Background
Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant salivary gland tumors, accounting for about 28% [
1,
2]. Five-year survival rates for patients with SACC are 50–90% but drop to 50% after 10 years, and 20% after 20 years. SACC patients usually suffered from metastatic relapse several or decades years after they had undergone radical surgery [
3,
4]. This phenomenon has become a puzzle for a long time till cancer dormancy was raised, which will have potential to explain this prevalent clinical behavior of SACC patients [
5].
Cancer dormancy, mentioned in 1864 [
6] and described in 1959 [
7], has been historically defined in clinical terms to describe the hypothetical state of cancer cells lying in wait over a period of time after treatment of the primary tumor, pending subsequent growth and clinical recurrence [
8]. The mitotic arrest actually got a real sense of dormancy, which precisely referred to cellular dormancy, suggesting that a G0/G1 arrest can exist in certain cancer cells [
9,
10]. Angiogenic dysfunction and immunologic regulation are responsible for tumor mass dormancy with a sound-equilibrium between dead cells and proliferative cells [
11‐
14]. In according with the properties of tumor dormancy including insensitivity to radiotherapy and chemotherapy, and escapable from immune-surveillance [
15,
16], it deems to be the “seeds” for tumor relapse and metastasis.
Recent studies have shed significant light on the molecular mechanisms governing the invasion and dissemination phase of metastasis through cancer dormancy. Kim et al. demonstrated that suppression of two dormancy genes, BHLHE41 and NR2F1, increased the growth of ER positive MCF7 cells in vivo [
17]. And disseminated ER positive tumor cells carrying a dormancy signature were more likely to undergo prolonged dormancy before resuming metastatic growth [
17]. Using computational tools, Adam et al. found that p38 transcriptionally regulated a core network of 46 genes that included 16 TFs in head and neck squamous cell carcinoma (HNSCC), which played key roles in tumor suppression and induction of tumor cell dormancy [
18]. Bragado et al. showed that TGF-β2 and TGF-β-RIII signaling through p38α/β regulated the dormancy of disseminated tumor cells (DTCs) and defined restrictive (BM) and permissive (lung) microenvironments for HNSCC metastasis [
19]. However, in spite of these significant advances, the mechanism of cancer dormancy elucidating the post-dissemination phase of metastasis has remained less understood.
NR2F1 (nuclear Receptor subfamily 2 group F member 1, or COUP-TF1) is one of NR2F family and modulates gene expression during cancer development and growth [
20]. Recently, NR2F1 has been shown to be associated with cancer cell dormancy in HNSCC [
21]. Here, we evaluated the correlations between NR2F1 expression and tumor cell dormancy, and the clinical pathological characteristics of SACC patients. SACC cells with NR2F1 over-expression and NR2F1 knockdown were used to investigate the differences of biological behaviors including proliferation, cell cycle, apoptosis, migration and invasion. Finally, the mechanism of NR2F1 contributing to cancer cell dormancy, invasion and metastasis of SACC cells was investigated. Our findings showed that in NR2F1 overexpressed tumor cells, proliferation and cell cycle could remain arrested, but invasive and metastatic properties could be enhanced. This observation might have important implications in the therapeutic options for SACC patients.
Methods
Tissue sample collection
The cohort was obtained from patients who were histologically diagnosed as SACC and underwent radical surgery at West China Hospital of Stomatology, Sichuan University from January, 2004 to December, 2007. Tumors were staged and graded according to the American Joint Committee on cancer. Exclusion criteria included recurrence, preoperative radiotherapy, chemotherapy or biotherapy, and incomplete medical records. Finally, 59 patients (28 males and 31 females; median age, 42 years, range from 22 to 77) were recruited in this study. Immunohistochemical analysis for the formalin-fixed, paraffin-embedded specimens from these patients. This study was approved by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China. Pathologic characteristics of the tumors and clinical data of the patients were summarized in Table
1.
