Background
Liver cancer is the fifth most common cancer worldwide and the second leading cause of cancer-related death worldwide [
1]. In primary liver cancers, most (70 to 90%) cancers are hepatocellular carcinoma (HCC) [
1]. The treatment efficacy of HCC is rather low mainly due to chemoresistance and metastasis which resulted in poor 5-year survival of less than 5% in advanced HCC [
2]. Cancer stem cells (CSCs) are considered to be one of the main mechanisms of chemoresistance and metastasis [
3‐
5]. Hepatic CSCs have been identified and isolated from HCC in previous reports [
2,
6‐
8]. To elucidate potential targetable molecular markers as well as signaling pathways of hepatic CSCs will be helpful in improving treatment efficacy in HCC.
Hepatitis B virus (HBV) or Hepatitis C virus (HCV) are hepatotropic, noncytopathic DNA viruses that cause acute and chronic necroinflammatory liver diseases and hepatocellular carcinoma [
9]. Type I interferons (IFNs) are pro-inflammatory cytokines that activate JAK-STAT signaling pathways leading to transcription of IFN-stimulated genes (ISGs) to protect cells against invading viral pathogens including HBV and HCV [
10‐
14]. Although hundreds of ISGs have been identified for the past decades, only a few have been characterized with antiviral activity. Using an overexpression screening approach, 380 human ISGs including interferon-induced protein 44-like (IF144L) gene were tested for their abilities to suppress the replication of viruses [
15].
IFI44L is a type I ISG and belongs to the IFI44 family [
16]. The IFI44L protein is 452 amino acid long, an approximately 47 kDa protein, and located on chromosome 1 at area p31 (GenBank AB000115). Increased expression of IFI44L was reported after treatment with IL-28A and IFN-α to inhibit HCV replication [
12]. In addition, the functions of miR-9 in some cancers are recently implicated in regulating proliferation, invasion, metastasis, epithelial–mesenchymal transition (EMT), apoptosis, and tumor angiogenesis [
17‐
19]. A previous study reported that overexpression of miR-9 significantly upregulated the expression of a lot of ISGs including IFI44L in nasopharyngeal carcinoma cells [
20]. These studies indicated the promising role of IFI44L not only in anti-viral aspects but also in cancer treatment.
An earlier report documented that a novel ISG, BATF2, as potent negative regulator of hepatocyte growth factor (HGF)/Met signaling in colorectal cancer and may serve as a prognostic tumor marker [
21]. IFN-α activates STAT signaling and downregulates Met in primary human hepatocytes was also reported [
22]. Blocking the HGF/Met pathway by Met inhibitors or monoclonal antibodies strongly inhibits tumor growth and tumorigenicity in many malignancies including HCC [
23]. Met has been known that is an upstream regulator of multiple pathways, including PI3K/Akt, Ras/MAPK, Src/Stat3, and NF-κB [
22]. In liver cancer, many studies have demonstrated that Met overexpression is associated with the development of distant metastases and a shorter metastasis-free survival [
23]. Consequently, Met activation is considered to be crucial for the acquisition of metastatic potential and the correlation between Met pathway and ISGs warrants further study.
In this study, we successfully enriched hepatic cancer stem-like cells and first identified that overexpression of IFI44L significantly reduces the chemoresistance towards doxorubicin and knockdown of IFI44L promotes sphere formation in HCC cells. Furthermore, we found that depletion of IFI44L expression promotes migration, invasion, and pulmonary metastasis in HCC cells. We first demonstrated that suppression of IFI44L leads to activation of Met/Src pathway. We also first identified that the expression of IFI44L decreased in tumor tissues and correlated with several poor clinical outcomes in HCC patients. Our data demonstrated that IFI44L is a potent negative regulator of Met/Src signaling pathway in modulating HCC cancer stemness and drug resistance and may serve as an important prognostic marker.
Methods
Patients
217 HCC tissue microarray slides were obtained from HCC patients receiving surgeries in Changhua Christian Hospital from July 2011 to November 2013 [
24]. Paraffin-embedded HCC samples were obtained from Changhua Christian Hospital under the approved Institutional Review Board (IRB) protocol. Clinical patterns and overall survival data were analyzed by SPSS software and chart review. The age of all patients was between twenty-nine and eighty-one years. The clinical characteristics of these 217 patients are shown in Table
1.
