shSOX4-1 before and after induction of the shSOX4. (B) Q-PCR to determine DNA enrichment

of the indicated regions of known SOX4 targets relative to the input. (C) Q-PCR to determine

DNA enrichment of the indicated regions of the new candidate targets relative to the input. The

locations of the SOX4-consensus sequences (black boxes) and that of the control regions

(CNTL) relative to the transcription start site (TSS) are included below each graph. Error bars,

SD from triplicates.

Figure 3. Relative levels of gene expression of SOX4 targets in lung cancer and in Sox4-/- mouse

embryonic fibroblasts.

(A) representative examples of real-time quantitative RT-PCRs to determine mRNA levels of

the indicated genes relative to GAPDH in lung tumors with high (H-SOX4; n=6) or low (L-

SOX4; n=6) levels of SOX4. The median level of gene expression for each group is also

indicated. Bars, SD from triplicates. (B) Left panel, heat map depicting the expression levels of

all the transcripts in the H-SOX4 and L-SOX4 tumors from dataset GSE8569. *P<0.05;

**P<0.01; ***P<0.005; NS, not significant. Mann-Whitney U test. Right panel Graphical

representation of the ranked gene lists derived from the comparison (using GSEA) of dataset

GSE8569 and the genes down-regulated in the H522Tr-shSOX4-1 clone upon induction of the

shSOX4. The corresponding P and false discovery rate (FDR) values are indicated. (C) Upper

panel, genotype of Sox4 in the MEFs from the Sox4+/+, Sox4+/- and Sox4-/- mice. Lower panel,

western blot of MEFs from the Sox4+/+, Sox4+/- and Sox4-/- mice. (D) Real-time quantitative RT-

PCR of the indicated genes in the MEFs from Sox4-/-, relative to Gapdh. The values depicted are

relative to the control MEFs (from Sox4+/+ mice). Bars, SD from triplicates. **P<0.01; *P<0.05.

Figure 4. Variation of Sox4 gene expression in ageing lung from mice.

(A) Sox4 and p16INK4a mRNA levels assessed by real-time quantitative RT-PCR normalized

with the internal control Gapdh in lung tissues from old (n=5) and young (n=4) C57BL6 mice.

The values were relative to the mean values of young mice. (B) mRNA levels assessed by real-

time quantitative RT-PCR relative to the internal control Gapdh and to the mean values of

young mice, of the indicated genes in lungs from young and old C57BL6 mice. Bars, mean ±SD

from the different groups of individuals. *P<0.05; **P<0.01; ***P<0.005

Figure 5. Characterization of gene expression in SOX family members among the different lung

cancer histopathologies.

(A) mRNA levels of SOX11 and SOX2 genes assessed by real-time quantitative RT-PCR,

relative to the internal control GAPDH, in lung cancer cell lines of different histopathologies and

in normal lung. SCLC, small cell lung cancer; SCC, squamous cell carcinoma; LCC, large cell

carcinoma; AC, adenocarcinoma; NL, normal lung. Bars, means ±SD from triplicates. (B)

Representative examples of SOX9-positive immunostaining (original magnification, 200x), in

two different lung primary tumors. Cells from normal tissue that do not express detectable SOX9

protein are included in the images and serve as negative controls. (P=0.01; Fisher’s exact test).

the indicated lung cancer cell lines. GAPDH is also shown for protein loading comparison. The

histopathological subtypes are also indicated.

Figure 6. Similarities between the SOX4 expression signature and the expression profile of

SCLC.

(A) Heat map depicting the comparison of gene expression levels of the indicated transcripts in

non-small cell lung cancer (squamous cell carcinoma tumor, SCC; adenocarcinoma, AC) and

SCLC. The arrows indicate two AC cell lines with a similar profile to those of the SCLC type.

(B) On the left panel, two-dimensional hierarchical clustering of the genes with greatest

differential expression between NSCLC and SCLC cell lines. On the right panel, graphical

representation of the ranked gene lists derived from the comparison (using GSEA) of the

indicated dataset and the genes down-regulated in the H522Tr-shSOX4-1 clone upon induction

of the shSOX4. The P and false discovery rate (FDR) values are indicated.

