Background
Salivary adenoid cystic carcinoma (SACC) is one of the most common malignancies characterized with slow growth, but high incidence of local recurrence and metastasis [
1,
2]. Although the 5-year disease-free survival rate generally reaches 90%, it drops to 10% after 20 years owing to potential local recurrence and hematogenous distant metastases [
3]. Therefore, it is necessary to elucidate the molecular mechanisms of recurrence and metastasis, which will provide molecular targets for the treatment of SACC patients.
Tumor dormancy was a period in tumor progression in which residual disease existed but remained asymptomatic clinically for years or even decades [
4]. It may appear during the formation of primary tumor, after dissemination of primary tumor cells or in the micrometastasis [
5,
6]. Tumor cell dormancy is defined at cellular level which is characterized with cells that are not divided and arrested in G0/G1 cell phase [
7]. The dormant tumor cells could escape immune surveillance and chemo-radiotherapies, remaining undetectable for long periods [
6]. Dormant tumor cells are emerging as a critical cause for recurrence and metastasis once they escape this state [
8]. However, how tumor cells enter into dormancy and what processes govern their exit in human cancers including SACC are still absent and remain unclear.
Differentiated embryonic chondrocyte gene 2 (DEC2, also known as BHLHE41/BHLHB3/Sharp1) is one of the basic helix-loop-helix (bHLH) transcription regulators, which has been demonstrated to play important roles in regulating circadian rhythms, cell proliferation, hypoxia reaction, immune responses as well as malignant tumor progression [
9‐
12]. Recent evidence demonstrated that inhibition of DEC2 and nuclear receptor subfamily 2 group F member 1(NR2F1) resulted in increased growth of breast cancer cells [
13] and downregulation of DEC2 interrupted tumor cell dormancy [
14], indicating DEC2 might involve in tumor dormancy. And TGF-β2 activated a [ERK/p38]
low signaling ratio, which resulted in induction of DEC2 and dormancy of malignant disseminated tumor cells (DTCs) in the bone marrow of head and neck squamous cell carcinoma (HNSCC). However, the role and its molecular mechanism of DEC2 in the dormancy and malignant progression of SACC remain unclear.
Here, we found that DEC2 induced tumor dormancy of the primary SACC and in the model of lung metastasis, DEC2 positive tumor cells manifested enhanced migration and invasion and formed more metastases and the level of DEC2 was reduced significantly with the resumption of cell proliferation. Then, DEC2 may associate with HIF1α in contributing to tumor dormancy, which might provide a possible cue to explain the different roles of DEC2 in primary and metastasis lesions. Our findings demonstrated that overexpression of DEC2 contributed to the dormancy of tumor and low expression reawakened cell dormancy of SACC, which may provide important implications for the therapy of patients.
Materials and methods
Xenografts
Lentivirus-transfected SACC-83 cells were subcutaneously injected s.c. (5 × 106cells/200 μL PBS/mouse) into the flank of 6-week-old nude female mice (Laboratory Animal Center of Sichuan University, Chengdu, China) and examined every 3 to 5 days for tumor appearance. Tumor growth was then measured once a week until 35 days after inoculation by determining the tumor volumes using caliper measurements. Lentivirus-transfected SACC-83 cells (5 × 106cells/200 μL PBS/mouse) were injected via tail vein of nude female mice to establish the model of lung metastasis. The nude mice were weighed weekly. Lung tissues were excised after 4 weeks for immumohistochemical staining. All animal experiments were approved by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China.
Immunohistochemistry
Paraffin-embedded sections were cut into 4um and deparaffinized in xylene and rehydrated, and endogenous peroxidase was blocked with 3%H2O2. Antigen retrieval was accomplished by 0.01 mol/L citrate buffer solution (pH 6.0) in a 700 W microwave oven for 15 min. After incubation with 5% normal goat serum for 20 min, the slides were exposed overnight at 4 °C to the rabbit anti-DEC2 (1:150; Proteintech), rabbit anti-Ki-67(1:800; Proteintech), rabbit anti-NR2F1 (1:200; Proteintech). Sections were then incubated with biotinylated goat anti-rabbit IgG (Zhongshan Goldenbridge Biotechnology) for 1 h, and streptavidin-peroxidase for 30 min. The 0.02% diaminobenzidine tetrahydrochloride was used as a chromogen, and the slides were counterstained with hematoxylin. The percentage of positive cells was estimated using an image analysis system (Leica).
