IFI44L is a novel tumor suppressor in human hepatocellular carcinoma affecting cancer stemness, metastasis, and drug resistance via regulating met/Src signaling pathway

IFI44L is a novel tumor suppressor in human hepatocellular carcinoma affecting cancer stemness,... Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The disease recurrent rate is relatively high resulted in poor 5-year survival in advanced HCC. Cancer stem cells (CSCs) have been considered to be one of the main mechanisms for chemoresistance, metastasis, and recurrent disease. Interferon-induced protein 44-like (IFI44L) gene is a type I interferon-stimulated gene (ISG) and belongs to the IFI44 family. Previous reports indicated antiviral activity against HCV in IFI44L, however, its precise role and function in HCC has not been unveiled. Methods: To explore the characteristics of hepatic CSCs, we successfully enriched hepatic cancer stem-like cells from three established liver cancer cell lines (Hep3B, HepG2, and PLC lines). Parental Hep3B and HepG2 cells and their sphere cells were treated with doxorubicin for 48 h and cell viability was measured by MTT assay. HCC tissue blocks from 217 patients were sampled for tissue microarray (TMA). Follow-up information and histopathological and clinical data including age, gender, tumor grade, advanced stages, HBV, HCV, tumor number, tumor size, relapse-free survival, and overall survival were obtained from the cancer registry and medical charts. The liver TMA was evaluated for IFI44L expression using immunohistochemical staining and scores. Results: These hepatic cancer stem-like cells possess important cancer stemness characteristics including sphere- forming abilities, expressing important HCC cancer stem cell markers, and more chemoresistant. Interestingly, we found that overexpression of IFI44L decreased chemoresistance towards doxorubicin and knockdown of IFI44L restored chemoresistance as well as promoted sphere formation. Furthermore, we found that depletion of IFI44L enhanced migration, invasion, and pulmonary metastasis through activating Met/Src signaling pathway. Clinically, the expression level of IFI44L significantly reduced in HCC tumor tissues. Low expression of IFI44L levels also correlated with larger tumor size, disease relapse, advanced stages, and poor clinical survival in HCC patients. (Continued on next page) * Correspondence: chu.peiyi@msa.hinet.net Wei-Chieh Huang and Shiao-Lin Tung contributed equally to this work. School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei, Taiwan Department of Pathology, Show Chwan Memorial Hospital, No.542, Sec.1, Chung-Shang Road, Changhua City, Changhua County 50008, Taiwan, Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Cancer (2018) 18:609 Page 2 of 10 (Continued from previous page) Conclusion: Taken together, we first demonstrated that IFI44L is a novel tumor suppressor to affect cancer stemness, metastasis, and drug resistance via regulating Met/Src signaling pathway in HCC and can be serve as an important prognostic marker. Keywords: IFI44L, Cancer stem cells, Hepatocellular carcinoma Background (HGF)/Met signaling in colorectal cancer and may serve Liver cancer is the fifth most common cancer worldwide as aprognostictumor marker [21]. IFN-α activates STAT signaling and downregulates Met in primary hu- and the second leading cause of cancer-related death worldwide [1]. In primary liver cancers, most (70 to 90%) man hepatocytes was also reported [22]. Blocking the cancers are hepatocellular carcinoma (HCC) [1]. The HGF/Met pathway by Met inhibitors or monoclonal antibodies strongly inhibits tumor growth and tumori- treatment efficacy of HCC is rather low mainly due to chemoresistance and metastasis which resulted in poor genicity in many malignancies including HCC [23]. Met 5-year survival of less than 5% in advanced HCC [2]. Can- has been known that is an upstream regulator of mul- tiple pathways, including PI3K/Akt, Ras/MAPK, Src/ cer stem cells (CSCs) are considered to be one of the main Stat3, and NF-κB[22]. In liver cancer, many studies mechanisms of chemoresistance and metastasis [3–5]. Hepatic CSCs have been identified and isolated from have demonstrated that Met overexpression is associ- ated HCC in previous reports [2, 6–8]. To elucidate potential with the development of distant metastases and a targetable molecular markers as well as signaling pathways shorter metastasis-free survival [23]. Consequently, Met of hepatic CSCs will be helpful in improving treatment ef- activation is considered to be crucial for the acquisition ficacy in HCC. of metastatic potential and the correlation between Met Hepatitis B virus (HBV) or Hepatitis C virus (HCV) pathway and ISGs warrants further study. are hepatotropic, noncytopathic DNA viruses that cause In this study, we successfully enriched hepatic cancer acute and chronic necroinflammatory liver diseases and stem-like cells and first identified that overexpression hepatocellular carcinoma [9]. Type I interferons (IFNs) are of IFI44L significantly reduces the chemoresistance to- pro-inflammatory cytokines that activate JAK-STAT signal- wards doxorubicin and knockdown of IFI44L promotes ing pathways leading to transcription of IFN-stimulated sphere formation in HCC cells.Furthermore,wefound genes(ISGs)toprotectcellsagainstinvading viralpatho- that depletion of IFI44L expression promotes migra- gens including HBV and HCV [10–14]. Although hundreds tion, invasion, and pulmonary metastasis in HCC cells. of ISGs have been identified for the past decades, only a We first demonstrated that suppression of IFI44L leads few have been characterized with antiviral activity. Using an to activation of Met/Src pathway. We also first identi- overexpression screening approach, 380 human ISGs in- fied that the expression of IFI44L decreased in tumor cluding interferon-induced protein 44-like (IF144L) gene tissues and correlated with several poor clinical out- were tested for their abilities to suppress the replication of comes in HCC patients. Our data demonstrated that viruses [15]. IFI44L is a potent negative regulator of Met/Src signal- IFI44L is a type I ISG and belongs to the IFI44 family ing pathway in modulating HCC cancer stemness and [16]. The IFI44L protein is 452 amino acid long, an ap- drug resistance and may serve as an important prog- proximately 47 kDa protein, and located on chromosome nostic marker. 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 Methods functions of miR-9 in some cancers are recently impli- Patients cated in regulating proliferation, invasion, metastasis, epi- 217 HCC tissue microarray slides were obtained from thelial–mesenchymal transition (EMT), apoptosis, and HCC patients receiving surgeries in Changhua Christian tumor angiogenesis [17–19]. A previous study reported Hospital from July 2011 to November 2013 [24]. that overexpression of miR-9 significantly upregulated the Paraffin-embedded HCC samples were obtained from expression of a lot of ISGs including IFI44L in nasopha- Changhua Christian Hospital under the approved Insti- ryngeal carcinoma cells [20]. These studies indicated the tutional Review Board (IRB) protocol. Clinical patterns promising role of IFI44L not only in anti-viral aspects but and overall survival data were analyzed by SPSS software also in cancer treatment. and chart review. The age of all patients was between An earlier report documented that a novel ISG, BATF2, twenty-nine and eighty-one years. The clinical character- as potent negative regulator of hepatocyte growth factor istics of these 217 patients are shown in Table 1. Huang et al. BMC Cancer (2018) 18:609 Page 3 of 10 Table 1 Relationship between clinical parameters and IFI44L anti-IFI44L (Abcam), p-Met (Cell signaling, Tyr1234/ expression in hepatocellular patients 1235), Met (Cell signaling), Src (Cell signaling) and p-Src IFI44L (Cell signaling, Tyr416). IFI44L-specific siRNAs were pur- chased from MDBio, Inc. Detailed sequences for IFI44L Variables N Low High p-value siRNA oligonucleotides were shown in Additional file 1: Age (years) Table S1. For cell sensitivity assays, HCC cells were < 65 100 49 (49%) 51 (51%) 0.892 pretreated with doxorubicin (Sigma-Aldrich) for 18 h ≧65 117 56 (48%) 61 (52%) (overnight) in serum-free culture medium. Gender Female 58 35 (60%) 23 (40%) 0.306 RNA extraction and qRT-PCR Male 159 75 (47%) 84 (53%) Quantitative RT-PCR (qRT-PCR) was used for gene detec- tion. Detailed procedure of reverse transcription reaction Differentiation was described elsewhere [25]. qRT-PCR was performed on Well 12 3 (25%) 9 (75%) 0.892 a CFX96 qPCR detection system (Bio-Rad) with a 1:10 Moderate 105 55 (52%) 50 (48%) dilution of cDNA by using KAPA SYBR FAST qPCR Kits Poor 94 47 (50%) 47 (50%) (KAPA Biosystems). The mRNA levels were