Table 1The association between NR2F1 expression and clinical pathologic characteristic of 59 patients with SACC
Age at diagnosis, yr |
≤ 55 | 27 | 22(81.81) | 5(18.19) | 0.3875 |
>55 | 32 | 23(71.87) | 9(28.13) |
Sex |
Male | 28 | 21(75) | 7(25) | 0.8273 |
Female | 31 | 24(77.42) | 7(22.58) |
Tumor site |
Major salivary glands | 14 | 8(57.14) | 6(42.86) | 0.1172 |
Small salivary glands | 45 | 37(82.22) | 8(17.78) |
T stage |
T1/T2 | 46 | 35(76.09) | 11(23.91) | 0.7592 |
T3/T4 | 13 | 10(76.92) | 3(23.08) |
Local invasion |
with | 31 | 26(83.87) | 5(16.13) | 0.1488 |
without | 28 | 19(67.86) | 9(32.14) |
Recurrence |
with | 10 | 5(50) | 5(50) | 0.0321 |
without | 49 | 40(81.63) | 9(18.37) |
Metastasis |
with | 3 | 0(0) | 3(100) | 0.0112 |
without | 56 | 45(80.36) | 11(19.64) |
Immunohistochemical staining
Anti-NR2F1 (1:200, abcam) and Ki-67 (1:400, Cell Signaling Technology) were used for Immunohistochemical staining. Negative was graded as 0 to 10% within 4–6 microscopic fields at × 400 magnification and positive was graded as more than 10% as well.
TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) Kit (KeyGEN) was to determine the cell apoptosis. Negative was graded as 0 to 10% within 4–6 microscopic fields at × 400 magnification and positive was graded as more than 10% as well.
Cell culture and transfection
SACC-83 and SACC-LM cell line have been purchased from Shanghai Life Science College Cell Resource Center, Chinese Academy of Sciences and conserved in State Key Laboratory of Oral Diseases. For in vitro assays, cells were seeded at 2 × 105/ml. For the NR2F1 induction experiment, SACC-83 and SACC-LM cells were grown in RPMI 1640 with 10% FBS and 1% P/S and transfected with pGS5-empty or pGS5-NR2F1.
NR2F1 transient siRNA knockdowns
SiRNAs targeting NR2F1 (NR2F1-Homo-2112 (siRNA-1), NR2F1-Homo-2838 (siRNA-2), Human NR2F1 (siRNA-3)) and control siRNA (siControl) were purchased from Genechem. The target sequence was: siRNA-1:GCCUCAAGAAGUGCCUCAATT, UUGAGGCACUUCUUGAGGCTT;siRNA-2:UCAUCGAGCAGCUCUUCUUTT,AAGAAGAGCUGCUCGAUGATT;siRNA-3:CUCUCAUCCGCGAUAUGUUTT,AACAUAUCGCGGAUGAGAGTT;siControl:UUCUCCGAACGUGUCACGUTT,ACGUGACACGUUCGGAGAATT. Transient transfection in SACC cells was performed using 20 μM of each siRNA with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Knockdown was verified by real time qRT-PCR.
Immunofluorescence
SACC cells were seeded into coverslips (1 × 104/ml) and cultured in a 12-well culture plate for 24 h. After washed in cold PBS, the cells were fixed in 4% paraformaldehyde for 20–25 min and blocked in 1% bovine serum albumin for 30 min at room temperature. Rabbit anti-NR2F1 (abcam, 1:200) and FITC-conjugated goat anti-rabbit IgG (1:500; Zhongshan Goldenbridge) were orderly used to incubate these cells. 4′ 6-diamidino-2-phenylindole (DAPI; 1 μg/μL) was used to determine the cell nucleus. The results were collected by a fluorescence microscope (Olympus).
Real time reverse transcriptase PCR (qRT-PCR)
One Step PrimeScript™ RT-PCR Kit (TaKaRa) was for Real time qPCR and the results were analyzed by Applied Biosystems ABI PRISM 7300. NR2F1/TF-COUP1: forward: GCCTCAAAGCCATCGTGCTG; reverse: CCTCACGTACTCCTCCAGTG. GAPDH was used as an internal control for the normalization of target gene expression.