Table 1
Relationship between clinical parameters and IFI44L expression in hepatocellular patients
Age (years) |
< 65 | 100 | 49 (49%) | 51 (51%) | 0.892 |
≧65 | 117 | 56 (48%) | 61 (52%) | |
Gender |
Female | 58 | 35 (60%) | 23 (40%) | 0.306 |
Male | 159 | 75 (47%) | 84 (53%) | |
Differentiation |
Well | 12 | 3 (25%) | 9 (75%) | 0.892 |
Moderate | 105 | 55 (52%) | 50 (48%) | |
Poor | 94 | 47 (50%) | 47 (50%) | |
Undifferentiation | 6 | 1 (17%) | 5 (83%) | |
Stage |
I, II | 180 | 84 (47%) | 96 (53%) | 0.029 |
III, IV | 37 | 25 (68%) | 12 (32%) | |
Hepatitis B surface antigen |
Negative | 106 | 52 (49%) | 54 (51%) | 0.892 |
Positive | 111 | 56 (51%) | 55 (49%) | |
Hepatits C virus |
Negative | 150 | 74 (49%) | 76 (51%) | 0.883 |
Positive | 67 | 34 (51%) | 33 (49%) | |
Tumor Number |
Single | 177 | 87 (49%) | 90 (51%) | 0.841 |
Multiple | 40 | 21 (53%) | 19 (47%) | |
Tumor size |
< 5 cm | 140 | 59 (42%) | 81 (58%) | 0.002 |
≧5 cm | 77 | 49 (64%) | 28 (36%) | |
Relapse |
- | 196 | 91 (46%) | 105 (54%) | 0.002 |
+ | 21 | 17 (81%) | 4 (19%) | |
Cell culture
The human liver cancer cell lines Hep3B (ATCC number: HB-8064), HepG2 (ATCC number: HB-8065) and PLC (ATCC number: HB-8024) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured at 37 °C under 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Biological Industries) and 100 units/ml of penicilium and streptomycin (Life Technologies, Carlsbad, CA, USA).
Vectors, antibodies, and reagents
For IFI44L-expressing vector, IFI44L coding sequence was amplified and cloned in pMSCV plasmid. Antibodies for western blotting and immunohistochemistry (IHC) are anti-IFI44L (Abcam), p-Met (Cell signaling, Tyr1234/1235), Met (Cell signaling), Src (Cell signaling) and p-Src (Cell signaling, Tyr416). IFI44L-specific siRNAs were purchased from MDBio, Inc. Detailed sequences for IFI44L siRNA oligonucleotides were shown in Additional file
1: Table S1. For cell sensitivity assays, HCC cells were pretreated with doxorubicin (Sigma-Aldrich) for 18 h (overnight) in serum-free culture medium.
RNA extraction and qRT-PCR
Quantitative RT-PCR (qRT-PCR) was used for gene detection. Detailed procedure of reverse transcription reaction was described elsewhere [
25]. qRT-PCR was performed on a CFX96 qPCR detection system (Bio-Rad) with a 1:10 dilution of cDNA by using KAPA SYBR FAST qPCR Kits (KAPA Biosystems). The mRNA levels were normalized to actin mRNA. The primers used for mRNA expression are listed in Additional file
1: Table S1.
Monolayer cells of three HCC cell lines (Hep3B, HepG2 and PLC cells) were cultured in a stem cell selective condition described previously to obtain spheres [
5]. Spheres comprised at least five cells were calculated by visual counts according to a previous report [
26].
Cell proliferation assay
The cell proliferation assay was measured by MTT assay (Promega, Madison, WI, USA). The assay was performed according to the manufacture’s protocol. Briefly, cells (with density around 3 X 103 per well) were seeded in 96-well plates and were incubated for 24 h. Cells were subsequently treated with various concentrations of doxorubicin and then were incubated for 48 h. Viable cells with active metabolism converted MTT into a formazan product, the quantity of which was measured at a wave length of 490 nm with 96-well plate reader and was directly proportional to the number of viable cells. The drug concentration required to reduce proliferation by 50% is defined as IC50. All the experiments were performed in triplicates and repeated three times.
Cell chemotatic migration and invasion assay
Migration and invasion abilities of HCC cells were carried out using the Falcon Cell Culture Inserts with or without Matrigel (BD Biosciences) coating as described previously [
27]. Detailed procedures were described elsewhere [
25].
Hep3B Cells (1 × 106) with indicated treatments were suspended in phosphate-buffered saline (PBS) and were injected individually into the tail vein of 6- to 8-week-old C.B-17 severe-combined immunodeficient (CB17-SCID) mice. All mice were monitored meticulously and were sacrificed after 40 days of implantation. Tumor growth was observed by live animal BLI (Caliper IVIS system, PerkinElmer).
Immunohistochemistry (IHC)
IHC was performed to detect IFI44L expression from paraffin-embedded HCC specimens. The slides were stained with anti-IFI44L antibody (Bethyl Labs, Montgomery, TX, USA) [
28]. The IFI44L antibody was purchased from ThermoFisher (Rock, USA). In liver cancer specimens, the detailed scores for IHC were defined as described previously [
24,
29].
