Background
Emerging evidence suggests that chronic stress in patients, including fear, anxiety, and depression, can promote cancer progression by inducing overactivity of the sympathetic nervous system (SNS) [
1‐
4]. Chronic stress has been shown to increase the production of stress hormones such as norepinephrine (NE) and epinephrine from the SNS, enhancing tumor growth, invasion, and metastases in multiple malignancies [
5‐
7]. However, the molecular mechanisms underlying the effects of chronic stress on cancer promotion are not fully understood.
Hepatocellular carcinoma (HCC) ranks fourth among cancer-related deaths worldwide [
8]. HCC often arises on a background of inflamed or fibrotic livers rich in activated hepatic stellate cells (HSCs) [
9‐
11]. As major sources of extracellular matrix (ECM) deposition, activated HSCs can profoundly alter the tumor microenvironment [
11‐
13], and interactions between HCC and activated HSCs greatly influence HCC onset and progression [
14‐
17]. In addition, it has been reported that hyperactive SNS contributes to the stress-induced aggravation of many liver diseases through its catecholamine neurotransmitters (including NE) [
2,
18,
19]. Further, several neuronal genes were found to be highly expressed in HSCs, including adrenergic receptors [
20‐
26]. This prompted us to hypothesize that chronic stress could affect HCC progression through stress hormone-modulating HSC activities.
In this study, we showed the crosstalk between chronic stress and HCC progression through NE-mediated activation of HSCs. Our observations included the following: (i) conditioned medium from NE-stimulated HSCs significantly enhanced the malignant phenotype of HCC cells; (ii) secreted frizzled-related protein 1 (sFRP1), a regulator of Wnt signaling, played a key role in the stress response of HSCs to NE and mediated the pro-tumoral effects of HSCs on HCC cells; (iii) sFRP1 enhanced Wnt16B expression to augment an autocrine feedback loop of Wnt16B/β-catenin signaling in HCC cells; (iv) expression of sFRP1 in HSCs accelerated HCC progression in an in vivo model under chronic restraint stress, which was significantly thwarted by the knockdown sFRP1. This study has therapeutic implications for the treatment of chronic stress-driven HCC progression.
Materials and methods
Cell lines and culture conditions
The human liver cell L02 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), human HCC cell lines PLC/PRF/5, HepG2 and Hep3B (ATCC, USA), SMMC7721 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MHCC97H and HCCLM3 (Liver Cancer Institute, Fudan University, Shanghai, China), and Huh7 (Japanese Cancer Research Resources Bank) were propagated in the corresponding culture medium RPMI-1640 or Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Invitrogen). Human hepatic stellate cell line LX-2 (a gift from SL. Friedman, Mount Sinai, New York) were grown in DMEM containing 2% FBS. Primary human hepatic stellate cells (pHSCs) (ScienCell, USA) were maintained in the provided complete stellate cell medium. All cell cultures were conducted at 37 °C in a thermostatic incubator containing 5% CO2.
Norepinephrine (NE) treatment
For NE treatments, LX-2 cells were seeded in 6-well plates (2 × 105 cells/well) (Corning Inc., USA) for 8 h to allow the cells to adherent, and then treated with increasing concentrations of NE (0.1, 1, 5 and 10 μM) (Sigma-Aldrich Co., St. Louis, MO) for 24 h.
Conditioned media (CM) collected from NE-treated HSCs were used to cultivate HCC cells. In brief, LX2 cells or sRFP1 knockdown LX2 cells (LX-2shRNA sFRP1) were treated with 10 μM NE for 24 h. The next day, the medium was exchanged with fresh DMEM supplemented with 2% FBS without NE, and the cells were cultured for another 24 h. The supernatant of cells was harvested, filtered and centrifuged with 3000 rpm for 15 min to remove debris. For individual experiments, HCCs were incubated with CM collected from NE-treated HSCs.