normalized Undifferentiation 6 1 (17%) 5 (83%) to actin mRNA. The primers used for mRNA expression Stage are listed in Additional file 1: Table S1. I, II 180 84 (47%) 96 (53%) 0.029 Sphere-forming assay III, IV 37 25 (68%) 12 (32%) Monolayer cells of three HCC cell lines (Hep3B, HepG2 Hepatitis B surface antigen and PLC cells) were cultured in a stem cell selective Negative 106 52 (49%) 54 (51%) 0.892 condition described previously to obtain spheres [5]. Positive 111 56 (51%) 55 (49%) Spheres comprised at least five cells were calculated by Hepatits C virus visual counts according to a previous report [26]. Negative 150 74 (49%) 76 (51%) 0.883 Cell proliferation assay Positive 67 34 (51%) 33 (49%) The cell proliferation assay was measured by MTT assay Tumor Number (Promega, Madison, WI, USA). The assay was performed Single 177 87 (49%) 90 (51%) 0.841 according to the manufacture’s protocol. Briefly, cells Multiple 40 21 (53%) 19 (47%) (with density around 3 X 10 per well) were seeded in Tumor size 96-well plates and were incubated for 24 h. Cells were subsequently treated with various concentrations of < 5 cm 140 59 (42%) 81 (58%) 0.002 doxorubicin and then were incubated for 48 h. Viable ≧5 cm 77 49 (64%) 28 (36%) cells with active metabolism converted MTT into a for- Relapse mazan product, the quantity of which was measured at a - 196 91 (46%) 105 (54%) 0.002 wave length of 490 nm with 96-well plate reader and + 21 17 (81%) 4 (19%) was directly proportional to the number of viable cells. The drug concentration required to reduce proliferation Cell culture by 50% is defined as IC . All the experiments were per- The human liver cancer cell lines Hep3B (ATCC number: formed in triplicates and repeated three times. HB-8064), HepG2 (ATCC number: HB-8065) and PLC (ATCC number: HB-8024) were obtained from the Ameri- Cell chemotatic migration and invasion assay can Type Culture Collection (ATCC, Manassas, VA). All Migration and invasion abilities of HCC cells were carried cells were cultured at 37 °C under 5% CO in Dulbecco’s out using the Falcon Cell Culture Inserts with or without modified Eagle medium (DMEM; Invitrogen) supple- Matrigel (BD Biosciences) coating as described previously mented with 10% fetal bovine serum (FBS; Biological [27]. Detailed procedures were described elsewhere [25]. Industries) and 100 units/ml of penicilium and strepto- mycin (Life Technologies, Carlsbad, CA, USA). In vivo metastasis assays Hep3B Cells (1 × 10 ) with indicated treatments were sus- Vectors, antibodies, and reagents pended in phosphate-buffered saline (PBS) and were For IFI44L-expressing vector, IFI44L coding sequence was injected individually into the tail vein of 6- to 8-week-old amplified and cloned in pMSCV plasmid. Antibodies for C.B-17 severe-combined immunodeficient (CB17-SCID) western blotting and immunohistochemistry (IHC) are mice. All mice were monitored meticulously and were Huang et al. BMC Cancer (2018) 18:609 Page 4 of 10 sacrificed after 40 days of implantation. Tumor growth cell selective condition described in ‘Methods’ to form was observed by live animal BLI (Caliper IVIS system, spheres. Most of the suspended cells underwent apop- PerkinElmer). tosis during the first 2 days of culturing, and the rest of survived cells gradually formed floating spheres. The Immunohistochemistry (IHC) spheres grew larger and often reached to 50–100 μMin IHC was performed to detect IFI44L expression from diameter after 4–8 days (Fig. 1a). Overexpression of paraffin-embedded HCC specimens. The slides were mRNA of HCC cancer stem cell markers was found in. stained with anti-IFI44L antibody (Bethyl Labs, Mont- Hep3B sphere cells compared with their parental cells. gomery, TX, USA) [28]. The IFI44L antibody was pur- These cancer stem cell markers, including CD24, CD44, chased from ThermoFisher (Rock, USA). In liver cancer CD117, CD133, ALDH, ABCG2, OCT4, and Nanog, specimens, the detailed scores for IHC were defined as were significantly higher in Hep3B sphere cells shown described previously [24, 29]. by qRT–PCR analysis (Fig. 1b)[7, 30–37]. Next, we examined the chemosensitivity of these sphere Statistical analysis cells. Parental Hep3B and HepG2 cells and their sphere The SPSS software (Version 13.0 SPSS Inc., Chicago, IL, cells were treated with doxorubicin for 48 h and cell USA) was used to conduct Chi-square analysis and viability was measured by MTT assay. Hep3B and HepG2 paired-samples t-test. Kaplan-Meier method was performed sphere cells are found to be more chemoresistant to con- for analyzing survival data. Variables related to survival tinuous exposure to various concentrations of doxorubicin were analyzed using Cox’s proportional hazards regression (Fig. 1c). Thus, we have successfully enriched HCC cancer model via SPSS software. Differences between experimental stem-like cells from Hep3B, HepG2, and PLC lines groups were calculated using the Mann–Whitney U test. displaying cancer stem cell characteristics including Differences with P values of < 0.05 are considered statisti- sphere-forming, expression of HCC cancer stem cell cally significant. markers, and more chemoresistant in accordance with established parameters of cancer stem-like cells [38–40]. Results Successful enrichment of human HCC cancer stem-like Overexpression of IFI44L restores chemosensitivity and cells from Hep3b, HepG2, and PLC lines knockdown of IFI44L promotes sphere formation In order to enrich for CSCs, parental Hep3B, HepG2, Since IFI44L was implied to be correlated with cancer and PLC cells from monolayer were cultured in a stem [20], we then investigated the impact of IFI44L on drug Fig. 1 HCC cancer stem-like cells were successfully enriched from Hep3B, HepG2, and PLC cell lines. a Formation of spheres under the stem cell selective condition on day 8 after culturing from parental Hep3B, HepG2, and PLC cells is shown. b The mRNA expression levels of HCC cancer stem cell markers in parental Hep3B cells and their sphere cells were analyzed by qRT–PCR with actin as an internal control. Histograms represent means ± s.d. from three independent experiments (*, P < 0.05; **, P < 0.01). c Dose-dependent growth inhibition of parental Hep3B and HepG2 cells and their sphere cells upon continuous exposure to the indicated concentrations of doxorubicin for 48 h was measured by MTT assay. Each dosage point represents the mean ± s.e. from three independent experiments (*, P < 0.05; **, P < 0.01) Huang et al. BMC Cancer (2018) 18:609 Page 5 of 10 resistance. Cells transfected with IFI44L expression plas- Next we tested whether sphere-forming ability of Hep3B, mid or control plasmid were tested their protein expres- HepG2, and PLC lines could be promoted by knockdown sion of IFI44L to confirm the transfection efficiency. of IFI44L. After 8 days culturing of Hep3b, HepG2, and Western blotting showed upregulation of IFI44L protein PLC cells in the stem cell selective condition, sphere num- level in Hep3B and HepG2 cells after transfection with ber was calculated by visual counting under microscope. the expression plasmid of IFI44L (IFI44L vector) (Add- Knockdown of IFI44L caused significant increase of itional file 2: Fig. S1). Our data indicated that Hep3b and sphere number (Fig. 2d). Thus, our data suggested that HepG2 cells became more chemosensitive to continuous IFI44L may play as a tumor suppressor role in restoring exposure to different doses of doxorubicin after transfec- chemosensitivity and affecting cancer stemness. tion with IFI44L vector (Fig. 2a), whereas IFI44L knock- down restored their chemoresistance (Additional file 3: Depletion of IFI44L expression promotes migration, Figure S2). These data suggested that overexpression of invasion and pulmonary metastasis and implicates in IFI44L significantly decreased chemoresistance of HCC met/Src signaling pathway in HCC lines towards doxorubicin. To assess whether IFI44L Furthermore, we evaluate the tumor suppressor role of level correlated with cancer stemness in HCC, we exam- IFI44L in regulating cancer metastasis. In Boyden cham- ined the protein expression level of IFI44L in HCC lines. ber assay, we found that depletion of IFI44L expression Decrease of IFI44L protein level in Hep3b and HepG2 significantly promotes Hep3B, HepG2 and PLC cell mi- sphere cells was found compared with their parental gration and invasion abilities (Fig. 3a and Additional file 5: cells by Western blotting analysis (Fig. 2b). We then Figure S4). To investigate whether IFI44L regulated can- investigated if suppression of IFI44L by its small interfer- cer cell metastasis in vivo, we employed an experimental ing RNAs (siRNA) could inhibit cancer stemness charac- metastasis model via tail vein injection in SCID mice. In teristics in HCC lines. Three specific IFI44L-siRNAs this model, knockdown of IFI44L significantly promoted were tested for their inhibitory efficacy by analyzing the lung metastasis of Hep3B cells compared with the IFI44L protein levels in Hep3B, HepG2 and PLC cells, control group (Fig. 3b). Since ISGs are implied to be cor- IFI44L-siRNA-2 showed the highest knockdown effect in related with Met pathway [11, 22, 23], we then explored inhibiting IFI44L protein and it was used in the subse- the role of IFI44L in Met signaling pathway. By Western quent experiments (Fig. 2c, Additional file 4: Figure S3). blotting analysis, we found that suppression of IFI44L Fig. 2 The effects of IFI44L on drug resistance and sphere formation. a Dose-dependent growth inhibition of Hep3B and HepG2 cells upon continuous exposure to the indicated concentrations of doxorubicin for 48 h was measured by MTT assay (*, P < 0.05; **, P < 0.01). Cells were transfected with 1 μg of pMSCV or pMSCV-IFI44L expression plasmids (IFI44L vector). b The expression levels of IFI44L in parental Hep3B and HepG2 cells and their sphere cells were measured by Western blotting. