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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 LopezRios4, Robin Lovell-Badge2 and Montse Sanchez-Cespedes1*
Authors´Affiliations:1Genes and Cancer Group, Cancer Epigenetics and Biology ProgramPEBC), 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]
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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 Sox4deficient 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.
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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 EGFRmutant 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 (1518). 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
3
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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.
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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
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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 reversetranscribed. 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-mouseIgG: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,
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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 nonimmune 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 crosslinks 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
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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 limmamoderated 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 KolmogorovSmirnoff 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 H522TrshSOX4-1 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 H522Tr8
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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 Cterminal 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 downregulated after SOX4 depletion were direct transcriptional targets of SOX4. The SOX4 HMGbox binds preferentially to the 4-bp DNA-motif 5´-ACAA-3´ sequence (34) and we searched for
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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 upregulated 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
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We extended our analysis to lung primary tumors selected on the basis of high (SOX4H) 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
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(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 realtime 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
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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
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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.
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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.
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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
16
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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.
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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.
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 shortterm 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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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
References
1.
Pao W, Girard N. New driver mutations in non-small-cell lung cancer. Lancet Oncol
2011; 12: 175-80.
2.
Medina PP, Castillo SD, Blanco S, Sanz-Garcia M, Largo C, Alvarez S, et al. The SRY-
HMG box gene, SOX4, is a target of gene amplification at chromosome 6p in lung cancer. Hum
Mol Genet 2009; 18: 1343-52.
3.
Prior HM, Walter MA. SOX genes: architects of development. Mol Med 1996; 2: 405-
12.
4.
Penzo-Mendez AI. Critical roles for SoxC transcription factors in development and
cancer. Int J Biochem Cell Biol 2010; 42: 425-8.
5.
Farr CJ, Easty DJ, Ragoussis J, Collignon J, Lovell-Badge R, Goodfellow PN.
Characterization and mapping of the human SOX4 gene. Mamm Genome 1993; 4: 577-84.
6.
van de Wetering M, Oosterwegel M, van Norren K, Clevers H. Sox-4, an Sry-like HMG
box protein, is a transcriptional activator in lymphocytes. Embo J 1993; 12: 3847-54.
7.
Wegner M. From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res
1999; 27: 1409-20.
8.
Aaboe M, Birkenkamp-Demtroder K, Wiuf C, Sorensen FB, Thykjaer T, Sauter G, et al.
SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization.
Cancer Res 2006; 66: 3434-42.
9.
Beer DG, Kardia SL, Huang CC, Giordano TJ, Levin AM, Misek DE, et al. Gene-
expression profiles predict survival of patients with lung adenocarcinoma. Nat Med 2002; 8:
816-24.
10.
Evans AJ, Gallie BL, Jewett MA, Pond GR, Vandezande K, Underwood J, et al.
Defining a 0.5-mb region of genomic gain on chromosome 6p22 in bladder cancer by
quantitative-multiplex polymerase chain reaction. Am J Pathol 2004; 164: 285-93.
11.
Garber ME, Troyanskaya OG, Schluens K, Petersen S, Thaesler Z, Pacyna-Gengelbach
M, et al. Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci U S A
2001; 98: 13784-9.
19
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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.
12.
Liu P, Ramachandran S, Ali Seyed M, Scharer CD, Laycock N, Dalton WB, et al. Sex-
determining region Y box 4 is a transforming oncogene in human prostate cancer cells. Cancer
Res 2006; 66: 4011-9.
13.
Pramoonjago P, Baras AS, Moskaluk CA. Knockdown of Sox4 expression by RNAi
induces apoptosis in ACC3 cells. Oncogene 2006; 25: 5626-39.
14.
Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q, Bos PD, et al. Endogenous
human microRNAs that suppress breast cancer metastasis. Nature 2008; 451: 147-52.
15.
Du Y, Spence SE, Jenkins NA, Copeland NG. Cooperating cancer-gene identification
through oncogenic-retrovirus-induced insertional mutagenesis. Blood 2005; 106: 2498-505.
16.
Lund AH, Turner G, Trubetskoy A, Verhoeven E, Wientjens E, Hulsman D, et al.
Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient
mice. Nat Genet 2002; 32: 160-5.