Transfection of Lentiviral vectors with shRNA for sFRP1
LX2 cells with stable sFRP1 knockdown were generated using lentivirus-mediated knockdown of sFRP1. Lentiviral vectors encoding shRNA targeting sFRP1 or scramble (negative control) were purchased from the GENECHEM gene company, Shanghai, China. LX2 cells were transfected by lentivirus particles (multiplicity of infections, MOI =5) and then selected in culture medium with 2 μg/ml puromycin. The efficiency of gene silencing was assayed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the cells or tissues using TRIzol reagent (Invitrogen, USA), Then, synthesis of cDNA reaction was carried out with 2 μg of total RNA using a PrimeScript RT Reagent Kit (Takara, Japan) following the manufacturer’s instructions. Subsequently, PCR amplification was done on cDNA using Maxinma SYBR Green qPCR Master Mix (Thermo Scientific). Quantification of target genes was performed with the 2
−ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin for normalization. Melting curve analysis was used to check the specificity of PCR products. The used primers are listed in Table S1 (Additional file
1: Table S1).
Western blot
Briefly, total proteins were extracted using RIPA (Radio-Immunoprecipitation Assay) lysis buffer containing 1 mM PMSF (Phenylmethanesulfonyl fluoride) (Beyotime, Beyotime Institute of Biotechnology, Shanghai, China) and 10% PhosSTOP phosphatase inhibitor Cocktail (Roche), and then resolved by electrophoresis through a 10% SDS-PAGE. The amount of separated proteins (20 μg per lane) was transferred onto 0.45 μM PVDF membranes (Millipore, USA) and incubated with primary antibody against α1A-adrenergic receptor (1:1000, Abcam), Collagen I (1:1000, Abcam), α-SMA (1:300, Abcam), α-AMPK (1:1000, CST, Cell Signal Technology), p- αAMPK (1:1000, CST), GSK-3β (1:1000, CST), p-GSK-3β (1:1000, Tyr 216) (Abcam), Cyclin D1 (1:10000, Abcam), c-Myc (1:10000, Abcam), Nanog (1:2000, CST), N-Cadherin(1:1000, Abcam), E-Cadherin(1:1000, Abcam), Vimentin (1:1000, Abcam), Snail (1:1000, Abcam), β-catenin (1:1000, CST), sFRP1 (1:1000, Abcam), Wnt 16B (1:1000, Bioss) or GAPDH (1:1000, Beyotime) and the corresponding HRP-conjugated secondary antibody (PeproTech). Protein band was developed using Ncm-ECL Ultra (New Cell & Molecular Biotech Co., Ltd., China).
Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 assay (CCK-8, Yeasen, Shanghai, China). Briefly, cells were seeded into 96-well plates (1 × 103 cells /well) and cultured for the indicated time periods. Then, 10 μl of CCK-8 reagent was added into each well for 1 h at 37 °C. The absorbance was measured using a Multiskan spectrophotometer at a wavelength of 450 nm.
Immunofluorescence assay
After fixed with 4% paraformaldehyde (Sangon, Shanghai, China), incubated with 0.3% Triton X-100 (Sangon, Shanghai, China) and blocked with 5% BSA (Sangon, Shanghai, China), the cells were incubated with primary antibody at 4 °C overnight, followed by incubation with the appropriate secondary antibody (Thermo Scientific). The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Yeasen, Shanghai, China). The intensity of fluorescence was detected using a confocal laser scanning microscopy (LSM510, Zeiss, Germany).
Migration and invasion assays
For migration analyzed by wound-healing assay, the cell monolayer was mechanically disrupted to produce a linear wound using a sterile 200 μl pipette tip. The distance migrated by cells was measured using a microscope equipped with an ocular micrometer. For invasion assay, 1 × 105 cells suspended in serum-free medium were seeded into the upper chamber coated with Matrigel (BD Biosciences, USA) in 24-well transwell plates (8-μm pore size, Corning, NY, USA), and 600 μl DMEM with 10% FBS was added into the lower chamber. After incubation for an indicated time points, the invading cells on the outer side of the upper chamber membrane were fixed with 4% paraformaldehyde, stained with crystal violet and counted under a light microscopy.
Flow cytometric analysis
For analysis of apoptosis using Alexa Fluor 488 Annexin V Kit (Invitrogen), the cells (1 × 106 cells/ml) were harvested, washed with PBS and centrifuged at 1000 rpm for 5 min. Then, the cell pellet was re-suspended in the annexin-binding buffer, incubated with annexin V and PI working solution for 15 min at room temperature. Cell apoptosis was determined using FACS caliber Flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA).
