SOX7 is down-regulated in lung cancer
© Hayano et al.; licensee BioMed Central Ltd. 2013
Received: 4 December 2012
Accepted: 6 March 2013
Published: 4 April 2013
SOX7 is a transcription factor belonging to the SOX family. Its role in lung cancer is unknown.
In this study, whole genomic copy number analysis was performed on a series of non-small cell lung cancer (NSCLC) cell lines and samples from individuals with epidermal growth factor receptor (EGFR) mutations using a SNP-Chip platform. SOX7 was measured in NSCLC samples and cell lines, and forced expressed in one of these lines.
A notable surprise was that the numerous copy number (CN) changes observed in samples of Asian, non-smoking EGFR mutant NSCLC were nearly the same as those CN alterations seen in a large collection of NSCLC from The Cancer Genome Atlas which is presumably composed of predominantly Caucasians who often smoked. However, four regions had CN changes fairly unique to the Asian EGFR mutant group. We also examined CN changes in NSCLC lines. The SOX7 gene was homozygously deleted in one (HCC2935) of 10 NSCLC cell lines and heterozygously deleted in two other NSCLC lines. Expression of SOX7 was significantly downregulated in NSCLC cell lines (8/10, 80%) and a large collection of NSCLC samples compared to matched normal lung (57/62, 92%, p= 0.0006). Forced-expression of SOX7 in NSCLC cell lines markedly reduced their cell growth and enhanced their apoptosis.
These data suggest that SOX7 is a novel tumor suppressor gene silenced in the majority of NSCLC samples.
KeywordsCNAG SNP-Chip Lung cancer SOX7 Promoter methylation
Lung cancer is the leading cause of cancer-related death in the world. If surgery is inadequate, further therapy is rarely curative. Understanding the genomic abnormalities in this disease affords the opportunity to identify new therapeutic targets. An example is the use of Gefitinib for patients whose non-small cell lung cancer (NSCLC) has an epidermal growth factor receptor (EGFR) mutation in either exon 19 or 21.
SOX7 is a member of the SOX (SRY-related high mobility group box) transcription factors . This protein, together with SOX17 and SOX18, comprises the SOX F subgroup  and helps mediate various developmental processes including a role in the regulation of hematopoiesis , cardiogenesis , vasculogenesis [5, 6], endoderm differentiation  and myogenesis . Recently, SOX7 has been proposed to function as a tumor suppressor in colorectal and prostate cancers [9, 10]. We provide evidence that SOX7 behaves as a tumor suppressor in lung tissue and its expression is either low or silenced in the majority of lung cancers.
Materials and methods
Cell lines and tissue samples
Ten human lung cancer cell lines (H23, H460, H820, H1299, H1975, HCC827, HCC2279, HCC2935, HCC4006, PC14) were cultured in RPMI medium with 10% FBS and kept in a humidified atmosphere of 5% CO2. After IRB consent, total DNA and RNA of normal and cancerous lung tissues were obtained from the National University of Singapore (NUH-NUS Tissue Repository). Also, sixty-two pairs of primary NSCLCs and their corresponding adjacent normal tissues, which were at least 5 cm away from the cancer, were obtained from NSCLC patients treated at Shanghai Chest Hospital (Shanghai, China), after their written informed consent. None of the patients received radio-chemotherapy prior to obtaining the tissues. Lung cancer cells stably expressing either GFP or SOX7 were generated by transducing them with PLKO.1 lentiviral vector system (Sigma). Briefly, cells were transduced with lentiviral vectors (SOX7 or GFP) at an MOI of 25 with 5 ug/ml polybrene added for 6 h. Twenty-four hours post-transduction, stable cells were selected using 1ug/ml puromycin for 2-3 weeks.
High-density single nucleotide polymorphism-array analysis
Genomic DNA from NSCLC cells were subjected to GeneChip Human mapping (1000 K array for the EGFR mutant lung cancer samples and 250 K array for the NSCLC cell lines). Both total and allelic-specific copy numbers (CN) were determined using CNAG software [11, 12].
Quantitative real-time polymerase chain reaction
Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed using Maxima® First Strand cDNA Synthesis Kit for RT-qPCR (Fermentas) according to the manufacturer’s protocol. The expression level of SOX7 mRNA in the samples was determined by quantitative real-time PCR (7500 Fast Real-Time PCR System, Applied Biosystems) using KAPA™ SYBR® FAST qPCR Kit Master Mix (2X) Universal (Kapa Biosystems). Levels of β-actin mRNA were used as an internal control. The delta threshold value (DCt) was calculated from the given threshold (Ct) value by the formula DCt = (Ct SOX7 – Ct β-actin) for each sample.
