SOSTDC1 is down-regulated in non-small cell lung cancer and contributes to cancer cell proliferation
- Lei Liu†1, 2,
- Shanshan Wu†1, 2,
- Yi Yang1, 3,
- Junchao Cai1, 2,
- Xun Zhu1, 2,
- Jueheng Wu1, 2,
- Mengfeng Li1, 2 and
- Hongyu Guan4Email author
© Liu et al. 2016
Received: 10 March 2016
Accepted: 4 April 2016
Published: 14 April 2016
Non-small cell lung cancer (NSCLC) is the most commonly diagnosed and fatal cancer worldwide. Sclerostin domain containing protein 1 (SOSTDC1) has been found to be tumor-suppressive in several types of cancers. However, the expression level and biological functions of SOSTDC1 in NSCLC remain unknown. Our current study aimed to identify the biological significance of SOSTDC1 in NSCLC.
We found that SOSTDC1 was significantly down-regulated in NSCLC. Moreover, patients with higher expression of SOSTDC1 had a significant better prognosis than those with lower SOSTDC1 expression. Ectopic expression of SOSTDC1 in NSCLC cell lines A549 and NCI-H520 could inhibit proliferation as shown by MTT, colony formation, soft agar and EdU incorporation assays in vitro. Furthermore, A549 cells stably expressing ectopic SOSTDC1 grew more slowly and formed smaller tumors than vector-control cells in vivo. Mechanistic studies demonstrated that SOSTDC1 over-expression led to increased p21Cip and p27Kip levels, thereby decreasing Rb phosphorylation status and E2F transcription activity.
SOSTDC1 is down-regulated in NSCLC, and its expression level is indicative of clinical outcome of patients with the disease. SOSTDC1 might represent a tumor suppressor through inhibiting the proliferation of NSCLC cells by regulating p21Cip and p27Kip, which in turn affects Rb-E2F signaling.
KeywordsSOSTDC1 Non-small cell lung cancer Proliferation p21Cip p27Kip
Lung cancer is one of the most commonly diagnosed cancer types worldwide and the leading cause of cancer-related death [1, 2]. Non-small cell lung cancer (NSCLC), which includes squamous cell carcinoma (SCC), adenocarcinoma (AD), large cell carcinoma (LCC), and other less frequently diagnosed histological types, accounts for about 80 % of lung cancer cases . While various therapeutic approaches, including surgical resection, chemo- and radio-therapies, have been applied in the management of NSCLC, the overall 5-year survival rate of NSCLC patients still remains at only 15 % . Better understanding of the genetic events and key molecules involved in NSCLC development and progression is needed for developing effective therapeutic strategies against the disease.
Sclerostin domain-containing protein 1 (SOSTDC1), an important regulator of cell signaling, has been found to contribute to several physiological and pathological processes [5, 6]. Accumulating evidence has revealed that SOSTDC1 might act as a tumor suppressor in many cancers. In wilms tumor, SOSTDC1 is lost as a result of a 7p21 homozygous deletion, which led to accelerate angiogenesis and activation of Wnt signaling . The expression of SOSTDC1 is down-regulated in gastric cancer, and ectopic over-expression of SOSTDC1 in gastric cancer cells suppressed cell proliferation, cell cycle progression and anchorage-independent growth . Clausen et al. found that the expression of SOSTDC1 reduced in breast cancer and such a reduction correlated to poor prognosis of patients with the disease . They also demonstrated that SOSTDC1 interfered with the signaling mediated by the Wnt3a, BMP-2, and BMP-7 pathways in breast cancer cells . Moreover, down-regulation of SOSTDC1 was observed in renal cancer, and SOSTDC1 could suppress the proliferation of renal cancer cells via regulating BMP and Wnt3a signaling . Nevertheless, the expression pattern and biological significance of SOSTDC1 in NSCLC remains largely unknown.