Microarray
The mRNA expression profiles were generated using Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA, US) according to the manufacturer’s instructions, as described previously [
27].
Enzyme-linked Immunosorbent assay (ELISA)
The sFRP1 level in conditioned media collected from cells treated with NE was measured by an ELISA kit (R&D Systems, Wiesbaden, Germany) following the manufacturer’s instructions. For NE, tissue was homogenized in 0.01 M HCl at 10% volume (ml) by tissue weight. NE in serum and tissue was quantified by ELISA (Elabscience, E-EL-0047c, China) according to the manufacturer’s protocol, as described previously [
28].
α-AMPK kinase blocking assay
For blocking experiments, LX2 cells were pre-incubated with Dorsomorphin (200 nM, 1 μM or 5 μM) (α-AMPK inhibitor, Selleck Chemicals, China) for 2 h prior to exposure to NE (10 μM) for another 4 h or 24 h.
Inhibition of GSK-3β kinase and β-catenin
HCC cells were pre-treated with GSK-3β inhibitor CHIR-99021 (10 μM) or β-catenin inhibitor XAV-939 (10 μM) (Selleck Chemicals, China) for 2 h, followed by the treatment of NE (10 μM) for another 3 h or 24 h.
Immunoprecipitation
To identify sFRP1-targeted molecules, co-immunoprecipitation was conducted as previously described. Briefly, HCC cells were lysed in immunoprecipitation buffer supplemented with a protease inhibitor for 2 h at 4 °C. The sample was centrifuged at 12,000 x g for 15 min, and the protein concentration was determined using the Bradford method (Beyotime, Beyotime Institute of Biotechnology, Shanghai, China). Precleared lysates with equivalent amounts of protein were incubated with a primary antibody overnight at 4 °C. Then, protein A- and G-Sepharose beads (Pierce Biotechnology, Rockford, IL, USA) were added to the immunoprecipitation (IP) mixture for 2 h. After that, the precleared lysate was incubated with a specific antibody coupled to protein A/G-agarose beads for 2 h at 4 °C. The precipitates were washed four times with immunoprecipitation buffer, resolved by 10% SDS-PAG, detected by western blot with specific antibody and visualized by enhanced chemiluminescence.
HCC cells were transfected (riboFECT™ CP Reagent, Guangzhou RiboBio Co., Ltd) with a plasmid containing a full-length sequence of Wnt16B promoter (2000 bp upstream of transcription start site) and subsequently assayed for luciferase reporter gene expression (Promega), as described previously [
27].
Immunohistochemistry
Immunohistochemistry was performed using the EnVision two-step Visualization System (GeneTech, Shanghai, China). Briefly, tumor specimens were removed, fixed in 10% neutral formalin and embedded with paraffin, and sliced into 5 mm thick sections. Sections were deparaffinized with xylene, followed by rehydration with a graduated series of ethanol, blocking endogenous peroxides with 3% H2O2, antigen-retrieval with microwave, blocking non-specific antibody binding. Slides were next incubated with primary antibodies against Cyclin D1 (1:50, Abcam), N-Cadherin(1:100, Abcam), E-Cadherin (1:100, Abcam), β-catenin (1:100, CST), sFRP1 (1:50, Abcam), overnight at 4 °C, followed by incubation with the secondary antibodies the next day, and visualized with 3,3-diaminobenzidine (DAB) as a chromogen. The slides were counterstained with hematoxylin.