Cell lines and cell culture
Two SACC cell lines, SACC-83 and SACC-LM, were obtained from the State Key Laboratory of Oral Disease, Sichuan University. Cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FCS (Hyclone), 2 mmol/L L-glutamine, 25 mmol/L HEPES, and 100 units/mL penicillin and streptomycin at 37°Cin 5% CO2. For hypoxic treatment, cells were exposed to 0.1% O2 with 5% CO2 at 37 °C with hypoxia chamber.
Cloning, Lentivirus preparation, and plasmids
The targeted cDNA of DEC2 was cloned into the pEZ-Lv201 and constructed into EX-W0115-Lv201 plasmid and negative control plasmid EX-NEG-LV201. After sequencing verification, it was packaged into virus. Human DEC2, BC_025968, total 1449 bp. P/Puro plasmid vector and transfected into cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The stable transfected cells were selected with puromycin.
Transient siRNA knockdowns
SiRNAs targeting DEC2 and Slug and their control siRNAs were purchased from Genechem. The target sequences were as following: DEC2 siRNA-1: CUCCCUAUAUCCCAAUGGATT, UCCAUUGGGAUAUAGGGAGTT; DEC2 siRNA-2: CGAGGAAGAACUAUGAACATT, UGUUCAUAGUUCUUCCUCGTT;
DEC2 siRNA-3: GAUGAAAGAAUUACCGAAUTT; AUUCGGUAAUUCUUUCA UCTT; Control siRNA CUCUCCGAACGUGUCACGUTT; GCGUGACACGUUCG GAGAATT. The above transient transfections in SACC cells were performed using 20 μM of each siRNA with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Knockdown was verified by qRT-PCR and Western Blot.
Quantitative real-time RT-PCR
Total RNA was extracted from cells using the trizol (Invitrogen, Carlsbad, CA) and were quantified with the NanoDrop ND-1000 Spectrophotometer (Thermo ScientificInc., Waltham, MA). PCR amplification of the cDNA template was done using Thunderbird SYBR qPCR mix (TOYOBO) on ABI PRISM 7300 sequence detection system (Applied Biosystems) according to the manufacturer’s protocol. The resulting cDNA was diluted and used as a template for Quantitative real-time PCR using LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). β-actin was used as the housekeeping gene to normalize the target gene expression. The sequences of PCR primers were showed in Supplementary Table
S1.
Western blot
Total proteins were extracted from the cultured cells with a total protein extraction kit (Keygen, Nanjing, China). The protein concentrations were detected by a BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were then separated by 6 and 8% sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS-PAGE) and blotted on polyvinylidenefluoride (PVDF) membranes. Membranes were blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk and incubated with the following primary antibodies: DEC2 (Proteintech Group, Chicago, USA), HIF-1α (Wanleibio, China), Slug (Proteintech Group, Chicago, USA), Snail (Proteintech Group, Chicago, USA), β-actin (Sigma-Aldrich). Horse radish peroxidase–conjugated anti-rabbit or anti-mouse IgG were used as the secondary antibody (TA322704 or TA326473, ZSGB-BIO, China, 1:1000). Subsequent visualization was detected using a densitometer (GS-700, Bio-Rad Laboratories).
Immunofluorescence staining
SACC cells were cultured in 12-well cell culture plates. Upon reaching 70% confluence, cells were washed in cold PBS and fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.25% Triton X-100 in PBS for 15 min, and blocked with 1% bovine serum albumin prepared in PBS for 30 min. Lastly, cells were incubated overnight with mouse anti-Ki-67 (1:100 dilution), and then incubated with FITC or TRITC-conjugated goat anti-mouse IgG (1:500; Zhongshan Goldenbridge Biotechnology) at 37 °C for 1 h. Cells were visualized using the Olympus Fluoview confocal microscope (Tokyo, Japan), and fluorescence images were taken.
Wound healing assay
SACC cells were seeded and cultured in 6-well plates. Upon reaching 80% confluence, cells were wounded by scratching with a pipette tip and incubated with medium containing no FBS for 24 h. Then, they were photographed under phase-contrast microscopy.
In vitro cell invasion assay
Invasion of cells was assessed using Matrigel-coated membrane (24-well insert, pore size, 8 μm; BD Biosciences). About 5 × 104 cells were plated in the top chamber in serum free medium, and medium with serum was used as a chemo-attractant in the lower chamber. After 48 h of incubation, cells remaining on the top chamber were removed using a cotton swab. Traversed cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 1% Crystal Violet; five fields per filter were counted.
Cell proliferation assays
The cell proliferation was assessed by Cell Counting Kit-8 (CCK-8, Dojindo) assay. Cells were seeded in 96-well plates in triplicate and the proliferation assay was performed after 24 h incubation. 10ul of CCK-8 solution was added into per well and the absorbance reading was measured at 450 nm after 30 min of incubation at 37 °C. The above experiments were repeated the next 4 days.