Western blot
Rabbit anti-NR2F1 (abcam, 1:1000) and 1:3000 dilution of anti-rabbit IgG secondary antibody (ZSGB-BIO, China, 1:1000) were to determine the protein expression. Rabbit anti-Lamin B (ZSGB-BIO, China, 1:1000) was used as an internal control. Images were acquired with a ChemiDoc Touch imager (Bio-Rad) and quantification was done using Quantity One 4.4.0 software.
Proliferation assay
The cell proliferation assay was performed by Cell Counting Kit (CCK)-8 assay according to the manufacturer’s protocol (DOJINDO, Japan).
Cell cycle analysis
Cells were collected by centrifuge with disposed upper layer and then fixed and stained for total DNA with propidium iodide (PI) using Cell Cycle Detection Kit (KeyGEN). Data was acquired with a Beckman Coulter flow cytometer.
Wound healing assay
SACC-83 and SACC-LM cells seeded and cultured in a 96-well plate (1000/ml) and were wounded by scratching with a pipette tip when reached 80% confluence, and incubated with medium containing no FBS for 24 h. Cells were photographed under phase-contrast microscopy (× 100) as previously described.
Transwell invasion assays
In vitro cell invasion assays were performed with QCM− 96-well cell invasion assay kit (Chemicon International, Temecula, CA, USA). After 24 h, the tumor cells were stained by Crystal violet and photographed under microscopy (× 100) as previously described.
Xenografts
Balb/c immunodeficient nude female mice (Laboratory Animal Center of Sichuan University, Chengdu, China), aged 3 weeks were used. 20 mice were randomized and divided into two groups (NR2F1high, negative control), 10 mice each. Tumor cells were then injected via subcutaneous (2.5 × 106 cells/100 μl PBS/mouse) on the back of nude mice. Tumor growth was then monitored using caliper measurements. The mice were euthanized with a dosage of 150-200 mg/kg Pentobarbital Sodium via intraperitoneal injection after 4 weeks and tumors were harvested after 4 weeks and fixed by 4% paraformaldehyde and then embedded by paraffin for hematoxylin-eosin (HE) staining and IHC analyses. Another 10 mice were grouped as above and tumor cells were injected via tail vein (1 × 105 cells/100 μl PBS/mouse). The lung tissues were excised after 4 weeks for HE staining to detect micro-metastasis.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed using a ChIP Assay Kit (Abcam) according to the manufacturer’s instructions. Briefly, cells were fixed, lysed, and sonicated to obtain DNA fragments in arranging in size from 200 to 1,000 bp. Chromatin was then precipitated with nonspecific IgG antibodies (Sigma), ChIP-grade rabbit anti-NR2F1 (Abcam), or ChIP-grade rabbit anti-H3 (Abcam). DNA was extracted and PCR was performed with primers for CXCL12, CXCR4 and CXCR7 promoter fragments.
Statistical analysis
All data are presented as the mean ± standard deviation of at least 3 independent experiments. Graph construction and statistical analysis were performed using SPSS 17.0 and GraphPad Prism 5.0. The correlation between NR2F1 and clinicopathologic parameters in all patients was analyzed through the Fisher’s exact test. P values were calculated to determine statistical significance of the results. *p < 0.05 and **p < 0.01 were considered statistically significant.
Discussion
Tumor dormancy has been demonstrated to empower the tumor recurrence and metastasis in many types of cancers, including breast cancer, prostate cancer, melanoma and HNSCC [
5,
19,
24]. In this study, we found that high expression of NR2F1 was strongly associated with recurrence, metastasis and dormancy of SACC patients. NR2F1 overexpression in SACC cells could reduce cell proliferation and arrest G0/G1 phases, as well as enhance migration and invasion activity. Mechanistically, overexpression of CXCL12 rescued the proliferation, migration, invasion activities induced by knockdown of NR2F1 in SACC cells, at least in part, indicating that the role of NR2F1 in regulation of SACC cell behaviors was mainly mediated by CXCL12/CXCR4. Collectively, NR2F1 may be a marker for SACC tumor cell dormancy and high expression of NR2F1 in SACC may be useful to identify patients at high risk for recurrence and metastasis.