17.
Shin MS, Fredrickson TN, Hartley JW, Suzuki T, Akagi K, Morse HC, 3rd. High-
throughput retroviral tagging for identification of genes involved in initiation and progression of
mouse splenic marginal zone lymphomas. Cancer Res 2004; 64: 4419-27.
18.
Suzuki T, Shen H, Akagi K, Morse HC, Malley JD, Naiman DQ, et al. New genes
involved in cancer identified by retroviral tagging. Nat Genet 2002; 32: 166-74.
19.
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, et al. Large-scale
meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic
transformation and progression. Proc Natl Acad Sci U S A 2004; 101: 9309-14.
20.
Reymann S, Borlak J. Transcription profiling of lung adenocarcinomas of c-myc-
transgenic mice: identification of the c-myc regulatory gene network. BMC Syst Biol 2008; 2:
46.
21.
Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K. Autocrine TGF-
beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box
factors. Cell Stem Cell 2009; 5: 504-14.
22.
Thein DC, Thalhammer JM, Hartwig AC, Crenshaw EB, 3rd, Lefebvre V, Wegner M,
et al. The closely related transcription factors Sox4 and Sox11 function as survival factors
during spinal cord development. J Neurochem; 115: 131-41.
20
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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.
23.
Lowry WE, Blanpain C, Nowak JA, Guasch G, Lewis L, Fuchs E. Defining the impact
of beta-catenin/Tcf transactivation on epithelial stem cells. Genes Dev 2005; 19: 1596-611.
24.
Angulo B, Suarez-Gauthier A, Lopez-Rios F, Medina PP, Conde E, Tang M, et al.
Expression signatures in lung cancer reveal a profile for EGFR-mutant tumours and identify
selective PIK3CA overexpression by gene amplification. J Pathol 2008; 214: 347-56.
25.
Conde E, Angulo B, Tang M, Morente M, Torres-Lanzas J, Lopez-Encuentra A, et al.
Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6K signaling.
Clin Cancer Res 2006; 12: 710-7.
26.
Pantoja C, Serrano M. Murine fibroblasts lacking p21 undergo senescence and are
resistant to transformation by oncogenic Ras. Oncogene 1999; 18: 4974-82.
27.
Schilham MW, Oosterwegel MA, Moerer P, Ya J, de Boer PA, van de Wetering M, et
al. Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking
Sox-4. Nature 1996; 380: 711-4.
28.
Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression
and hybridization array data repository. Nucleic Acids Res 2002; 30: 207-10.
29.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al.
Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide
expression profiles. Proc Natl Acad Sci U S A 2005; 102: 15545-50.
30.
Bergsland M, Werme M, Malewicz M, Perlmann T, Muhr J. The establishment of
neuronal properties is controlled by Sox4 and Sox11. Genes Dev 2006; 20: 3475-86.
31.
Bhattaram P, Penzo-Mendez A, Sock E, Colmenares C, Kaneko KJ, Vassilev A, et al.
Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal
progenitors. Nat Commun 2010; 1: 9.
32.
Dy P, Penzo-Mendez A, Wang H, Pedraza CE, Macklin WB, Lefebvre V. The three
SoxC proteins--Sox4, Sox11 and Sox12--exhibit overlapping expression patterns and molecular
properties. Nucleic Acids Res 2008; 36: 3101-17.
33.
Hoser M, Potzner MR, Koch JM, Bosl MR, Wegner M, Sock E. Sox12 deletion in the
mouse reveals nonreciprocal redundancy with the related Sox4 and Sox11 transcription factors.
Mol Cell Biol 2008; 28: 4675-87.
21
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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.
34.
Scharer CD, McCabe CD, Ali-Seyed M, Berger MF, Bulyk ML, Moreno CS. Genome-
wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells. Cancer
Res 2009; 69: 709-17.
35.
Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, et al.
MatInspector and beyond: promoter analysis based on transcription factor binding sites.
Bioinformatics 2005; 21: 2933-42.
36.
Yokota S, Hirayama T, Hirano K, Kaneko R, Toyoda S, Kawamura Y, et al.