Apoptosis detection by FCM
Cell apoptosis was performed by combined application of Annexin V–FITC and propidium iodide (BD Biosciences Clontech, USA). Cells were washed by PBS and adjusted to1 × 106 cells/ml with 4 °C PBS. One hundred microliter of suspensions was added to each labeled Falcon tube (12 mm × 75 mm, polystyrene round-bottom); 10 μl of Annexin V–FITC and 10 μl propidium iodide (20 μg/ml) were added into the above labeled tube, which was incubated for 30 min at room temperature in the dark environment; and then 400 μl PBS binding buffer was added to each tube which was analyzed using FCM analysis (BD Biosciences Clontech, USA).
Glucose consumption test
Glucose consumption was detected using a glucose assay kit (Nobio, China). About 1 × 105 cells/well was seeded in 6-well plates. The test was performed according to the manufacturer’s protocol. The experiments were performed at least three times.
Cell senescence detection
Senescent cells were measured using a senescence β-galactosidase staining kit (Beyotime, China). Cells were seeded in 6-well plates (1 × 105 cells/well). The staining was performed according to the manufacturer’s instructions. The cells were then observed under an Olympus BX51 microscope and were analyzed using ImageJ software.
Clinical samples collection and study
The cohort was assembled from 70 patients who were histologically diagnosed with SACC and underwent resection of their tumors at West China Hospital of Stomatology, Sichuan University, between 2005 and 2015. Exclusion criteria included preoperative chemotherapy, hormone therapy or radiotherapy. All samples of SACC were collected at the time of surgery. All the paraffin-embedded sections were confirmed histologically with blind method by two pathologists. The protocol of the study was approved by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China. The pathologic characteristics of the tumors and clinical data of the patients were summarized in Table
1.
Table 1
Clinical-pathologic characteristic of 70 patients with SACC, and the association between DEC2 expression and these variables
Age (years) | ≤55 | 34 | 25 | 9 | 0.684 |
>55 | 36 | 28 | 8 | |
Gender | Male | 31 | 23 | 8 | 0.795 |
Female | 39 | 30 | 9 | |
Tumor site | Major salivary gland | 21 | 18 | 3 | 0.043 |
Minor salivary gland | 49 | 30 | 9 | 8 |
T stage | T1/T2 | 25 | 16 | 9 | 0.091 |
T3/T4 | 45 | 37 | 8 | |
Histological subtype | Cribriform/Tubular | 58 | 38 | 20 | 0.009 |
Solid | 12 | 3 | 9 | |
Recurrence | With | 33 | 20 | 13 | 0.005 |
Without | 37 | 33 | 4 | |
metastasis | With | 31 | 18 | 13 | 0.002 |
Without | 39 | 35 | 4 | |
TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) Kit (KeyGEN) was to test cell apoptosis. Negative was graded as 0 to 10% within 4–6 microscopic fields at × 400 magnification; and the positive was graded as more than 10% as well.
Statistical analyses
All statistical analyses were conducted using GraphPad Prism. Data was plotted with GraphPad Prism software. A value of P < 0.05 was considered statistically significant. All experiments were performed independently at least three times.
Discussion
Tumor dormancy, mentioned in 1864 and described in 1954 by Hadfield as a temporary arrest in mitosis [
25] has been defined as a clinical term [
26]. It has been demonstrated that tumor dormancy was implicated in the invasion and metastasis of EMT program in many types of tumors. Here, we demonstrated that DEC2 participated in SACC dormancy under a high expression condition in xenograft of nude mice. And the formation of lung metastases was accompanied by low level of DEC2, which then reawakened dormancy and promoted proliferation of metastases. Then, we further addressed these different roles of DEC2 in primary and metastasis lesions and found that high expression of DEC2 involved in CoCl
2-induced dormancy, indicating that different hypoxia states in primary and metastasis lesion may regulate DEC2-induced dormancy to keep or exit cell dormancy state. Further, overexpression of DEC2 induced the entry of SACC into dormancy, mediated by activating the expression of Slug, which then drived EMT program, contributing to growth arrest and dormancy, as well as enhanced migration and invasion capabilities. Our current finding of low level of DEC2 inducing SACC cells to exit dormancy in the second lesion reveals the role of DEC2 in regulating tumor cell dormancy has involved in hypoxia condition and EMT program.