FIGURE 3

A C ** *** *** MEFs genotype

+/+ +/+ +/+ +/- -/- +/- +/- Sox4 wt-

over GAPDH GAPDH over Knock out- Fold change of expression of expression change Fold Sox4-

Gapdh- *** *** *** over GAPDH GAPDH over Fold change of expression of expression change Fold

B D H-SOX4 L-SOX4 Lung primary tumors (GSE8569). *** SOX4 CCNG2 Samples stratified according to the SOX4 status *** +/+ * DOCK4 (6 samples of each SOX4-H and SOX4-L) NS MLLT11 NS MYB Sox4 100% NS RBP1 * TMEFF1 NS EGR3 ** ** * ** ** NS GPC2 * MTA2 ** TEAD2

NS TUBB3 FDR= 0.006 Expression over *** VASH2 P<0.0001 NS RNF122 0% * PCDHB2 NS PCDHB5 ** PCDHB7 *** PCDHB11 *** PCDHB14 *** PCDHB9

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2011 American Association for Cancer Research. FIGURE 6 Downloaded from Author ManuscriptPublishedOnlineFirstonNovember14,2011;DOI:10.1158/0008-5472.CAN-11-3506 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited.

A H2009 H2887 SCC AC SCLC

GENE

SOX4 142,2 84,1 271,2 1316,0 1832,4 3123,5 2027,68,2 1373,1 195,1 736,6 119,4 1435,8 132,8 1957,2 182,3 267,2 1049,6 2167,1 687,1 849,6 1102,6 613,3 323,6 2775,91426,5 1676,4 1180,5 1628,9 1053,82056,2 259,4606,0 2906,7462,1 1590,8385,3 475,2 1631,8 1628,7918,62204,5 218,4 363,1776,0241,8 129,7 1666,7 1290,0847,1 2813,71519,8 1647,6 1226,1 1495,81014,6 4246,1 10449,7 7403,2 4584,7 3514,4 7810,8 7573,0 3260,21016,4 2518,8 3695,6 9952,2 1459,3 10794,5

CCNG2 12,1 224,6 43,3 74,7 108,7 56,770,1 214,9 71,2 113,6 54,5 168,7 248,9 179,146,5 51,9 279,2 460,5 101,9 453,1 162,571,4106,9 444,1 34,1 420,5 95,099,774,1 194,6 283,5 539,8 53,9 44,4 611,5 18,3170,0 214,8 253,8 115,8108,4 142,9 493,5 7,3 98,8 140,6 39,5 238,2 272,8227,7 220,6449,8 183,2151,9 347,4 315,1109,3210,3 432,7 142,131,9 408,3 793,9 76,4 282,9241,5 405,9 247,8 286,3 364,4

DLG3 24,9 32,3 113,4 23,2 133,0 119,3 99,98,2105,4 56,5 231,120,8 82,1 23,176,2 36,3 19,3 31,430,6 158,7 126,5105,1 285,0 132,1106,9 279,4 188,4 229,4 121,0 139,1 173,440,4 128,5 366,0 10,8202,537,4104,2 160,1 173,9229,8 121,0 227,3 23,1 126,3 130,9 73,2 155,9 198,288,5 192,9216,9 12,8 275,6 435,4 164,1 228,1 338,2 505,7 248,0156,6 320,0 249,7 251,7 213,9194,8 280,5 486,8 281,3 228,2 cancerres.aacrjournals.org

DOCK4 92,8 39,7 38,6 275,9 146,4 36,4200,972,5 69,3 136,2 182,3 157,7 51,3 145,558,4 51,8 99,7 68,9105,440,9 36,6 142,9 75,9 14,7 278,8 132,332,7 686,4 33,9 41,4 173,0 66,9 283,4 169,7 60,374,2273,2 137,3 71,745,342,9 65,810,9 602,8 309,3 33,3 29,230,1 88,2 314,5 64,085,0140,4 72,8 86,8 192,9 309,1 79,992,0 299,8 692,6658,3 246,4 88,0108,3148,982,9 114,8 311,6 80,9