Statistical analysis
The SPSS software (Version 13.0 SPSS Inc., Chicago, IL, USA) was used to conduct Chi-square analysis and paired-samples t-test. Kaplan-Meier method was performed for analyzing survival data. Variables related to survival were analyzed using Cox’s proportional hazards regression model via SPSS software. Differences between experimental groups were calculated using the Mann–Whitney U test. Differences with P values of < 0.05 are considered statistically significant.
Discussion
HCC has been a global health problem with rising incidence in Western countries recently [
1]. In the West, around 40% of patients are diagnosed as early Barcelona Clinic Liver Cancer (BCLC) stages and are eligible for potential curative treatment such as surgical resection, radiofrequency ablation, microwave ablation, percutaneous alcohol injection, and liver transplantation [
9,
41‐
43]. However, the probability of disease recurrence is around 50% within 3 years after successful treatment [
2]. Hepatic CSCs exhibit multidrug and radio-resistant properties and are considered as in part the main mechanism of chemoresistance and recurrent disease [
2,
4]. In our study, we successfully enriched cancer stem-like cells via sphere-forming method in nonadhesive culture plates with serum-free culture medium from three hepatic cancer cell lines. These cancer stem-like cells express important hepatic CSC markers such as CD24, CD44, CD117, CD133, ALDH, ABCG2, Oct4, and Nanog which were extensively reported before [
7,
30‐
37]. They also reveal significant chemoresistance towards doxorubicin in accordance with previous reports [
2]. To find specific molecules to target these cancer stem-like cells would be very important in treating HCC.
Type I IFNs are a family of cytokines to directly activate the transcription of ISGs to exert anti-viral, anti-proliferative, and immunomodulatory activities [
10,
11]. IFI44L, one of the type I ISG, exhibits a low antiviral activity against HCV and is indicated to be correlated with some cancer recently although the reports are scarce [
12,
20,
44]. In present study, our data showed that overexpression of IFI44L restores chemosensitivity towards doxorubicin whereas decreased expression of IFI44L promotes sphere formation in HCC cell lines. Depletion of IFI44L also enhanced migration, invasion, and lung metastasis in HCC cells. According to the above results, IFI44L was proposed as a novel tumor suppressor modulating cancer stemness, drug resistance, migration and invasion, as well as pulmonary metastasis in HCC. Although one recent study indicated that upregulation of IFI44L was significantly correlated with shorter overall survival and shorter median survival time in pancreatic ductal adenocarcinoma [
44], our data revealed that low expression of IFI44L was found in HCC tumor samples and was correlated with larger tumor size, more disease relapse, advanced stages as well as significant poorer RFS and OS. Although some study identified that IFI44L overexpressed in pancreatic ductal adenocarcinoma and correlated with worse clinical prognosis, this conclusion is only made from statistics of databases collecting from gene expression profiling and TCGA database but lacks in vitro and in vivo experimental confirmation [
44]
. The functional role of IFI44L in different cancers still warrants further study.
In advanced stages of HCC, conventional chemotherapy such as doxorubicin, cisplatin, and 5-fluorouracil were generally introduced but the response rate was very low (from 15 to 20%) and these chemotherapeutic agents failed to prolong survival [
2,
41]. Sorefenib, a small molecule multikinase inhibitor that inhibits tumor-cell proliferation and tumor angiogenesis, is the first targeted therapy to reveal survival benefit in patients with advanced HCC [
41]. Other new molecular pathways including.
Ras/Raf/MEK/ERK (MAPK) pathway, wnt/catenin pathway, PI3K/Akt/mTOR pathway, VEGF pathway, and HGF/Met pathway etc. were extensively explored in HCC patients [
9,
23,
45]. The efficacy of new targeted therapies such as lenvatinib, nivolumab, ramucirumab, tivantinib, and cabozantinib etc. are still under evaluation in large clinical trials [
9]. Of the above mentioned pathways, the HGF/Met pathway has been implicated in tumor cell migration, invasion, proliferation, and angiogenesis [
23]. High expression of Met and HGF was reported to be correlated with early recurrence of HCC after hepatectomy and shorter survival in HCC patients [
23]. Several studies indicated that IFN regulates multiple STAT signaling and downregulates Met resulting in suppression of HGF-induced signals and cell proliferation [
14,
22]. In our study, we first identified that suppression of IFI44L leads to the activation of Met/Src pathway. Thus, the phenomenon that suppression of IFI44L promotes cancer stemness, migration, invasion, and pulmonary metastasis in HCC cells and overexpression of IFI44L results in restoring chemosensitivity observed in our study might be regulated via affecting Met/Src signaling pathway.