NSCLC cells were lysed with ProteoJET™ Mammalian Cell Lysis Reagent (Fermentas). Immunoblotting was performed using either anti-SOX7 antibody (Sigma, HPA009065) or anti-β-actin antibody (Sigma, AC-15) and either secondary anti-Rabbit IgG antibody (GE Healthcare, NA934) or anti-murine IgG antibody (GE Healthcare, NA931), respectively. SOX7 or β-actin bands were detected using Pierce® Fast Western Blot Kit, SuperSignal® West Femto Substrate (Thermo SCIENTIFIC) and SuperSignal® West Pico Chemiluminescent Substrate (Thermo SCIENTIFIC), respectively.
Genomic DNA was modified by sodium bisulfite using the CpGenome™ Turbo Bisulfite Modification Kit (MILLIPORE). The following PCR primers were used for bisulfite-modified genomic DNA :
Region (-687 to -440): 5’-TTAATTAGGTGGTTGAGAATTAGAA and 5’-TAACCATAAACCCCTCAAAACA
Region (-71 to +251): 5’-TTTTGGAGAGTTATTGGAGGA and 5’-CCTTAACCCAAACCATAAAAA
PCR products were cloned into either the pGEM-T or pGEM-T easy vector (Promega), and at least four clones from each sample were sequenced.
Methylation specific PCR (MSP) assay
Primers specific for the unmethylated (U) and methylated (M) sequences were designed by using Meth Primer . Primers sequences are as follows:
MSP-U (-683 to -493): 5'-TAGGTGGTTGAGAATTAGAATGAT G and 5'-CTTTCAAAAATAACCAAACTTCAAC
MSP-M (-683 to 493): 5'-TTAGGTGGTTGAGAATTAGAACGAC and 5'-TCGAAAATAACCGAACTTCGA
MSP-U (+192 to +321): 5'-ATAAGGGTTTTGAGAGTTGTATTTG and 5'-ACTCACCCAACATCTTACTAAACTCA
MSP-M (+192 to +321): 5'-ATAAGGGTTTCGAGAGTCGTATTC and 5'-TCACCCAACATCTTACTAAACTCG
H23 and H1975 cells were seeded at 5 × 103 per well in 96-well plates. H1299 cells were seeded at 1.5 × 103 per well in 96-well plates. MTT reagents were added to each well, and absorbance was measured according to the manufacturer’s instructions (Promega).
Cell cycle analysis by flow cytometry
2x106 cells stably expressing either SOX7 or GFP were seeded into 6-well plates for 24 h. Cells were harvested and washed twice with cold phosphate-buffered saline (PBS) and fixed in 75% ethanol (precooled at -20°C) for 24 h at 4°C. The fixed cells were washed twice with 2 ml of cold PBS. Cells then were stained with 500 ul of propidium iodide (PI) staining solution (50 ug/ml PI, 0.1%Triton X-100, 200 mg/ml DNase-free RNase in PBS) for 30 min at room temperature in the dark. Ten thousand events per sample were acquired using a LSR-II flow cytometer (Becton-Dickinson, San Jose, CA, USA), and the percentage of cells in G0/G1, S, G2/M and Sub-G2/M phases of the cell cycle were determined using FACS DIVA software (Becton-Dickinson).
Annexin V and propidium iodide (Annexin V–PI) staining apoptosis test
4× 105 cells were seeded into each well of a 6-well plate for 48 h. The staining was carried out according to the instructions provided by the manufacturer of PE Annexin V Apoptosis Detection Kit I, BD Pharmingen (BD Biosciences, USA). Briefly, cells were washed with PBS, suspended in 1X binding buffer and then added with annexin-V APC and propidium iodide (PI) for 15 min. The samples were then analyzed by LSR-II flow cytometer (Becton-Dickinson, San Jose, CA, USA).
Whole genomic copy number analysis using high resolution SNP-Chips in NSCLC samples and cell lines
Common copy number genomic alterations in NSCLC found in two cohorts: TCGA and EGFR mutant, non-smoking Asians
Region of Chromosome
Candidate target genes
Gain of 1q21.1q-24.2
Gain of 5p13.2
Gain of 7p11.2
Gain of 8q24.3
Gain of 8q24.21
Gain of 8q24.12
Loss of 8p23.1
UNQ9391, RP1|1, SOX7
Gain of 11q13.2-13.3
CYCLIN D1, FGF3, FGF4, FGF19
Gain of 12q13.3-14.1
Gain of 12q12.1
Gain of 12q11.21
Gain of 17q12
Gain of 17q25.3
Copy number genomic alterations that predominant in NSCLC from non-smoking Asians with mutant EGFR compared to TCGA database
Region of Chromosome
NSCLC with mutant EGFR (n=8)
NSCLC from TCGA data base (n=56)
Potential target gene(s)
Gain of 1p36.32-36.31
Gain of Ch2p
Gain of Ch3q
Loss of 6q22.3-27
Loss of 9p21.3
Gain of 15q23-26.3
Gain of 19q12
Gain of 20q11.21
BCL2L1, TPX2, MYLK2, DUSP15
Cell lines enhance the opportunity to discover homozygous deletions because they are not contaminated with normal cells. A homozygous deletion often marks the position of a tumor suppressor gene that may be deleterious for either development or progression of cancer. A small homozygous deletion at 8p23.1 was found in one (HCC2935) of 10 NSCLC cell lines. The SOX7 was located in this small homologously deleted region together with 2 other genes (UNQ9391 and RP1L1) (Figure 1C; Table 1).