MLLT11 2796,7 156,4 243,9 2241,5 284,2 620,5 1412,8208,1 1163,8 322,7 1813,1 2321,2 115,5 632,6 229,0 233,9 323,2 3 00,3404,8 93,7 1099,8 128,0 539,8 61,5122,6206,688,2 3370,468,7 110,6 2372,2 258,8 57,9 85,5134,9230,2680,5 252,21004,8 114,2 140,7 618,8 939,6 1885,7570,6199,8909,5 70,4 237,9852,5 674,23132,8 7785,3 4378,6 4437,0 5651,1 2326,4 7569,0 7258,7 1185,2 5944,86392,4 12945,5 3506,2 7824,7 2371,84038,4 3594,8 4099,7 2960,7

MYB 12,9 49,2105,4 9 44,3 86,7 244,655,1 24,5 75,5 93,1 35,8 49,9 85,2 146,7 43,8 262,1 135,1 89,6 63,5120,293,6 77,7 64,1121,177,4 150,938,592,8 89,5 38,8 72,5 48,3102,245,958,6109,4 89,2 57,68549,2109 80,9 74,1 168,5 325,5 17,5 72,9 64,664,4 230,7213,7 180,2 296,7 373,9 182,5 396,7355,6 252,5 470,4 127,4 442,5 204,8 3 00,7 341 219,5 261,6 355,1 335,2500

NBEA 20,5 32,9 34,3 235,5 141,3 135,1 161,551,2 67,9 117,9 76,2 35,9 63,7 35,8 123,8 172,2 55,5 125,0 16,7 94,7 37,8105,5 125,0 29,6184,380,968,6 165,747,1 77,2 97,9 42,2 117,5 132,745,651,170,9 113,2 74,533,931,9 388,6 83,3 203,1 49 0,8 108,8 118,462,5 124,971,186,4124,8 124,7 98,7 325,5 193,4 582,9 415,9423,3 198,9 361,0307,7 162,4 720,6 194,3 21 4 ,0 408,6 496,8 521,6 90,5

PCDHB6 9,4 22,9 14,7 59,3 93,6 17,8 73,153,6 91,6 79,9 97,6 35,1758860,270,5 55,8 25,2 123,2 71,3 47,2 14,4 14,3 56 67,972,910,5 43,157,6 41,320,1 21,9 35,6 36,1 99,7 6,9 50 41,5 91,7 12,330,4 25,228,2 90,3 116,7 57,3 48,8 94,4 153 45 78,130,3 53,6 33,7 117,9 31,1 61,2 19,7 79 ,7 106,9 72,2 117,8 90,560,3103,54446,8 168,5 77,3 116,3

PCDHB8 15 26,41,5 35,1 32,25,3 23,69,3 43,4 45,75,5 18,9 27,8 21,914,20,85,3 13,8 60,3 12,8 14,53,56,5 0,8 40,832,56,33,713,8 19,89,63,77,83,71,134,93,53,42,92,853,75,58,9324,12,6 16,261,6 46,2 101,8 9,726,8 8,612,4 22,2 166,7 18,4 60,658,7 38,1 24 82 22,9 10,69,8 23 ,1 71,5 16,7 45,9 5,2

PCDHB11 5,96,754,28,23,37,89,8 26,97,24,77,9 14,7 17,45,46,56,76,8 15,85,7 7,73,34,92,94,222,28,2746,277,93,73,917,975,7 11,86,4 3,1 8,25,112,27,3 24,24,2712,2 117,3 38,632,111,4 31,88,2 27,820,676,420,1 55,2 19,2 63,1 19,9 6,95,5 15 ,823,5 23,6 52,5 9,8