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 (Windows version of NIH Image, http://rsb.info.nih.gov/ij/) and was represented with normalized mean ± s.e. (n =3) below each band. c Western blotting analysis of three different siRNAs against IFI44L in Hep3B cells. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band. d Sphere formation under stem cell selective condition was examined on day 8 after culturing of the cells transfected with the indicated IFI44L-siRNA. The original magnification was 40X. Histograms represent means ± s.d. from three independent experiments (*, P < 0.05, **, P < 0.01) Huang et al. BMC Cancer (2018) 18:609 Page 6 of 10 Fig. 3 IFI44L functions as a tumor suppressor affecting metastasis implicates with Met/Src signaling. a Analysis of the effect of IFI44L on Hep3B and HepG2 cell migration and invasion using Boyden chamber assay. Quantitative data are shown by histograms and representative photographs of the migrated/invaded cells from different treatments are shown. b Representative xenograft tumors formed by 5 × 10 Hep3B sphere cells in the SCID mice. Tumor growth was monitored by BLI. Representative BLIs are shown on day 30 after implantation. c The protein expression levels of the signaling components of the Met/Src signaling in Hep3B and HepG2 cells are shown by Western blotting. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band enhances the phosphorylation of Met and Src in significantly higher in normal liver tissues compared with Hep3B and HepG2 cells (Fig. 3c). To further assess tumor tissues (Fig. 4a). Western blotting analysis also the role of IFI44/Met/Src axis in regulating cancer revealed that all of ten pairs of matched HCC tumor metastasis, we performed additional Western blotting tissues expressed lower level of IFI44L in comparison with analysis as well as migration and invasion assay. We the matched normal tissues (Fig. 4b). Downregulation of found that overexpression of IFI44L decreased phos- IFI44L expression found in HCC tumor tissues is compat- phorylation of Met as well as migration and invasion ible with the tumor suppressor role in HCC we discovered abilities in Hep3B cell line, whereas ectopic expression above. of Met reversed IFI44L-mediated inhibition of migration and invasion abilities approximately 50% (Additional file 6: Dowregulation of IFI44L expression levels significantly Figure S5). Taken together, these findings reinforced correlated with larger tumor size, disease relapse, that the functional role of IFI44L as a tumor suppressor advanced stages, and poor clinical survival in HCC and it could implicate in Met/Src signaling pathway patients in HCC. Furthermore, the correlation between clinicopathological characteristics and IFI44L of these 217 patients were an- The expression level of IFI44L significantly decreased in alyzed in Table 1. Among these parameters, age, gender, HCC tumor tissues tumor differentiation, HBV surface antigen, anti-HCV To evaluate the correlation of IFI44L with clinical sam- antibody, and the tumor number were not significantly ples, the expression of IFI44L in 217 pairs of normal liver different in patients with low versus high expression and HCC tumor tissues were analyzed by IHC and West- levels of IFI44L (Table 1). However, low expression of ern blotting analysis. The IHC score of IFI44L was IFI44L was observed in only 47% (84/180) of the early Huang et al. BMC Cancer (2018) 18:609 Page 7 of 10 Fig. 4 The expression level of IFI44L decreased in HCC tumor tissues. a The level of IFI44L was examined by IHC staining in 217 pairs of HCC tumor tissues and their adjacent normal tissues (**, P < 0.01). b Images of Western blotting analyses of IFI44L protein level in ten matched pairs of HCC tumor tissues and adjacent normal tissues. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band stages (stage I/II) HCC patents whereas 68% (25/37) of cells. In clinic data, we also found that patients with low the late stages (stage III/IV) HCC patients expressed low expression level of IFI44L had significantly higher relapse levels of IFI44L (P = 0.029) (Table 1). IHC staining also rate (81% vs 19%, P = 0.002) and shorter relapse-free sur- confirmed that IFI44L protein level decreased markedly vival (RFS) (p = 0.0012) than patients with high expression in advanced stages in HCC samples (Fig. 5a). Moreover, level of IFI44L (Table 1,Fig. 5b). higher percentage of HCC patients with low expression In survival analysis, the influence of clinicopathologi- level of IFI44L had larger tumor size then patients with cal characteristics including IFI44L on patients’ overall high expression level of IFI44L (64% vs 36%, P = 0.002) survival (OS) was statistically examined by univariate (Table 1). Since CSCs are indicated to be associated with analysis shown in Table 2. Four parameters including cancer recurrence [2, 38], our previous experiments also advanced stages, larger tumor size, disease relapse, and indicated that IFI44L affects cancer stemness in HCC low expression of IFI44L are significant correlated with Fig. 5 The expression level of IFI44L correlates with clinical staging, RFS, and OS in HCC patients. a Representative examples of the expression levels of IFI44L protein determined by IHC of clinical specimens. b The mRNA expression level of IFI44L correlates with RFS in 217HCC patients. c The mRNA expression level of IFI44L correlates with OS in 217 HCC patients Huang et al. BMC Cancer (2018) 18:609 Page 8 of 10 Table 2 Univariate analysis of influence of clinical characteristics alcohol injection, and liver transplantation [9, 41–43]. on overall survival in hepatocellular patients However, the probability of disease recurrence is around OS 50% within 3 years after successful treatment [2]. Hepatic CSCs exhibit multidrug and radio-resistant properties and Characteristics N Median survival Survival Log-rank (months) (%) are considered as in part the main mechanism of Age (years) chemoresistance and recurrent disease [2, 4]. In our study, we successfully enriched cancer stem-like cells < 65 100 36.83 75.00% 0.282 via sphere-forming method in nonadhesive culture ≧65 117 41.42 80.34% plates with serum-free culture medium from three hepatic Gender cancer cell lines. These cancer stem-like cells express Female 58 38.37 76.27% 0.631 important hepatic CSC markers such as CD24, CD44, Male 159 40.49 78.48% CD117, CD133, ALDH, ABCG2, Oct4, and Nanog which Differentiation were extensively reported before [7, 30–37]. They also reveal significant chemoresistance towards doxorubicin in Well, Moderate 117 40.69 78.45% 0.731 accordance with previous reports [2]. To find specific Undifferentitation, Poor 100 39.26 77.23% molecules to target these cancer stem-like cells would be Stage very important in treating HCC. I, II 180 42.22 82.49% < 0.001 Type I IFNs are a family of cytokines to directly III, IV 37 30.13 57.50% activate the transcription of ISGs to exert anti-viral, Hepatitis B surface antigen anti-proliferative, and immunomodulatory activities [10, 11]. IFI44L, one of the type I ISG, exhibits a low Negative 106 40.33 79.25% 0.617 antiviral activity against HCV and is indicated to be Positive 111 39.08 76.58% correlated with some cancer recently although the reports Hepatits C virus are scarce [12, 20, 44]. In present study, our data showed Negative 150 39.14 76.67% 0.489 that overexpression of IFI44L restores chemosensitivity Positive 67 41.1 80.60% towards doxorubicin whereas decreased expression of Tumor Number IFI44L promotes sphere formation in HCC cell lines. Depletion of IFI44L also enhanced migration, invasion, Single 177 40.25 77.78% 0.926 and lung metastasis in HCC cells. According to the above Multiple 40 37.17 78.38% results, IFI44L was proposed