Animal experiments
All animal experiments were approved by the Ethical Committee on Animal Experiments of Animal Care Committee of Zhongshan Hospital of Fudan University, Shanghai, China and carried out according to the Shanghai Medical Experimental Animal Care Commission Guidelines. All Male BALB/c nude mice (4–6 weeks old and weighing 18–20 g) were purchased from SLAC Laboratory Animal Co., Ltd., Shanghai, China and housed in a pathogen-free condition, and all efforts were made to minimize animal suffering. In one experiment, mice were injected subcutaneously with a cell suspension containing 3× 10
7 Huh7 cells with or without 1× 10
7 LX-2 cells into the upper right flank portion of each mouse. After 5 days of tumor inoculation, mice were assigned to four groups: (a) Huh7 control group (
n = 5); (b) Huh7 + daily stress group (
n = 5); (c) Huh7/LX2 cells control group (
n = 5); (d) Huh7/LX2 cells + daily stress group (
n = 5). In the chronic stress, the mouse is restrained in a movement-restricted space using an acrylic cylindrical animal restrainer, which restricts the movement of the limbs with unlimited breathing. After restraint for 2 h, the mice were returned to their home cages and allowed access to food and water. The mice were subjected to daily restraint stress for 3 weeks as described previously [
7]. In the second experiment, cell suspensions containing 3× 10
7 Huh7 cells with 1× 10
7 sFRP1 shRNA LX-2 (LX-2
shRNA sFRP1) or scramble shRNA LX-2 (LX-2
shRNA NC) cells were injected subcutaneously into mice (
n = 5 for each group). After 1 week of inoculation, mice were subjected to chronic stress for 3 weeks as described above. The length and width of the tumors were measured twice per week, and the tumor volume was calculated according to the formula: (length × width
2)/2. After 1 month, mice were sacrificed, and tumors, livers, and lungs were harvested, fixed with 10% formalin or frozen in liquid nitrogen for the following analyses.
Human samples
Ethical approval from the Zhongshan Hospital of Fudan University (Shanghai, China) Research Ethics Committee and patient written informed consents were obtained from each patient. HCC and matched nontumor liver tissues were collected from 26 patients who underwent curative resection at the Liver Cancer Institute, Zhongshan Hospital of Fudan University (Shanghai, China) in 2015. The pathologic diagnosis of HCC was confirmed. Clinicopathological information was retrieved from the medical records.
Statistical analysis
Data were expressed as means ± standard deviation (SD) from three independent experiments. All statistical analyses were performed using the GraphPad Prism Software (GraphPad Software, San Diego, CA). The unpaired Student’s t test, one-way analysis of variance (ANOVA) or Fisher’s exact test was conducted for comparison between groups wherever appropriate. All statistical tests were two-sided and a P < 0.05 was considered statistically significant.
Discussion
Associations between chronic stress and cancer progression have been studied [
37,
38], and persistent SNS activation during chronic stress is known to contribute to the onset and progression of various diseases, including cancers [
38]. In this study, we present a new evidence that chronic restraint stress promotes HCC progression through the activation of HSCs by the stress hormone NE. Specifically, NE-stimulated HSCs secrete sFRP1 to enhance HCC progression by augmenting an autocrine feedback loop of Wnt16B/β-catenin signaling. Our study suggests that sFRP1 may be a new potential therapeutic target for the treatment of chronic stress-induced HCC progression.
Several physiological problems, including fear, depression, and anxiety, are often seen in patients with cancer and can chronically activate stress pathways. Continuous exposure to stress enhances the progression of cancers, and this is partly mediated by NE and epinephrine released during increased sympathetic nervous activity as part of the body’s fight-or-flight stress response [
39]. During stress, NE is locally secreted from nerve endings in various tissues and then into the blood, while epinephrine is secreted from the adrenal medulla [
40‐
42]. In many types of tumors, NE, epinephrine, and activation of adrenergic receptors are involved in tumorigenesis, cancer survival, proliferation, angiogenesis, tumor progression, and metastasis through adrenergic receptor signaling-coupled intracellular molecular pathways [
43,
44] or immunosuppression in the tumor microenvironment [
45,
46]. Epidemiological data indicate the correlation between long-term survival of patients with cancer and β-blocker use (which antagonize NE/epinephrine receptors), suggesting a new class of potential cancer therapeutics [
47]. Catecholamines, such as NE, affect not only cancer cells that express adrenergic receptors but also stromal cells within the tumor microenvironment. Different from previous studies [
43], which focused on adrenergic receptor signaling in tumor cells, our study examined the effects of the stress hormone NE on HSCs, a type of stromal cells rich in the fibrotic tumor environment of HCC. Electron-microscopic studies have shown that HSCs, the major fibrogenic cells in the liver, are in contact with nerve fibers in the human liver [
22]. In addition, several neuronal genes, including adrenergic receptors, are expressed in HSCs [
48], and catecholamines are known to aggravate stress-induced liver diseases (e.g., cirrhosis) [
49]. In this study, sFRP1 was chosen as the central molecule for the following reasons. CM from NE-treated HSCs promoted the malignant behaviors of HCC cells and increased β-catenin activity, suggesting that NE-stimulated HSCs may enhance HCC malignancies by a secreted/paracrine factor, which has the role in the alteration of Wnt/β-catenin pathway. After conducting differential gene expression analysis of NE-treated versus vehicle-treated LX-2 cells, we limited our search for candidate genes related to a secreted protein whose expression can modulate Wnt/β-catenin activity. Therefore, we selected sFRP1 as a downstream effector of NE-treated HSCs for its known roles in mediating Wnt/β-catenin signaling. To the best of our knowledge, this is the first study to report that NE up-regulates sFRP1 expression in HSCs through the activation of α1A-ADR signaling and that sFRP1 signaling from NE-stimulated HSCs promotes malignant characteristics of HCC cells (EMT, parameters of proliferation-related genes, and cancer stem cell markers). This study suggests sFRP1 as a potential target for interruption of chronic stress-promoted HCC progression.