PCDHB12 11,6 86,1 17,4 12,6 21,5 15,3 21,235,2 46,9 34,3 22,9 24,4 81,2 71,146,66,5 24,9 12,3 74,8 23,9 53,615,5 14,43,625,119,232,78,242,4 28,1 21,8 22 16,4 14,947,218,818,7 35,1 11,1 8,711,4 18,3 17,4 52,3 53,816,3 37,124,1 111,3 34,136,112,7 21,941,3 47 79,559,8 8,23373,2 18,6 157,9 122,8 8,58,7 18 ,338,8 53,7 114,8 55,2

PCDHB13 3,55,02,86,54,43,27,84,6 57,58,03,27,55,4 18,18,93,76,14,55,35,29,42,83,3 2,7 4,312,12,94,62,4 34,5 5,63,72,46,810,05,26,09,87,12,75,9 16,07,9 12,66,013,8 3,8 58,4 79,4 83,7 61,9 5,9 22,7 6,7 12,520,522,8 4,725,7 70,5 12,6 128,1 21,3 4,322,4 12 ,720,6 5,7 80,0 4,8

PCDHB17 0,9 6,64,14,71,651,817,8 4,46,952,1241,72,34,91,51,83,41,34,6 12,2 4,9 14,4 4,60,92,56,74,13,10,81,65,62,1 5,4 16,3 1,81,8 8,8 1,43,12,22,12,61,522,1 26,4 7,83,5 4,7 26,7 3,4 4,92,1 3,8 11 ,8 1,3 5,4 22,5 5,4 14,66,27,4 12 3,4 3,9

PLEKHB1 5,6 54,710,9 24,4 67,5 334,1 78,626,5 39,9 120,0 32,7 47,9 85,0 11,050,490,1 25,4 66,9 73,4 38,4195,068,920,8100,369,7114,7 110,75,2209,0101,5 329,5 159,1 19,6 48,430,2101,948,5 55,160,1 199,082,8 192,5106,2 67,1 88,631,190,837,4 153,6117,5202,959,1 90,0 20,6 304,7 187,3 651,1 237,1375,5246,6 84,8 437,3 52,6 368,8 119,3252,0 233,4 502,8 331,8 29,4

RBP1 209,9 235,2 436,0 36,2 51,8 99,2 317,053,3 11,2 58,6 13,9 89,6 145,0 84,091,07,3 115,8 128,5 32,8 33,9 47,637,3 73,9 85,4 8,6174,925,436,977,4 95,39,9 56,1 47,3501,4219,323,291,9 247,5 39,432,2109,8 84,0 381,9167,080,830,1107,854,8 69,1 3535,6 314,5 3365,6 154,7 2834,8 258,5 724,6496,8 1651,2 621,4 688,2249,1 2558,0 1138,1 16,5 1784,4 619,3 494,5 771,5 1961,6 2332,9

RNF122 62,2 320,5 195,6205,7 127,3 214,7 177,2 122,8 166,3 350,6 175,2 379,7 245,5 140,1 178,6 113,5 330,7 220,3 98 77,5 167 131,8 150,9 145,1125,9 637,4 135,8 112,770,1108,3 306,6 224,8102,8 170,9164,7101,5 238,5 57 0,5 168,2130,2 315,8205,2250,3 164,1 224,7 1 00,5 99,9 194,9 164,6 417 473,7 31 3 ,9 398,7287,7 246,9 287,4 100,7 224,8 436,9 125,9132,6 131,9 324,1 124,8 308,1238,4 480,7 333,7 138,8 197,3

SOX2 18,3 65,8 127,9 67,020,4 45,4 41,79,3 14,3 21,9 23,3 65,9 417,5 76,242,1 47,4 75,4 172,1 67,8 57,1660,452,1 71,7 29,8165,9338,8167,2 31,813,7 53,6 219,56,340,7 134,952,991,115,2 16,578,040,2225,4 85,866,3 22,6 186,5 540,8 237,629,3 447,2 387,3 1688,9 8,4 33,1 4,2 1142,6 92,834,8 489,0 44 ,5 516,2 274,1 576,3 74,010,215,7 621,2 95,0 380,9 1226,8 395,7