as a novel tumor suppressor Tumor size modulating cancer stemness, drug resistance, migration < 5 cm 140 49.79 86.43% < 0.001 and invasion, as well as pulmonary metastasis in HCC. ≧5 cm 77 33.12 62.34% Although one recent study indicated that upregulation of Relapse IFI44L was significantly correlated with shorter overall survival and shorter median survival time in pancreatic - 196 41.31 81.54% < 0.001 ductal adenocarcinoma [44], our data revealed that low + 21 28.79 45.00% expression of IFI44L was found in HCC tumor samples and was correlated with larger tumor size, more disease shorter median OS (P <0.001) (Table 2). Kaplan–Meier relapse, advanced stages as well as significant poorer RFS survival analysis of these 217 patients also revealed that and OS. Although some study identified that IFI44L low expression level of IFI44L correlated with poor OS overexpressed in pancreatic ductal adenocarcinoma and (P < 0.001) (Fig. 5c). These results suggested that down- correlated with worse clinical prognosis, this conclusion is regulation of IFI44L expression levels significantly only made from statistics of databases collecting from correlated with larger tumor size, disease relapse, ad- gene expression profiling and TCGA database but lacks in vanced stages, and poor clinical survival in HCC patients vitro and in vivo experimental confirmation [44]. The and could serve as an important prognostic marker. functional role of IFI44L in different cancers still warrants further study. Discussion In advanced stages of HCC, conventional chemother- HCC has been a global health problem with rising inci- apy such as doxorubicin, cisplatin, and 5-fluorouracil dence in Western countries recently [1]. In the West, were generally introduced but the response rate was around 40% of patients are diagnosed as early Barcelona very low (from 15 to 20%) and these chemotherapeutic Clinic Liver Cancer (BCLC) stages and are eligible for agents failed to prolong survival [2, 41]. Sorefenib, a potential curative treatment such as surgical resection, ra- small molecule multikinase inhibitor that inhibits diofrequency ablation, microwave ablation, percutaneous tumor-cell proliferation and tumor angiogenesis, is the Huang et al. BMC Cancer (2018) 18:609 Page 9 of 10 first targeted therapy to reveal survival benefit in pa- Additional file 5: Figure S4. Analysis of the effect of IFI44L on PLC cell tients with advanced HCC [41]. Other new molecular migration and invasion using Boyden chamber assay. Quantitative data are shown by histograms and representative photographs of the pathways including. migrated/invaded cells from different treatments are shown. Histograms Ras/Raf/MEK/ERK (MAPK) pathway, wnt/catenin represent means ± s.d. from 3 independent experiments (**, P < 0.01). pathway, PI3K/Akt/mTOR pathway, VEGF pathway, and (TIF 252 kb) HGF/Met pathway etc. were extensively explored in Additional file 6: Figure S5. Ectopic expression of Met significantly restored IFI44L expression-mediated inhibition of migration and invasion HCC patients [9, 23, 45]. The efficacy of new targeted abilities. Left, overexpression of IFI44L reduced the phosphorylation of therapies such as lenvatinib, nivolumab, ramucirumab, Met, which could be partially rescued by transfecting Met vector. The tivantinib, and cabozantinib etc. are still under evalu- actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. ation in large clinical trials [9]. Of the above mentioned (n = 3) below each band. Right, the migration and invasion abilities pathways, the HGF/Met pathway has been implicated in affected by overexpression of IFI44L and ectopic expression of Met in tumor cell migration, invasion, proliferation, and angio- Hep3B cell line. Quantitative data are shown by histograms and representative photographs of the migrated/invaded cells from genesis [23]. High expression of Met and HGF was re- different treatments are shown. Histograms represent means ± s.d. ported to be correlated with early recurrence of HCC from 3 independent experiments (*, P < 0.05; **, P < 0.01). (TIF 437 kb) after hepatectomy and shorter survival in HCC patients [23]. Several studies indicated that IFN regulates mul- Abbreviations tiple STAT signaling and downregulates Met resulting in CSCs: Cancer stem cells; EMT: Epithelial–mesenchymal transition; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; suppression of HGF-induced signals and cell prolifera- HGF: Hepatocyte growth factor; IF144L: Interferon-induced protein 44-like; tion [14, 22]. In our study, we first identified that IFNs: Type I interferons; ISG: Type I interferon-stimulated gene; siRNA: small suppression of IFI44L leads to the activation of Met/Src interfering RNAs pathway. Thus, the phenomenon that suppression of Funding IFI44L promotes cancer stemness, migration, invasion, This study was funded by grants MOST 103–2314-B-442-002-MY3 and MOST and pulmonary metastasis in HCC cells and overexpres- 106–2314-B-442-001-MY3 from Ministry of Science and Technology, Taiwan; RB17004 from Show Chwan Memorial Hospital, Taiwan. The funding bodies sion of IFI44L results in restoring chemosensitivity had no role in the design of the study and collection, analysis, and observed in our study might be regulated via affecting interpretation of data and in writing the manuscript. Met/Src signaling pathway. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conclusion Our study has demonstrated that IFI44L as a novel tumor Authors’ contributions suppressor in HCC through perturbation of Met/Src sig- Conception and design: WCH and PYC. Development of methodology: WCH and SLT. Acquisition of data: WCH, SLT, and PYC. Analysis and interpretation naling. Clinical relevance of low expression of IFI44L with of data: WCH, PMC, SLT and YLC. Study supervision: PYC. All authors read larger tumor size, disease relapse, advanced stages, and and approved the final manuscript. poor outcomes in HCC patients was also first identified. The IFI44L could serve as a prognostic biomarker and a Ethics approval and consent to participate Ethics approval was obtained from the Changhua Christian Hospital (CCH promising therapeutic target in the treatment of HCC. IRB No. 120504), Taiwan. The Written informed consent was provided by participants to be included in the study. The animal experiment protocols (NHRI-IACUC-104045A) were reviewed and approved by the Institutional Additional files Animal Care and Use Committee of National Health Research Institutes. Competing interests Additional file 1: Table S1. siRNA sequences and qRT-PCR primers used The authors declare they have no conflicts of interest. in this study. (TIF 523 kb) Additional file 2: Figure S1. The protein expression levels as reflected by Western blotting of IFI44L in Hep3B and HepG2 cells transfected with Publisher’sNote the IFI44L expression vector are shown. The actin was used as an internal Springer Nature remains neutral with regard to jurisdictional claims in control. Relative band intensity was quantified by ImageJ 1.42 (Windows published maps and institutional affiliations. version of NIH Image,http://rsb.info.nih.gov/ij/) and was represented with normalized mean ± s.e. (n = 3) below each band. (TIF 105 kb) Author details Graduate Institute of Integrated Medicine, China Medical University, Additional file 3: Figure S2. Dose-dependent growth inhibition of Taichung, Taiwan. Department of Hematology and Oncology, Ton-Yen Hep3B and HepG2 cells upon continuous exposure to the indicated General Hospital, Hsinchu, Taiwan. School of Medicine, Kaohsiung Medical concentrations of doxorubicin for 48 h was measured by MTT assay. University, Kaohsiung, Taiwan. Department of General Surgery, Changhua Cells were transfected with 20 nM of control (NC-siRNA) or IFI44L-siRNA Christian Hospital, Changhua, Taiwan. Taiwan Agricultural Chemicals and (*, P < 0.05; **, P < 0.01). (TIF 160 kb) Toxic Substances Research Institute, Council of Agriculture, Taichung, Taiwan. Additional file 4: Figure S3. Western blotting analysis of three different School of Medicine, College of Medicine, Fu Jen Catholic University, New siRNAs against IFI44L in HepG2 and PLC cells. The actin was used as an Taipei, Taiwan. Department of Pathology, Show Chwan Memorial Hospital, internal control. Relative band intensity was quantified by ImageJ 1.42 No.542, Sec.1, Chung-Shang Road, Changhua City, Changhua County 50008, and was represented with normalized mean ± s.e. (n = 3) below each Taiwan, Republic of China. National Institute of Cancer Research, National band. (TIF 168 kb) Health Research Institutes, Tainan, Taiwan. Huang et al. 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IFI44L is a novel tumor suppressor in human hepatocellular carcinoma affecting cancer stemness, metastasis, and drug resistance via regulating met/Src signaling pathway