Deregulation of cell cycle progression represents a key characteristic in many types of cancer . Under physiologically normal conditions, the cell cycle progression is elegantly regulated by cyclins and cyclin-dependent kinases (CDK) . Of note, the activity of these molecules is restricted by the Cip/Kip family members [14–16], among which p21Cip and p27Kip are key functional cell cycle inhibitors containing an N-terminal CDK inhibitory domain and prevent CDKs from phosphorylating Rb, thus restricting the E2F transcription activity [14–17]. Given that E2F is an essential transcription factor for the production of cell cycle regulatory genes, p21Cip and p27Kip induces G1 arrest. Moreover, p21Cip could directly interfere with DNA replication by inhibiting PCNA . Therefore, p21Cip and p27Kip are widely recognized as tumor suppressors, and their inactivation is frequently observed in various types of cancer, including NSCLC [19, 20]. While functional inactivation of p21Cip and p27Kip occur frequently in cancer cells, the mechanisms involved in the reduction or loss of p21Cip and p27Kip functions remain to be further understood.
In the context of understanding the possible role of SOSTDC1 in NSCLC development and progression, our current study led to findings that the protein is down-regulated in NSCLC and might play a tumor-suppressive role.
The expression of SOSTDC1 is down-regulated in NSCLC
Clinicopathologic characteristics of patients enrolled in the study
No of cases (%)
Squamous cell carcinoma
Correlation between the clinical pathologic features and expression of SOSTDC1
Squamous cell carcinoma
Ectopic over-expression of SOSTDC1 inhibits the proliferation of NSCLC cells
Ectopic over-expression of SOSTDC1 induces p21Cip and p27Kip expression and decreases the transcriptional activity of E2F
SOSTDC1 suppresses tumor growth in vivo
In our current study, we find that SOSTDC1 expression is down-regulated in NSCLC, and that such an alteration of SOSTDC1 expression is associated with a poor clinical outcome of NSCLC patients. We also demonstrate that SOSTDC1 might inhibit the proliferation of NSCLC cells, possibly through up-regulating p21Cip and p27Kip and subsequent suppression of Rb-E2F signaling.
It has been well demonstrated that SOSTDC1 is involved in the process of fetal development [22–25]. Of note, accumulating studies have revealed that SOSTDC1 acts as a tumor suppressor in several types of cancers. For example, Clausen et al. found that expression of SOSTDC1 was down-regulated in breast cancer but a high SOSTDC1 expression correlates to better prognosis . Furthermore, Blish et al. showed that SOSTDC1was expressed in human kidney tissues and significantly decreased in renal clear cell cancer . Moreover, down-regulation of SOSTDC1 was also observed in thyroid cancer . In NSCLC, several lines of evidence provided by our current study, including analysis of the public TCGA dataset, assessment of our fresh as well as archived clinical specimens and functional tests, has shown that SOSTDC1 is a down-regulated tumor suppressor in NSCLC cells.
Of note, various mechanisms have been proposed to contribute to the down-regulation of SOSTDC1 in cancers. For example, previous reports have shown hyper-methylation in SOSTDC1 promoter CpGs and epigenetic silencing of SOSTDC1 transcription via increased methylation in prostate cancer [9, 28]. Moreover, E4BP4, a transcriptional repressor, could bind to the promoter of SOSTDC1 gene and induce CpG hyper-methylation, thereby leading to repressed SOSTDC1 expression in breast cancer . Intriguingly, genomic deletion also contributes to decreased SOSTDC1 expression. Specifically, a 7p21 homozygous deletion has been identified in 9 of 97 Wilms tumor, resulting in loss of SOSTDC1 , and loss of heterozygosity at 7p has also been reported to cause SOSTDC1 reduction in clear cell renal cell carcinoma . Nevertheless, the exact molecular mechanism underlying the observed SOSTDC1 down-regulation in NSCLC is yet to be elucidated. Further studies are required to understand whether the aforementioned genomic and epigenetic alterations may also be responsible for the down-regulation of this important tumor suppressor in NSCLC, which are underway in our laboratory.