In this study, we show that compared to the normal salivary gland, SACC samples contained smaller amounts of NR2F1, which was in accordance with NR2F1 expression in mammary tumor and HNSCC [
21,
25]. However, in prostate cancer, esophageal cancer and melanoma, NR2F1 exhibited a higher expression compared with non-tumor samples [
26‐
28]. This difference might attribute to different kinds of human carcinoma and different sample sources. We further found that the expression of NR2F1 was associated with local recurrence and metastasis according to the results from pathological section staining of SACC patients. This is in line with the present reports that NR2F1 has been demonstrated to serve as a critical regulator in angiogenesis and lymphangiogenesis to promote tumor invasion and metastasis [
29‐
31]. Huang et al. found that the expression of lncRNA NR2F1-AS1 was up-regulated in chemo-resistant hepatocellular carcinoma and could promote the invasion, migration and drug-resistant in vitro [
32]. Jiang et al. demonstrated that dietary supplements could suppress metastatic behavior of prostate cancer cells by down-regulating the expression of NR2F1 [
33].
Then, we showed that both in SACC samples and SACC cell lines, NR2F1
high cancer cells displayed neither a proliferative nor an apoptotic state, namely a state of dormancy. As expected, NR2F1 silencing stimulated SACC cell growth in vitro. Intriguingly, we noticed that NR2F1
high cancer cells preformed an enhanced invasion and migration in vitro and an advanced metastasis in vivo. These not only identify NR2F1 as a marker of SACC dormancy, but also a mediator for the process of tumor metastasis. Many studies have identified NR2F1 as a marker of tumor cell dormancy in breast cancer, HNSCC, prostate cancer, etc. In breast cancer, Borgen et al. [
34,
35] analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients, and found NR2F1 as a marker of dormancy in breast cancer. Cackowski et al. [
36] demonstrated that MERTK, one of TAM family of receptor tyrosine kinases, being knockdown could induce a G0/G1 arrest in prostate cancer cells via increasing expression of NR2F1 and ratio of p38 to pERK1/2, which was reversed by p38 inhibitor. Sosa and his colleagues [
21] suggested a NR2F1-dependent dormancy via SOX-9/RARβ axis in HNSCC and breast cancer. Additionally, NR2F1 could induce global chromatin repression and act as a key gene which contributes to dormancy of DTCs in the bone marrow, while the effect of NR2F1 on growth arrest was reversed by siRNA or knockdown. The results further affirmed that NR2F1 was a critical node in dormancy induction.
The CXCL12/CXCR4 signaling is composed of the chemokine CXCL12 (also called SDF-1 for stromal cell-derived factor 1) and its receptors CXCR4 and CXCR7, playing pivotal roles in the cell migration, angiogenesis, proliferation, and survival of many cancer cells, including SACC [
22,
23]. Here, we found that the high expression of NR2F1 promoted the expression of CXCL12 and CXCR4, and overexpression of CXCL12 rescued SACC cell behaviors inhibited by NR2F1 silencing. This is supported by the data of Boudot group, who detected that NR2F1 stimulated the metastatic cascade via CXCL12/CXCR4 pathway by activating epithelial growth factor (EGF) and EGF receptor in breast cancer [
37]. This indicated that NR2F1 may contribute to cancer cell dormancy, invasion and metastasis of salivary adenoid cystic carcinoma by activating CXCL12/CXCR4 pathway.
Targeting the tumor dormancy is far from clinical application, but the NR2F1 regulation on tumor dormancy comprises several therapeutic insights both in clinical use and under clinical trials [
36,
38]. William and his group have launched a clinical trial in combination treatment of 5-Aza and AtRA for patients with recurrent prostate cancer. 20 participants were randomly recruited and treated with reprogramming therapy, which utilizing a combination of 5-Aza and AtRA to elicit a NR2F1-regulartory cancer dormancy process. Although the results are waiting to be published, it is anticipated to decrease the rate of disease progression-free and to suffer a low percentage of adverse events.
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