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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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Abstract

Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The disease recurrent rate is relatively high resulted in poor 5-year survival in advanced HCC. Cancer stem cells (CSCs) have been considered to be one of the main mechanisms for chemoresistance, metastasis, and recurrent disease. Interferon-induced protein 44-like (IFI44L) gene is a type I interferon-stimulated gene (ISG) and belongs to the IFI44 family. Previous reports indicated antiviral activity against HCV in IFI44L, however, its precise role and function in HCC has not been unveiled. Methods: To explore the characteristics of hepatic CSCs, we successfully enriched hepatic cancer stem-like cells from three established liver cancer cell lines (Hep3B, HepG2, and PLC lines). Parental Hep3B and HepG2 cells and their sphere cells were treated with doxorubicin for 48 h and cell viability was measured by MTT assay. HCC tissue blocks from 217 patients were sampled for tissue microarray (TMA). Follow-up information and histopathological and clinical data including age, gender, tumor grade, advanced stages, HBV, HCV, tumor number, tumor size, relapse-free survival, and overall survival were obtained from the cancer registry and medical charts. The liver TMA was evaluated for IFI44L expression using immunohistochemical staining and scores. Results: These hepatic cancer stem-like cells possess important cancer stemness characteristics including sphere- forming abilities, expressing important HCC cancer stem cell markers, and more chemoresistant. Interestingly, we found that overexpression of IFI44L decreased chemoresistance towards doxorubicin and knockdown of IFI44L restored chemoresistance as well as promoted sphere formation. Furthermore, we found that depletion of IFI44L enhanced migration, invasion, and pulmonary metastasis through activating Met/Src signaling pathway. Clinically, the expression level of IFI44L significantly reduced in HCC tumor tissues. Low expression of IFI44L levels also correlated with larger tumor size, disease relapse, advanced stages, and poor clinical survival in HCC patients. (Continued on next page) * Correspondence: chu.peiyi@msa.hinet.net Wei-Chieh Huang and Shiao-Lin Tung contributed equally to this work. School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei, Taiwan Department of Pathology, Show Chwan Memorial Hospital, No.542, Sec.1, Chung-Shang Road, Changhua City, Changhua County 50008, Taiwan, Republic of China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Cancer (2018) 18:609 Page 2 of 10 (Continued from previous page) Conclusion: Taken together, we first demonstrated that IFI44L is a novel tumor suppressor to affect cancer stemness, metastasis, and drug resistance via regulating Met/Src signaling pathway in HCC and can be serve as an important prognostic marker. Keywords: IFI44L, Cancer stem cells, Hepatocellular carcinoma Background (HGF)/Met signaling in colorectal cancer and may serve Liver cancer is the fifth most common cancer worldwide as aprognostictumor marker [21]. IFN-α activates STAT signaling and downregulates Met in primary hu- and the second leading cause of cancer-related death worldwide [1]. In primary liver cancers, most (70 to 90%) man hepatocytes was also reported [22]. Blocking the cancers are hepatocellular carcinoma (HCC) [1]. The HGF/Met pathway by Met inhibitors or monoclonal antibodies strongly inhibits tumor growth and tumori- treatment efficacy of HCC is rather low mainly due to chemoresistance and metastasis which resulted in poor genicity in many malignancies including HCC [23]. Met 5-year survival of less than 5% in advanced HCC [2]. Can- has been known that is an upstream regulator of mul- tiple pathways, including PI3K/Akt, Ras/MAPK, Src/ cer stem cells (CSCs) are considered to be one of the main Stat3, and NF-κB[22]. In liver cancer, many studies mechanisms of chemoresistance and metastasis [3–5]. Hepatic CSCs have been identified and isolated from have demonstrated that Met overexpression is associ- ated HCC in previous reports [2, 6–8]. To elucidate potential with the development of distant metastases and a targetable molecular markers as well as signaling pathways shorter metastasis-free survival [23]. Consequently, Met of hepatic CSCs will be helpful in improving treatment ef- activation is considered to be crucial for the acquisition ficacy in HCC. of metastatic potential and the correlation between Met Hepatitis B virus (HBV) or Hepatitis C virus (HCV) pathway and ISGs warrants further study. are hepatotropic, noncytopathic DNA viruses that cause In this study, we successfully enriched hepatic cancer acute and chronic necroinflammatory liver diseases and stem-like cells and first identified that overexpression hepatocellular carcinoma [9]. Type I interferons (IFNs) are of IFI44L significantly reduces the chemoresistance to- pro-inflammatory cytokines that activate JAK-STAT signal- wards doxorubicin and knockdown of IFI44L promotes ing pathways leading to transcription of IFN-stimulated sphere formation in HCC cells.Furthermore,wefound genes(ISGs)toprotectcellsagainstinvading viralpatho- that depletion of IFI44L expression promotes migra- gens including HBV and HCV [10–14]. Although hundreds tion, invasion, and pulmonary metastasis in HCC cells. of ISGs have been identified for the past decades, only a We first demonstrated that suppression of IFI44L leads few have been characterized with antiviral activity. Using an to activation of Met/Src pathway. We also first identi- overexpression screening approach, 380 human ISGs in- fied that the expression of IFI44L decreased in tumor cluding interferon-induced protein 44-like (IF144L) gene tissues and correlated with several poor clinical out- were tested for their abilities to suppress the replication of comes in HCC patients. Our data demonstrated that viruses [15]. IFI44L is a potent negative regulator of Met/Src signal- IFI44L is a type I ISG and belongs to the IFI44 family ing pathway in modulating HCC cancer stemness and [16]. The IFI44L protein is 452 amino acid long, an ap- drug resistance and may serve as an important prog- proximately 47 kDa protein, and located on chromosome nostic marker. 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 Methods functions of miR-9 in some cancers are recently impli- Patients cated in regulating proliferation, invasion, metastasis, epi- 217 HCC tissue microarray slides were obtained from thelial–mesenchymal transition (EMT), apoptosis, and HCC patients receiving surgeries in Changhua Christian tumor angiogenesis [17–19]. A previous study reported Hospital from July 2011 to November 2013 [24]. that overexpression of miR-9 significantly upregulated the Paraffin-embedded HCC samples were obtained from expression of a lot of ISGs including IFI44L in nasopha- Changhua Christian Hospital under the approved Insti- ryngeal carcinoma cells [20]. These studies indicated the tutional Review Board (IRB) protocol. Clinical patterns promising role of IFI44L not only in anti-viral aspects but and overall survival data were analyzed by SPSS software also in cancer treatment. and chart review. The age of all patients was between An earlier report documented that a novel ISG, BATF2, twenty-nine and eighty-one years. The clinical character- as potent negative regulator of hepatocyte growth factor istics of these 217 patients are shown in Table 1. Huang et al. BMC Cancer (2018) 18:609 Page 3 of 10 Table 1 Relationship between clinical parameters and IFI44L anti-IFI44L (Abcam), p-Met (Cell signaling, Tyr1234/ expression in hepatocellular patients 1235), Met (Cell signaling), Src (Cell signaling) and p-Src IFI44L (Cell signaling, Tyr416). IFI44L-specific siRNAs were pur- chased from MDBio, Inc. Detailed sequences for IFI44L Variables N Low High p-value siRNA oligonucleotides were shown in Additional file 1: Age (years) Table S1. For cell sensitivity assays, HCC cells were < 65 100 49 (49%) 51 (51%) 0.892 pretreated with doxorubicin (Sigma-Aldrich) for 18 h ≧65 117 56 (48%) 61 (52%) (overnight) in serum-free culture medium. Gender Female 58 35 (60%) 23 (40%) 0.306 RNA extraction and qRT-PCR Male 159 75 (47%) 84 (53%) Quantitative RT-PCR (qRT-PCR) was used for gene detec- tion. Detailed procedure of reverse transcription reaction Differentiation was described elsewhere [25]. qRT-PCR was performed on Well 12 3 (25%) 9 (75%) 0.892 a CFX96 qPCR detection system (Bio-Rad) with a 1:10 Moderate 105 55 (52%) 50 (48%) dilution of cDNA by using KAPA SYBR FAST qPCR Kits Poor 94 47 (50%) 47 (50%) (KAPA Biosystems). The mRNA levels were normalized Undifferentiation 6 1 (17%) 5 (83%) to actin