The observed down-regulation of SOSTDC1 in NSCLC implicates that SOSTDC1 is likely to be biologically involved in the development and progression of the disease. Indeed, our current data demonstrate that SOSTDC1 may be involved in regulating the proliferative capability of NSCLC cells, and that such an anti-proliferative function is associated with an upregulation of cell cycle-inhibitory factors p21Cip and p27Kip. In the light that p21Cip and p27Kip are both well recognized regulators of the RB-E2F signaling and indeed our present study has indicated a possible role of SOSTDC1 in modulating this cell-cycle regulatory pathway, it will be of great interest to further elucidate whether the observed changes of p21Cip and p27Kip is essential to the inhibitory effects of SOSTDC1 on cell proliferation. On the other hand, more systemic studies would be needed to explore whether other molecules or pathways are also involved in mediating the tumor-suppressive function of SOSTDC1 in NSCLC.
SOSTDC1 is down-regulated and suppresses tumor growth in NSCLC. Down-regulation of SOSTDC1 is indicative of poor prognosis of the disease. The tumor suppressive function of SOSTDC1 is associated with upregulation of p21Cip and p27Kip, which may interfere with Rb-E2F signaling and disrupt the transactivating activity of E2F.
Lung cancer cell lines, including A549 and H520, were obtained from American Type Culture Collection (ATCC), and maintained in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (HyClone, Logan, UT) and 1 % penicillin/streptomycin (Invitrogen, Carlsbad, CA), as previously reported . The authenticity of the cell lines were verified by short tandem repeat fingerprinting at Medicine Laboratory of Forensic Medicine Department of Sun Yat-Sen University (Guangzhou, China).
RNA extraction and real-time PCR
Total RNA from tissues was extracted by the TRizol reagent (Life Technologies, Gaithersburg, MD), and reverse transcription (RT) reactions and real-time polymerase chain reaction (PCR) were performed as described previously . Primers were synthesized according to the following sequences: SOSTDC1 forward, 5′-CACGTTGAATCAAGCCAGAA-3′ and reverse, 5′-GATGTATTTGGTGGAACGCA-3′; and GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′ and reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′.
Plasmids and transfection
SOSTDC1 expression plasmid was generated by PCR sub-cloning human SOSTDC1 coding sequence into retroviral transfer plasmid pQCXIP (Clontech, Palo Alto, CA) to generate plasmid pQCXIP-SOSTDC1. Retroviral production and infection were performed as we previously described, and stable cell lines were selected by treatment with 0.5 μg/ml puromycin for 10 days, beginning at 48 h after infection [27, 30].
Western blotting analysis
Western blotting analysis was performed according to a standard method previously described , using anti-SOSTDC1, anti-cyclin B1, anti-cyclin D3 (Abcam, Cambridge, MA), anti-cyclin A2, anti-cyclin D1, anti-cyclin E1, anti-CDK4, anti-CDK6 (Epitomics, Burlingame, California), anti-cyclin D2, anti-CDK2 (BD Pharmingen, San Diego, CA), anti-cyclin E2, anti-p21Cip1, anti-p27Kip1, anti-p-Rb Ser608, anti-p-Rb Ser807, anti-Rb (Cell Signaling, Beverly, MA). When re-probing, blotted membranes were stripped and re-blotted with an anti-α-tubulin mouse monoclonal antibody (Sigma–Aldrich, St. Louis, MO) as a loading control.
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay
According to previous studies , cell viability was determined using an MTT assay. The cells were seeded at a density of 5 × 103 cells per well in 96-well plates. Subsequently at 1, 2, 3, 4, 5 and 6 days, 20 μl MTT (Sigma–Aldrich, St. Louis, MO) was added to each well and incubated for 4 h. The culture medium was removed, and 200 μl dimethyl sulfoxide (DMSO) (Amresco, Solon, Ohio) was added to each well. The plates were then shaken for 30 min, and the optical density (OD) at 490 nm was measured using a microplate reader. Each experiment was performed in triplicates.
Colony formation assay
For the colony formation assay, cells were plated into 6-well plates at the density of 500 per well. The cells were allowed to grow for 10 days and stained with crystal violet. The plates were photographed and the numbers of colonies formed by indicated cells were quantified using the Quantity One software package (Bio-Rad, Hercules, CA). Each experiment was repeated for three times.