Expression of SOX7 in NSCLC
Upstream region of SOX7 gene in lung cancer cell lines was highly methylated
Summary of methylation analysis of SOX7
BS (-687 and -493)
MSP (-683 and -493)
BS (-71 to +251)
MSP (+192 and +321)
Forced-expression of SOX7 in NSCLC cells slowed their proliferation
Effect of SOX7 expression on cell cycle regulation
Forced expression of SOX7 induces apoptosis in H23 and H1299 cell lines
We initially performed CN analysis of 9 NSCLC samples and 8 NSCLC cell lines, each with an EGFR mutation. Their pattern of genome alterations were compared to the SNP-Chip copy number changes found in 56 NSCLC in the TCGA data base. Our samples were from non-smoking Asians who had EGFR mutations. The TCGA samples were composed of predominantly Caucasians who smoked and therefore less than 7% of samples would be expected to contain an EGFR mutation . Remarkably, their genomic landscape of copy number change was very similar. All the samples had increase in CN throughout the genome (predominantly 3N), especially at 1q, 5p, 7p, 8q, 11q, 12q, 14q, 17q. However, although sample numbers were small, eight genome regions had notable difference in copy number changes between the NSCLC samples with EGFR mutation compared to those in the TCGA data base samples (Table 2) including 1p36.31-36.32 [8/9 (89%) versus 15/56 (27%)] and 19q21.3, [5/9 (56%) versus 6/56 (11%)], respectively. Further studies are required to clarify what the target genes are in these regions (Table 1).
One of the NSCLC cell lines (HCC2935) had a homozygous mutation at 8p23.1 which encompassed the SOX7 gene (Figure 1). Interestingly, 8p is one of the few regions in the NSCLC samples associated with deletions. Homozygous deletion usually represents the loss of a tumor suppressor gene deleted by the tumor. Our further studies focused on SOX7. Expression levels of SOX7 mRNA and protein were diminished in eight of 10 NSCLC cell lines (Figure 2), as well as in fifty-seven of 62 (92%) NSCLC patient samples compared with their matched normal tissues. Expression level of SOX7 in NSCLC samples was correlated with their histology, with levels being lower in adenocarcinomas compared with adenosquamous and squamous carcinomas (Figure 3). Furthermore, force-expression of SOX7 in several NSCLC lines (H23, H1299, and H1975) having constitutively low level of SOX7, suppressed their cellular proliferation and enhanced their apoptosis (tested with H23and H1299) (Figure 5, 6 and 7).
Recent studies of SOX7 in colorectal and prostate cancers showed that levels of this transcription factor were low in these cancers in part due to aberrant DNA methylation of the gene, and the protein behaved as a tumor suppressor gene in these cancers [10, 15]. We found that the upstream region (-687 to -440) of SOX7 was highly methylated in eight of 10 NSCLC cell lines (Table 3). Paradoxically, expression of SOX7 and methylation as measured by MSP analysis were not correlated in the H460 and PC14 cells, and only one of 5 fresh NSCLC samples was highly methylated in the promoter region of SOX7. This suggests that additional epigenetic changes are required for silencing of this gene in a proportion of NSCLC.
In summary, our study suggests that SOX7 is a tumor suppressor in the lung. One or occasionally both alleles are lost in the lung cancer. Other times the upstream CpG island of the SOX7 gene is robustly methylated, associated with low expression of the gene. SOX7 levels were nearly undetectable in seven of 9 (78%) highly methylated NSCLC cell lines, and levels were low in 57 of 62 (92%) NSCLC samples compared to adjacent normal tissues. Loss of SOX7 expression appears to provide a growth advantage to NSCLC cells.
This work was funded by the Singapore Ministry of Health’s National Medical Research Council under its Singapore Translational Research (STaR) Investigator Award to H. Phillip Koeffler, and NIH grants R01CA026038-32, as well as, the Cancer Science Institute of Singapore internal grant awarded to Patrick Tan. We are grateful to Dr. Eng Chon Boon (Head of NUH-NUS Tissue Repository) and his team who provided DNA and total RNA of normal and cancerous lung tissue.
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