mRNA. The primers used for mRNA expression Stage are listed in Additional file 1: Table S1. I, II 180 84 (47%) 96 (53%) 0.029 Sphere-forming assay III, IV 37 25 (68%) 12 (32%) Monolayer cells of three HCC cell lines (Hep3B, HepG2 Hepatitis B surface antigen and PLC cells) were cultured in a stem cell selective Negative 106 52 (49%) 54 (51%) 0.892 condition described previously to obtain spheres [5]. Positive 111 56 (51%) 55 (49%) Spheres comprised at least five cells were calculated by Hepatits C virus visual counts according to a previous report [26]. Negative 150 74 (49%) 76 (51%) 0.883 Cell proliferation assay Positive 67 34 (51%) 33 (49%) The cell proliferation assay was measured by MTT assay Tumor Number (Promega, Madison, WI, USA). The assay was performed Single 177 87 (49%) 90 (51%) 0.841 according to the manufacture’s protocol. Briefly, cells Multiple 40 21 (53%) 19 (47%) (with density around 3 X 10 per well) were seeded in Tumor size 96-well plates and were incubated for 24 h. Cells were subsequently treated with various concentrations of < 5 cm 140 59 (42%) 81 (58%) 0.002 doxorubicin and then were incubated for 48 h. Viable ≧5 cm 77 49 (64%) 28 (36%) cells with active metabolism converted MTT into a for- Relapse mazan product, the quantity of which was measured at a - 196 91 (46%) 105 (54%) 0.002 wave length of 490 nm with 96-well plate reader and + 21 17 (81%) 4 (19%) was directly proportional to the number of viable cells. The drug concentration required to reduce proliferation Cell culture by 50% is defined as IC . All the experiments were per- The human liver cancer cell lines Hep3B (ATCC number: formed in triplicates and repeated three times. HB-8064), HepG2 (ATCC number: HB-8065) and PLC (ATCC number: HB-8024) were obtained from the Ameri- Cell chemotatic migration and invasion assay can Type Culture Collection (ATCC, Manassas, VA). All Migration and invasion abilities of HCC cells were carried cells were cultured at 37 °C under 5% CO in Dulbecco’s out using the Falcon Cell Culture Inserts with or without modified Eagle medium (DMEM; Invitrogen) supple- Matrigel (BD Biosciences) coating as described previously mented with 10% fetal bovine serum (FBS; Biological [27]. Detailed procedures were described elsewhere [25]. Industries) and 100 units/ml of penicilium and strepto- mycin (Life Technologies, Carlsbad, CA, USA). In vivo metastasis assays Hep3B Cells (1 × 10 ) with indicated treatments were sus- Vectors, antibodies, and reagents pended in phosphate-buffered saline (PBS) and were For IFI44L-expressing vector, IFI44L coding sequence was injected individually into the tail vein of 6- to 8-week-old amplified and cloned in pMSCV plasmid. Antibodies for C.B-17 severe-combined immunodeficient (CB17-SCID) western blotting and immunohistochemistry (IHC) are mice. All mice were monitored meticulously and were Huang et al. BMC Cancer (2018) 18:609 Page 4 of 10 sacrificed after 40 days of implantation. Tumor growth cell selective condition described in ‘Methods’ to form was observed by live animal BLI (Caliper IVIS system, spheres. Most of the suspended cells underwent apop- PerkinElmer). tosis during the first 2 days of culturing, and the rest of survived cells gradually formed floating spheres. The Immunohistochemistry (IHC) spheres grew larger and often reached to 50–100 μMin IHC was performed to detect IFI44L expression from diameter after 4–8 days (Fig. 1a). Overexpression of paraffin-embedded HCC specimens. The slides were mRNA of HCC cancer stem cell markers was found in. stained with anti-IFI44L antibody (Bethyl Labs, Mont- Hep3B sphere cells compared with their parental cells. gomery, TX, USA) [28]. The IFI44L antibody was pur- These cancer stem cell markers, including CD24, CD44, chased from ThermoFisher (Rock, USA). In liver cancer CD117, CD133, ALDH, ABCG2, OCT4, and Nanog, specimens, the detailed scores for IHC were defined as were significantly higher in Hep3B sphere cells shown described previously [24, 29]. by qRT–PCR analysis (Fig. 1b)[7, 30–37]. Next, we examined the chemosensitivity of these sphere Statistical analysis cells. Parental Hep3B and HepG2 cells and their sphere The SPSS software (Version 13.0 SPSS Inc., Chicago, IL, cells were treated with doxorubicin for 48 h and cell USA) was used to conduct Chi-square analysis and viability was measured by MTT assay. Hep3B and HepG2 paired-samples t-test. Kaplan-Meier method was performed sphere cells are found to be more chemoresistant to con- for analyzing survival data. Variables related to survival tinuous exposure to various concentrations of doxorubicin were analyzed using Cox’s proportional hazards regression (Fig. 1c). Thus, we have successfully enriched HCC cancer model via SPSS software. Differences between experimental stem-like cells from Hep3B, HepG2, and PLC lines groups were calculated using the Mann–Whitney U test. displaying cancer stem cell characteristics including Differences with P values of < 0.05 are considered statisti- sphere-forming, expression of HCC cancer stem cell cally significant. markers, and more chemoresistant in accordance with established parameters of cancer stem-like cells [38–40]. Results Successful enrichment of human HCC cancer stem-like Overexpression of IFI44L restores chemosensitivity and cells from Hep3b, HepG2, and PLC lines knockdown of IFI44L promotes sphere formation In order to enrich for CSCs, parental Hep3B, HepG2, Since IFI44L was implied to be correlated with cancer and PLC cells from monolayer were cultured in a stem [20], we then investigated the impact of IFI44L on drug Fig. 1 HCC cancer stem-like cells were successfully enriched from Hep3B, HepG2, and PLC cell lines. a Formation of spheres under the stem cell selective condition on day 8 after culturing from parental Hep3B, HepG2, and PLC cells is shown. b The mRNA expression levels of HCC cancer stem cell markers in parental Hep3B cells and their sphere cells were analyzed by qRT–PCR with actin as an internal control. Histograms represent means ± s.d. from three independent experiments (*, P < 0.05; **, P < 0.01). c Dose-dependent growth inhibition of parental Hep3B and HepG2 cells and their sphere cells upon continuous exposure to the indicated concentrations of doxorubicin for 48 h was measured by MTT assay. Each dosage point represents the mean ± s.e. from three independent experiments (*, P < 0.05; **, P < 0.01) Huang et al. BMC Cancer (2018) 18:609 Page 5 of 10 resistance. Cells transfected with IFI44L expression plas- Next we tested whether sphere-forming ability of Hep3B, mid or control plasmid were tested their protein expres- HepG2, and PLC lines could be promoted by knockdown sion of IFI44L to confirm the transfection efficiency. of IFI44L. After 8 days culturing of Hep3b, HepG2, and Western blotting showed upregulation of IFI44L protein PLC cells in the stem cell selective condition, sphere num- level in Hep3B and HepG2 cells after transfection with ber was calculated by visual counting under microscope. the expression plasmid of IFI44L (IFI44L vector) (Add- Knockdown of IFI44L caused significant increase of itional file 2: Fig. S1). Our data indicated that Hep3b and sphere number (Fig. 2d). Thus, our data suggested that HepG2 cells became more chemosensitive to continuous IFI44L may play as a tumor suppressor role in restoring exposure to different doses of doxorubicin after transfec- chemosensitivity and affecting cancer stemness. tion with IFI44L vector (Fig. 2a), whereas IFI44L knock- down restored their chemoresistance (Additional file 3: Depletion of IFI44L expression promotes migration, Figure S2). These data suggested that overexpression of invasion and pulmonary metastasis and implicates in IFI44L significantly decreased chemoresistance of HCC met/Src signaling pathway in HCC lines towards doxorubicin. To assess whether IFI44L Furthermore, we evaluate the tumor suppressor role of level correlated with cancer stemness in HCC, we exam- IFI44L in regulating cancer metastasis. In Boyden cham- ined the protein expression level of IFI44L in HCC lines. ber assay, we found that depletion of IFI44L expression Decrease of IFI44L protein level in Hep3b and HepG2 significantly promotes Hep3B, HepG2 and PLC cell mi- sphere cells was found compared with their parental gration and invasion abilities (Fig. 3a and Additional file 5: cells by Western blotting analysis (Fig. 2b). We then Figure S4). To investigate whether IFI44L regulated can- investigated if suppression of IFI44L by its small interfer- cer cell metastasis in vivo, we employed an experimental ing RNAs (siRNA) could inhibit cancer stemness charac- metastasis model via tail vein injection in SCID mice. In teristics in HCC lines. Three specific IFI44L-siRNAs this model, knockdown of IFI44L significantly promoted were tested for their inhibitory efficacy by analyzing the lung metastasis of Hep3B cells compared with the IFI44L protein levels in Hep3B, HepG2 and PLC cells, control group (Fig. 3b). Since ISGs are implied to be cor- IFI44L-siRNA-2 showed the highest knockdown effect in related with Met pathway [11, 22, 23], we then explored inhibiting IFI44L protein and it was used in the subse- the role of IFI44L in Met signaling pathway. By Western quent experiments (Fig. 2c, Additional file 4: Figure S3). blotting analysis, we found that suppression of IFI44L Fig. 2 The effects of IFI44L on drug resistance and sphere formation. a Dose-dependent growth inhibition of Hep3B and HepG2 cells upon continuous exposure to the indicated concentrations of doxorubicin for 48 h was measured by MTT assay (*, P < 0.05; **, P < 0.01). Cells were transfected with 1 μg of pMSCV or pMSCV-IFI44L expression plasmids (IFI44L vector). b The expression levels of IFI44L in parental Hep3B and HepG2 cells and their sphere cells were measured by Western blotting. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 (Windows version of NIH Image, http://rsb.info.nih.gov/ij/) and was represented with normalized mean ± s.e. (n =3) below each band. c Western blotting analysis of three different siRNAs against IFI44L in Hep3B cells. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band. d Sphere formation under stem cell selective condition was examined on day 8 after culturing of the cells transfected with the indicated IFI44L-siRNA. The original magnification was 40X. Histograms represent means ± s.d. from three independent experiments (*, P < 0.05, **, P < 0.01) Huang et al. BMC Cancer (2018) 18:609 Page 6 of 10 Fig. 3 IFI44L functions as a tumor suppressor affecting metastasis implicates with Met/Src signaling. a Analysis of the effect of IFI44L on Hep3B and HepG2 cell migration and invasion using Boyden chamber assay. Quantitative data are shown by histograms and representative photographs of the migrated/invaded cells from different treatments are shown. b Representative xenograft tumors formed by 5 × 10 Hep3B sphere cells in the SCID mice. Tumor growth was monitored by BLI. Representative BLIs are shown on day 30 after implantation. c The protein expression levels of the signaling components of the Met/Src signaling in Hep3B and HepG2 cells are shown by Western blotting. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band enhances the phosphorylation of Met and Src in significantly higher in normal liver tissues compared with Hep3B and HepG2 cells (Fig. 3c). To further assess tumor tissues (Fig. 4a). Western blotting analysis also the role of IFI44/Met/Src axis in regulating cancer revealed that all of ten pairs of matched HCC tumor metastasis, we performed additional Western blotting tissues expressed lower level of IFI44L in comparison with analysis as well as migration and invasion assay. We the matched normal tissues (Fig. 4b). Downregulation of found that overexpression of IFI44L decreased phos- IFI44L expression found in HCC tumor tissues is compat- phorylation of Met as well as migration and invasion ible with the tumor suppressor role in HCC we discovered abilities in Hep3B cell line, whereas ectopic expression above. of Met reversed IFI44L-mediated inhibition of migration and invasion abilities approximately 50% (Additional file 6: Dowregulation of IFI44L expression levels significantly Figure S5). Taken together, these findings reinforced correlated with larger tumor size, disease relapse, that the functional role of IFI44L as a tumor suppressor advanced stages, and poor clinical survival in HCC and it could implicate in Met/Src signaling pathway patients in HCC. Furthermore, the correlation between clinicopathological characteristics and IFI44L of these 217 patients were an- The expression level of IFI44L significantly decreased in alyzed in Table 1. Among these parameters, age, gender, HCC tumor tissues tumor differentiation, HBV surface antigen, anti-HCV To evaluate the correlation of IFI44L with clinical sam- antibody, and the tumor number were not significantly ples, the expression of IFI44L in 217 pairs of normal liver different in patients with low versus high expression and HCC tumor tissues were analyzed by IHC and West- levels of IFI44L (Table 1). However, low expression of ern blotting analysis. The IHC score of IFI44L was IFI44L was observed in only 47% (84/180) of the early Huang et al. BMC Cancer (2018) 18:609 Page 7 of 10 Fig. 4 The expression level of IFI44L decreased in HCC tumor tissues. a The level of IFI44L was examined by IHC staining in 217 pairs of HCC tumor tissues and their adjacent normal tissues (**, P < 0.01). b Images of Western blotting analyses of IFI44L protein level in ten matched pairs of HCC tumor tissues and adjacent normal tissues. The actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. (n = 3) below each band stages (stage I/II) HCC patents whereas 68% (25/37) of cells. In clinic data, we also found that patients with low the late stages (stage III/IV) HCC patients expressed low expression level of IFI44L had significantly higher relapse levels of IFI44L (P = 0.029) (Table 1). IHC staining also rate (81% vs 19%, P = 0.002) and shorter relapse-free sur- confirmed that IFI44L protein level decreased markedly vival (RFS) (p = 0.0012) than patients with high expression in advanced stages in HCC samples (Fig. 5a). Moreover, level of IFI44L (Table 1,Fig. 5b). higher percentage of HCC patients with low expression In survival analysis, the influence of clinicopathologi- level of IFI44L had larger tumor size then patients with cal characteristics including IFI44L on patients’ overall high expression level of IFI44L (64% vs 36%, P = 0.002) survival (OS) was statistically examined by univariate (Table 1). Since CSCs are indicated to be associated with analysis shown in Table 2. Four parameters including cancer recurrence [2, 38], our previous experiments also advanced stages, larger tumor size, disease relapse, and indicated that IFI44L affects cancer stemness in HCC low expression of IFI44L are significant correlated with Fig. 5 The expression level of IFI44L correlates with clinical staging, RFS, and OS in HCC patients. a Representative examples of the expression levels of IFI44L protein determined by IHC of clinical specimens. b The mRNA expression level of IFI44L correlates with RFS in 217HCC patients. c The mRNA expression level of IFI44L correlates with OS in 217 HCC patients Huang et al. BMC Cancer (2018) 18:609 Page 8 of 10 Table 2 Univariate analysis of influence of clinical characteristics alcohol injection, and liver transplantation [9, 41–43]. on overall survival in hepatocellular patients However, the probability of disease recurrence is around OS 50% within 3 years after successful treatment [2]. Hepatic CSCs exhibit multidrug and radio-resistant properties and Characteristics N Median survival Survival Log-rank (months) (%) are considered as in part the main mechanism of Age (years) chemoresistance and recurrent disease [2, 4]. In our study, we successfully enriched cancer stem-like cells < 65 100 36.83 75.00% 0.282 via sphere-forming method in nonadhesive culture ≧65 117 41.42 80.34% plates with serum-free culture medium from three hepatic Gender cancer cell lines. These cancer stem-like cells express Female 58 38.37 76.27% 0.631 important hepatic CSC markers such as CD24, CD44, Male 159 40.49 78.48% CD117, CD133, ALDH, ABCG2, Oct4, and Nanog which Differentiation were extensively reported before [7, 30–37]. They also reveal significant chemoresistance towards doxorubicin in Well, Moderate 117 40.69 78.45% 0.731 accordance with previous reports [2]. To find specific Undifferentitation, Poor 100 39.26 77.23% molecules to target these cancer stem-like cells would be Stage very important in treating HCC. I, II 180 42.22 82.49% < 0.001 Type I IFNs are a family of cytokines to directly III, IV 37 30.13 57.50% activate the transcription of ISGs to exert anti-viral, Hepatitis B surface antigen anti-proliferative, and immunomodulatory activities [10, 11]. IFI44L, one of the type I ISG, exhibits a low Negative 106 40.33 79.25% 0.617 antiviral activity against HCV and is indicated to be Positive 111 39.08 76.58% correlated with some cancer recently although the reports Hepatits C virus are scarce [12, 20, 44]. In present study, our data showed Negative 150 39.14 76.67% 0.489 that overexpression of IFI44L restores chemosensitivity Positive 67 41.1 80.60% towards doxorubicin whereas decreased expression of Tumor Number IFI44L promotes sphere formation in HCC cell lines. Depletion of IFI44L also enhanced migration, invasion, Single 177 40.25 77.78% 0.926 and lung metastasis in HCC cells. According to the above Multiple 40 37.17 78.38% results, IFI44L was proposed as a novel tumor