Soft agar colony formation assay
Two milliliters of 0.66 % agar medium was added to each well of six-well plates to form bottom agar. Three thousand A549 cells and H520 cells were mixed with 2 ml of 0.33 % agar medium and then layered onto the bottom agar and incubated at 37 °C in 5 % CO2, and 0.5 ml of culture medium was added every week to keep the soft agar from drying and to supply nutrition. After 2 weeks of culture, the numbers of colonies were counted using a Zeiss microscope (Carl Zeiss, Jena, Germany).
5-ethynyl-2′-deoxyuridine (Edu) incorporation assay
To examine the degree of DNA synthesis, the Cell Light EdU DNA imaging kit (RiboBio Co., Guangzhou, China) was used. Briefly, cells were seeded in 24-well plates and exposed to EdU for 2 h, followed by fixation in 4 % paraformaldehyde and permeabilization in 0.5 % Triton X-100. Images were taken using a fluorescent microscope at 488 nm excitation. Each experiment was repeated independently for three times.
Luciferase reporter assay
The pE2F-TA-Luc reporter plasmid was purchased from the Clonetech Inc. (Mountain View, CA). Luciferase assay was performed as we previously described . NSCLC cells were seeded in triplicate wells in 48-well plates and allowed to settle for 24 h. Two hundred nanogram of luciferase reporter plasmid or the control-luciferase plasmid, plus 5 ng of pRL-TK renilla plasmid (Promega, Madison, WI), was transfected into cells accompanied with indicated plasmid using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) by following the manufacturer’s protocol. Dual Luciferase Reporter Assays were performed to test luciferase and renilla signals after 48 h according to a protocol provided with the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) by the manufacturer.
Tumor specimens from patients
This study was conducted on a total of 128 paraffin-embedded NSCLC specimens, which were histopathologically and clinically diagnosed at the Sun Yat-Sen University Cancer Center from 2001 to 2006. The 18 NSCLC specimens and matched adjacent non-cancerous lung tissues were frozen and stored in liquid nitrogen until further use, according to our previous reports [29, 33]. For the use of these clinical materials for research purposes, prior patients’ consents and approval from the Institutional Research Ethics Committee were obtained.
Immunohistochemistry assays (IHC)
IHC analysis was performed to study altered protein expression in human paraffin-embedded NSCLC specimens. The procedure was carried out similarly to previously described methods . Immunostaining evaluations were performed independently by experimenters blinded to sample identity. The staining intensity was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percent positivity was also scored according to four categories 0 (<5 %), 1 (5–25 %), 2 (>25–50 %), 3 (>50–75 %) and 4 (>75 %). Then the value of percent positivity score was multiplied by staining intensity score to generate final expression scores of SOSTDC1, which ranged from 0 to 12. The staining scores were defined as follows: low expression (score ≤ 2) and high expression (score ≥ 3).
In vivo tumorigenesis assay
Animal protocols were approved by the Ethical Committee of Sun Yat-sen University. BALB/c nude mice were injected sc with 8 × 106 indicated cells in 0.1 ml of PBS in the right flank. Tumor volumes were measured with calipers and calculated by the formula: 0.52 × length in millimeters × (width in millimeters)2. Thirty days later, mice were killed, and tumors were excised and weighed.
All statistical analyses were carried out using the SPSS 19. 0 statistical software package. The Kaplan–Meier method was used to establish survival curves, and log-rank test was applied for comparative analysis of differences in patient survival. All values represent at least three independent experiments and are expressed as the mean ± SD. Comparisons between groups for statistical significance were performed with a two-tailed paired Student’s t test. In all cases, p < 0. 05 was considered statistically significant.
LL and SW participated in the design of the study and carried out experiments. YY, JC, JW and XZ assisted with the experiments and data analysis. ML participated in data analysis and critical review of the manuscript. HG conceived of the study, and participated in its design and coordination and critical review of the manuscript. All authors were involved in writing the paper and had final approval of the submitted and published versions. All authors read and approved the final manuscript.
This work was supported by The Natural Science Foundation of China (No. 81272417, 81572624).
The authors declare that they have no competing interests.
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