DEC2 is one of the basic helix- loop-helix-Orange transcription factors, featured with a basic DNA binding domain, a helix-loop-helix (HLH) dimerization domain, and Orange extended dimerization domain [
27]. And it has been implicated in regulating many types of biochemical processes, including circadian rhythm [
28], cell proliferation and differentiation [
29], apoptosis, hypoxia response, and EMT of tumor cells [
28,
30]. It was demonstrated that DEC2 inhibited tumor cells proliferation in esophageal cancer and osteosarcoma [
31,
32]. In this study, we demonstrated that DEC2 induced tumor dormancy of SACC both in vivo and vitro. It was supported by previous publication, which showed that the expression of DEC2 drived tumor dormancy in HNSCC and breast cancer [
13,
33]. Our previous study has demonstrated that atRA treatment could also drive tumor dormancy of SACC by upregulating NR2F1 [
15]. Sosa et al. proposed that NR2F1 was an important node in tumor dormancy induction and maintenance [
34]. In the present study, we found that DEC2 was also participated in atRA induced dormancy. DEC2 was increased in atRA treated dormant cells and these cells were activated partially after DEC2 knockdown. But the expression changes of DEC2 did not affect NR2F1. The reason for this may be that this dormant process was mediated by complicated signal networks including DEC2 and NR2F1, but they did not regulate each other directly.
Hypoxia was a poor-prognosis microenvironmental feature of solid tumor, and Fluegen et al. proposed that primary tumor hypoxic microenvironment promoted the production of dormant tumor cells and resulted in chemo-radiotherapy resistance [
16,
35]. It has also been demonstrated that hypoxic stress induced breast cancer dormancy, but the relationship and molecular mechanism within hypoxia and dormancy of SACC is still ambiguity [
22]. Previous studies have shown that the expression of DEC2 and HIF1α were positively correlated during the progression of human osteosarcoma [
36]. We found that high expression levels of HIF1α and P27 were all participated in the dormancy process regulated by DEC2 in SACC. And we also verified the validity of CoCl
2 -based model in vitro for researching the relationship between tumor progression and hypoxic stress. We next detected the relationship between hypoxia and dormancy of SACC and the function of DEC2 during this process. The results showed that CoCl
2 induced hypoxia–mimicking microenvironments can drive dormancy in SACC cells and high expression of DEC2 was necessary for the above dormant state. Furthermore, the dormant tumor cells could be reawakened when the microenvironment changed from hypoxia to normal oxygen. Consistent with the above results, we proposed that in mouse xenograft model, DEC2-overexpresed dormant SACC cells transferred into lung tissue and reactivated colony growth because of the abundant oxygen microenvironment. Exiting from dormancy always accompanied by DEC2 downregulation and resulted in the formation of numerous lung metastases and poor prognosis.
It has been proposed that EMT-positive cells may enter into dormant state [
5,
20,
37], so we investigated the role of DEC2 in EMT inducing dormancy. In the present study, we initially found that DEC2 overexpressed dormant tumor cells displayed upregulation of Slug, one of the EMT transcription factors. Knockdown of Slug reversed their dormant state and suppressed migration and invasion. Therefore, we hypothesized that the clock gene DEC2 could drive tumor dormancy through inducing EMT program and thus promoting tumor cells ability of migration and invasion in SACC. Consistently, it was shown that tumor cells with the features of invasiveness which have undergone EMT always manifested characteristics of dormancy. And re-expression of E-cadherin always accompanied by proliferative activity which further indicated the critical roles of EMT during tumor dormancy [
38]. Jiang et al. have also proposed that PRRX1 can drive the transition of EMT, and dormant state of cancer cells through miR-642b-3p in head and neck squamous cell carcinoma [
39]. Therefore, cellular phenotype affects the process of tumor dormancy. Furthermore, it was reported that DEC2, one of the clock genes, promoted tumor metastasis and drived tumor dormancy [
40]. To our knowledge, this is the first research suggesting that DEC2 induced EMT process to facilitate tumor dormancy through the control of Slug.
Additionally, we also investigated the expression of DEC2 in the primary SACC patients and normal salivary gland. The results showed that high expression of DEC2 was related with tumor dormancy and tumor site, histological subtype, and recurrence. We proposed the primary reason of poor prognosis caused by tumor dormancy is that dormant tumor cells are unstable and can resume proliferation as the external microenvironment changes. And just because of that, the purpose of tumor therapy might attempt to maintain the dormant state of tumor cells and prevent dormancy exit and growth resumption. Neophytou et al. proposed that the ultimate goal is to prolong the dormant period of metastatic tumor cells in breast cancer [
41]. And it was also demonstrated that during tumor treatment, the Hippo signaling pathway contributes the initiation and stabilization of tumor dormancy [
42]. But there are still questions that need to be answered to detect how DEC2 affect the growth state of tumor cells in different microenvironment.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.