Identification of the Cluster Control Region for the Protocadherin-{beta} Genes Located beyond
the Protocadherin-{gamma} Cluster. J Biol Chem 2011; 286: 31885-95.
37.
Reppe S, Rian E, Jemtland R, Olstad OK, Gautvik VT, Gautvik KM. Sox-4 messenger
RNA is expressed in the embryonic growth plate and regulated via the parathyroid
hormone/parathyroid hormone-related protein receptor in osteoblast-like cells. J Bone Miner
Res 2000; 15: 2402-12.
38.
Krishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, et al.
p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 2006; 443:
453-7.
39.
Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, et al.
Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during
ageing. Nature 2006; 443: 448-52.
40.
Bass AJ, Watanabe H, Mermel CH, Yu S, Perner S, Verhaak RG, et al. SOX2 is an
amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nat
Genet 2009; 41: 1238-42.
41.
Liao YL, Sun YM, Chau GY, Chau YP, Lai TC, Wang JL, et al. Identification of SOX4
target genes using phylogenetic footprinting-based prediction from expression microarrays
suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma.
Oncogene 2008; 27: 5578-89.
42.
Mariani TJ, Reed JJ, Shapiro SD. Expression profiling of the developing mouse lung:
insights into the establishment of the extracellular matrix. Am J Respir Cell Mol Biol 2002; 26:
541-8.
22
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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.
43.
Maeda Y, Dave V, Whitsett JA. Transcriptional control of lung morphogenesis. Physiol
Rev 2007; 87: 219-44.
44.
Bangur CS, Switzer A, Fan L, Marton MJ, Meyer MR, Wang T. Identification of genes
over-expressed in small cell lung carcinoma using suppression subtractive hybridization and
cDNA microarray expression analysis. Oncogene 2002; 21: 3814-25.
45.
Sutherland KD, Proost N, Brouns I, Adriaensen D, Song JY, Berns A. Cell of origin of
small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung.
Cancer Cell 2011; 19: 754-64.
23
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 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.
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 H522TrshSOX4-1 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 (LSOX4; n=6) levels of SOX4. The median level of gene expression for each group is also
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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 RTPCR 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 realtime 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
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protein are included in the images and serve as negative controls. (P=0.01; Fisher’s exact test).
(C) Western blot of SOX4, SOX2 and from the neural marker NSE (neuron-specific enolase) of
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.
26
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FIGURE 3
A
Fold change of expression
over GAPDH
**
C
***
***
MEFs genotype
+/+ +/+ +/+ +/- -/- +/- +/Sox4 wtKnock out-
Fold change of expression
over GAPDH
Sox4Gapdh-
***
***
***
B
***
***
*
NS
NS
NS
*
NS
NS
*
**
NS
***
NS
*
NS
**
***
***
***
SOX4
CCNG2
DOCK4
MLLT11
MYB
RBP1
TMEFF1
EGR3
GPC2
MTA2
TEAD2
TUBB3
VASH2
RNF122
PCDHB2
PCDHB5
PCDHB7
PCDHB11
PCDHB14
PCDHB9
L-SOX4
Lung primary tumors (GSE8569).
Samples stratified according to the SOX4 status
(6 samples of each SOX4-H and SOX4-L)
FDR= 0.006
P<0.0001
Expression over Sox4+/+
D
H-SOX4
100%
**
0%
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** *
** **
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H2009
H2887
A
SCC
GENE
SOX4
CCNG2
DLG3
DOCK4
MLLT11
MYB
NBEA
PCDHB6
PCDHB8
PCDHB11
PCDHB12
PCDHB13
PCDHB17
PLEKHB1
RBP1
RNF122
SOX2
SOX11
STMN1
TMEFF1
TUBB3
VASH2
B
1
2
7
4
2
,2
1
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AC
2
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8
2
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SCLC
1
6
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1
8
9
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NSCLC
1
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7
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1
SCLC
2
1
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9
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3
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7
7
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1
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2
5
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1
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9
6
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3
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4
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-0.62
1.49
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4
7
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3
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1
Lung cancer cell lines (dataset GSE4824)
Samples stratified according
to histopathology: NSCLC versus SCLC
FDR= 0.06
P<0.05
-3
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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.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2011 American Association for Cancer Research.
FIGURE 6
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.
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