on September 29, 2021. © 2011American Association for Cancer SOX11 14,94,9 18,0 2,43,3 3,530,04,97,5 14,3 14,54,1 18,16,64,8 24,6 41,930,13,7 28,23,414,33,5 13,6458,2115,535,44,916,75,55,69,1 14,7 12,218,97,727,7 11,15,28,614,3 65,957,77,91021,23,39,88,07,029,6 7,1 1556,6 139,8 1830,8 1551,3 484,4253,2 1736,4 3156,97,0 1671,3 1149,7 1885,0 16,5 789,613,7 1845,0 930,9 402,9 177,4

STMN1 519,3 344,9 367,7 132,9 325,6 331,1 382,2440,9 260,5 226,1 94,5 687,5 195,2 255,0 234,6 416,2 851,0 327,3 262,4220,3207,0 224,6306,4 134,6304,3 530,1436,2101,3 372,1 241,8 217,2 340,6 143,2 332,3562,6 275,5 163,0 599,6 279,5 161,3283,8 279,5510,7 252,3 528,8392,5 673,7380,9 290,7392,6 521,6 1456,7 509,3 980,6 713,5 542,3423,6 3022,9 1790,3994,0 2095,42187,1 1348,9 799,9 1772,6 908,9503,9 1245,1803,5 1007,3

TMEFF1 108,7 92,5 19,8 1 00,9 221,3 134,0 23,278,0 189,7 18,9 52,7 21,3 29,9 41,149,0 68,5 25,9 39,3107,3 32,4 12,126,1 36,7 33,9 37,086,223,2 143,151,9 24,61,7 27,2 129,0 39,170,030,040,1 11,53,023,730,0 15,961,9110,5 26,728,4 315,6 153,750,39,7 80,7147,8 128,6 81,4 747,7 309,7 719,3 413,4 903,8 301,0 774,4 1889,0 689,2805,1 301,887,3 397,6 629,3 993,5 216,2 Research.

TUBB3 1970,1 1667,8 1405,1 1193,2 2467,4 1364,8 3088,6 1709,5 1326,6 899,7 483,9 2364,0 1059,8 1383,7 1767,7 1693,1 530,7 965,4 2704,3 1517,3 1969,1 724,0 1513,0 2392,2 1128,31563,3 1644,4 1 03 0,3 2410,4 2373,31303,4 920,1 949,6 1749,8 1639,9 1755,8618,4 3111,5 1621,91063,4 1452,1 2763,6 3172,2 947,6 2239,2 2104,2 1793,5 2525,3 1382,2 4369,92217,4 5034,1 2369,1 6478,9 2823,2 2415,42749,3 7937,6 6230,5 2135,0 8784,6 4755,5 7683,7 2557,1 4604,4 4754,3 4572,7 6906,8 3386,9 3522,2

VASH2 11,8 23,0 12,4 13,220,5 20,0 54,212,8 26,940,7 13,0 24,3 44,52,320,81,8 268,5 27,5 42,7 28,8 16,716,59,4 38,2 20,060,71,7 8,84,1 16,3 61,7 14,63,410,521,329,227,3 73,7 16,220,09,1 32,239,9 6,4 15,38,0 22,321,9 17,4 31,6 85,8108,0 75,0 172,1 224,7 246,5 66,4131,5 152,7 47,427,4 64,9 211,0 41,9 191,6 27 ,7 153,7 122,3 37,7 93,0

B SCLC NSCLC Lung cancer cell lines (dataset GSE4824) Samples stratified according to histopathology: NSCLC versus SCLC

FDR= 0.06 P<0.05

-3 -0.62 1.49 Author Manuscript Published OnlineFirst on November 14, 2011; DOI: 10.1158/0008-5472.CAN-11-3506 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Novel transcriptional targets of the SRY-HMG box transcription factor SOX4 link its expression to the development of small cell lung cancer

Sandra D Castillo, Ander Matheu, Niccolo Mariani, et al.

Cancer Res Published OnlineFirst November 14, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-11-3506

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