Wnt signaling has been implicated in development, homeostasis, and disease [
36] and sFRP1, a Wnt signaling regulator, is often considered a Wnt pathway inhibitor. Loss or downregulation of sFRP1 expression plays an important role in the development and progression of various cancer, including colon cancer [
50], lung cancer [
51], and HCC [
52,
53]. However, sFRP1 also negatively regulates tumorigenesis and cancer progression [
31,
54]. For example, during the development of prostate cancer, sFRP1 secreted from tumor stroma can provide a pro-proliferative signal to adjacent prostate epithelial cells [
55]; in gastric cancer, crosstalk of sFRP1 with TGFβ signaling promotes cell proliferation, EMT and invasion. Furthermore, high levels of sFRP1 indicate aggressiveness in some subgroups of gastric cancer and poor survival of patients [
56]. In this study, we presented the evidence that in the tumor microenvironment affected by NE, stromal HSCs within the tumor microenvironment of HCC acquire the capacity to secrete sFRP-1 and thereby promote phenotypic progression in HCC cells. Moreover, we found that sFRP1 from NE-stimulated HSCs augments an autocrine feedback loop of Wnt16B/β-catenin signaling in HCC cells by increasing the interaction of Wnt16B with the receptor FZD7 and enhancing Wnt16B expression, thereby inducing cell migration, invasion, EMT, and expression of proliferation-related markers and cancer stem cell markers to promote HCC progression. Regarding the mechanism, we found that following Wnt16B-mediated Wnt/β-catenin activation, sFRP1 further increased the nuclear translocation of β-catenin and subsequent enhancement of β-catenin/TCF4 signaling. Transcriptional activation of WNT16B was promoted by sFRP1-induced β-catenin/TCF4 signaling. Chronic restraint stress causes anxiety- and depression-like statuses in animal models. Our in vivo experiments further confirmed that sFRP1 in HSCs accelerated HCC progression in restraint-stressed mice. In accordance with the previous report that the density of α1A-ADR was increased in nontumoral liver tissues [
57], we found that NE, α1A-ADR, α-SMA, COL1A1 (markers of HSCs activation) and sFRP1 were simultaneously over-expressed in nontumoral liver tissues, suggesting that HSCs in the peritumoral tissue may be the target of NE and the main source of sFRP1, whereas EMT, parameters of proliferation-related markers, and cancer stem cell markers, Wnt16B, and β-catenin were highly expressed in HCC tissues, suggesting that sFRP1 from HSCs in nontumoral liver tissues promotes HCC malignant characteristics. Due to a relatively small sample size of clinical HCC tissues (
n = 26), the findings about the role of sFRP1 in HCC tissues need to be cautiously interpreted. Taken together, the in vitro and in vivo experiments and evaluation of clinical tumors demonstrate that NE-induced sFRP1 and subsequent initiation of Wnt16B/β-catenin signaling play significant roles in the effect of chronic stress on HCC progression, suggesting sFRP1 as a new target for blocking chronic stress-induced tumor progression.
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