suppressor Tumor size modulating cancer stemness, drug resistance, migration < 5 cm 140 49.79 86.43% < 0.001 and invasion, as well as pulmonary metastasis in HCC. ≧5 cm 77 33.12 62.34% Although one recent study indicated that upregulation of Relapse IFI44L was significantly correlated with shorter overall survival and shorter median survival time in pancreatic - 196 41.31 81.54% < 0.001 ductal adenocarcinoma [44], our data revealed that low + 21 28.79 45.00% expression of IFI44L was found in HCC tumor samples and was correlated with larger tumor size, more disease shorter median OS (P <0.001) (Table 2). Kaplan–Meier relapse, advanced stages as well as significant poorer RFS survival analysis of these 217 patients also revealed that and OS. Although some study identified that IFI44L low expression level of IFI44L correlated with poor OS overexpressed in pancreatic ductal adenocarcinoma and (P < 0.001) (Fig. 5c). These results suggested that down- correlated with worse clinical prognosis, this conclusion is regulation of IFI44L expression levels significantly only made from statistics of databases collecting from correlated with larger tumor size, disease relapse, ad- gene expression profiling and TCGA database but lacks in vanced stages, and poor clinical survival in HCC patients vitro and in vivo experimental confirmation [44]. The and could serve as an important prognostic marker. functional role of IFI44L in different cancers still warrants further study. Discussion In advanced stages of HCC, conventional chemother- HCC has been a global health problem with rising inci- apy such as doxorubicin, cisplatin, and 5-fluorouracil dence in Western countries recently [1]. In the West, were generally introduced but the response rate was around 40% of patients are diagnosed as early Barcelona very low (from 15 to 20%) and these chemotherapeutic Clinic Liver Cancer (BCLC) stages and are eligible for agents failed to prolong survival [2, 41]. Sorefenib, a potential curative treatment such as surgical resection, ra- small molecule multikinase inhibitor that inhibits diofrequency ablation, microwave ablation, percutaneous tumor-cell proliferation and tumor angiogenesis, is the Huang et al. BMC Cancer (2018) 18:609 Page 9 of 10 first targeted therapy to reveal survival benefit in pa- Additional file 5: Figure S4. Analysis of the effect of IFI44L on PLC cell tients with advanced HCC [41]. Other new molecular migration and invasion using Boyden chamber assay. Quantitative data are shown by histograms and representative photographs of the pathways including. migrated/invaded cells from different treatments are shown. Histograms Ras/Raf/MEK/ERK (MAPK) pathway, wnt/catenin represent means ± s.d. from 3 independent experiments (**, P < 0.01). pathway, PI3K/Akt/mTOR pathway, VEGF pathway, and (TIF 252 kb) HGF/Met pathway etc. were extensively explored in Additional file 6: Figure S5. Ectopic expression of Met significantly restored IFI44L expression-mediated inhibition of migration and invasion HCC patients [9, 23, 45]. The efficacy of new targeted abilities. Left, overexpression of IFI44L reduced the phosphorylation of therapies such as lenvatinib, nivolumab, ramucirumab, Met, which could be partially rescued by transfecting Met vector. The tivantinib, and cabozantinib etc. are still under evalu- actin was used as an internal control. Relative band intensity was quantified by ImageJ 1.42 and was represented with normalized mean ± s.e. ation in large clinical trials [9]. Of the above mentioned (n = 3) below each band. Right, the migration and invasion abilities pathways, the HGF/Met pathway has been implicated in affected by overexpression of IFI44L and ectopic expression of Met in tumor cell migration, invasion, proliferation, and angio- Hep3B cell line. Quantitative data are shown by histograms and representative photographs of the migrated/invaded cells from genesis [23]. High expression of Met and HGF was re- different treatments are shown. Histograms represent means ± s.d. ported to be correlated with early recurrence of HCC from 3 independent experiments (*, P < 0.05; **, P < 0.01). (TIF 437 kb) after hepatectomy and shorter survival in HCC patients [23]. Several studies indicated that IFN regulates mul- Abbreviations tiple STAT signaling and downregulates Met resulting in CSCs: Cancer stem cells; EMT: Epithelial–mesenchymal transition; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; suppression of HGF-induced signals and cell prolifera- HGF: Hepatocyte growth factor; IF144L: Interferon-induced protein 44-like; tion [14, 22]. In our study, we first identified that IFNs: Type I interferons; ISG: Type I interferon-stimulated gene; siRNA: small suppression of IFI44L leads to the activation of Met/Src interfering RNAs pathway. Thus, the phenomenon that suppression of Funding IFI44L promotes cancer stemness, migration, invasion, This study was funded by grants MOST 103–2314-B-442-002-MY3 and MOST and pulmonary metastasis in HCC cells and overexpres- 106–2314-B-442-001-MY3 from Ministry of Science and Technology, Taiwan; RB17004 from Show Chwan Memorial Hospital, Taiwan. The funding bodies sion of IFI44L results in restoring chemosensitivity had no role in the design of the study and collection, analysis, and observed in our study might be regulated via affecting interpretation of data and in writing the manuscript. Met/Src signaling pathway. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Conclusion Our study has demonstrated that IFI44L as a novel tumor Authors’ contributions suppressor in HCC through perturbation of Met/Src sig- Conception and design: WCH and PYC. Development of methodology: WCH and SLT. Acquisition of data: WCH, SLT, and PYC. Analysis and interpretation naling. Clinical relevance of low expression of IFI44L with of data: WCH, PMC, SLT and YLC. Study supervision: PYC. All authors read larger tumor size, disease relapse, advanced stages, and and approved the final manuscript. poor outcomes in HCC patients was also first identified. The IFI44L could serve as a prognostic biomarker and a Ethics approval and consent to participate Ethics approval was obtained from the Changhua Christian Hospital (CCH promising therapeutic target in the treatment of HCC. IRB No. 120504), Taiwan. The Written informed consent was provided by participants to be included in the study. The animal experiment protocols (NHRI-IACUC-104045A) were reviewed and approved by the Institutional Additional files Animal Care and Use Committee of National Health Research Institutes. Competing interests Additional file 1: Table S1. siRNA sequences and qRT-PCR primers used The authors declare they have no conflicts of interest. in this study. (TIF 523 kb) Additional file 2: Figure S1. The protein expression levels as reflected by Western blotting of IFI44L in Hep3B and HepG2 cells transfected with Publisher’sNote the IFI44L expression vector are shown. The actin was used as an internal Springer Nature remains neutral with regard to jurisdictional claims in control. Relative band intensity was quantified by ImageJ 1.42 (Windows published maps and institutional affiliations. version of NIH Image,http://rsb.info.nih.gov/ij/) and was represented with normalized mean ± s.e. (n = 3) below each band. (TIF 105 kb) Author details Graduate Institute of Integrated Medicine, China Medical University, Additional file 3: Figure S2. Dose-dependent growth inhibition of Taichung, Taiwan. Department of Hematology and Oncology, Ton-Yen Hep3B and HepG2 cells upon continuous exposure to the indicated General Hospital, Hsinchu, Taiwan. School of Medicine, Kaohsiung Medical concentrations of doxorubicin for 48 h was measured by MTT assay. University, Kaohsiung, Taiwan. Department of General Surgery, Changhua Cells were transfected with 20 nM of control (NC-siRNA) or IFI44L-siRNA Christian Hospital, Changhua, Taiwan. Taiwan Agricultural Chemicals and (*, P < 0.05; **, P < 0.01). (TIF 160 kb) Toxic Substances Research Institute, Council of Agriculture, Taichung, Taiwan. Additional file 4: Figure S3. Western blotting analysis of three different School of Medicine, College of Medicine, Fu Jen Catholic University, New siRNAs against IFI44L in HepG2 and PLC cells. The actin was used as an Taipei, Taiwan. Department of Pathology, Show Chwan Memorial Hospital, internal control. Relative band intensity was quantified by ImageJ 1.42 No.542, Sec.1, Chung-Shang Road, Changhua City, Changhua County 50008, and was represented with normalized mean ± s.e. (n = 3) below each Taiwan, Republic of China. National Institute of Cancer Research, National band. (TIF 168 kb) Health Research Institutes, Tainan, Taiwan. Huang et al. 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BMC CancerSpringer Journals

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