Cancers, Vol. 14, Pages 5781: Canonical WNT Signaling Activated by WNT7B Contributes to L-HBs-Mediated Sorafenib Resistance in Hepatocellular Carcinoma by Inhibiting Mitophagy

1. IntroductionDue to its increased prevalence and rapid progression, hepatocellular carcinoma (HCC) is the world’s sixth most prevalent malignancy and the third most lethal cancer. HCC accounts for 8.2% of all tumor-related deaths worldwide [1]. Hepatitis B virus (HBV) is one of seven viruses recognized as Group 1 human carcinogens according to the evaluation of the International Agency for Research on Cancer (IARC) [2]. Chronic HBV infection is responsible for at least half of all HCC cases [3]. HBV has a 3.2 kb DNA genome that is largely double-stranded and contains four open reading frames that encode HBV surface antigen (HBs), core antigen (HBc), e antigen (HBe), polymerase (P), and X protein (HBx). HBs is synthesized in three forms: large (L-HBs), medium (M-HBs), and small (S-HBs), all of which surround an HBc nucleocapsid [4]. Serum HBs levels have been linked to HCC risk in patients with low viral loads [5,6].Systemic chemotherapy is the main treatment strategy for advanced HCC. However, tolerance to chemotherapy drugs finally leads to poor prognosis in all patients [7]. Sorafenib, a multikinase inhibitor that promotes apoptosis while inhibiting tumor-cell proliferation and angiogenesis, has been licensed for the treatment of advanced liver cancer since 2007 [8]. It is an effective first-line targeted therapy for late-stage HCC, with a median overall survival of 10.7 months. However, after 18 months, the majority of patients developed sorafenib resistance [8,9]. A phase III randomized controlled study of sorafenib discovered that HBV-positive HCC patients had a worse survival benefit than HBV-negative patients [10], showing that HBV may play a vital role in a lower likelihood of survival caused by sorafenib resistance. Mechanism studies showed that HBx and HBc were correlated to chemoresistance [11,12]. However, it is unknown what role HBs play in the development of HCC and sorafenib resistance.The WNT signaling pathway, which encompasses the CTNNB1-dependent (canonical) and CTNNB1-independent (non-canonical) pathways, is hyperactivated in HCC [13]. WNT7B, a WNT ligand, is involved in a number of cancers and contributes to drug resistance in several types of malignancies by activating canonical WNT signaling, including gemcitabine resistance in pancreatic cancer, cis-platinum resistance in cervical cancer, and doxorubicin resistance in osteosarcoma [14,15,16]. Whether WNT7B is involved in sorafenib resistance in HBV-associated HCC via canonical WNT signaling is uncertain.Mitophagy, a type of autophagy, regulates mitochondrial homeostasis in response to cellular stresses [17]. The outer mitochondrial membrane kinase ubiquitin-dependent PTEN-induced putative kinase 1 (PINK1) is triggered by mitochondrial depolarization and recruits the cytosolic ubiquitin E3 ligase PARKIN to damaged mitochondria, resulting in mitophagy activation [18]. Sorafenib treatment has been demonstrated to generate mitochondrial stress, which induces mitophagy in order to degrade sorafenib-damaged mitochondria [19,20]. Nonetheless, the significance of mitophagy in HBV-HCC sorafenib resistance is unknown.

In this study, WNT7B was discovered to be overexpressed in HBV-associated HCC tissues and cell lines. L-HBs enhanced the expression of WNT7B and its receptor frizzled-4 (FZD4) in HCC cells. Furthermore, L-HBs activated CTNNB1/TCF-dependent proto-oncogene transcription via WNT7B/FZD4. The CCK-8 assay, foci formation assay, wound healing assay, and Transwell assay all demonstrated that WNT7B played an oncogenic role in the development of HCC. Intriguingly, L-HBs reduced sorafenib-induced apoptosis by WNT7B-inhibited mitophagy. As a result, our findings revealed that L-HBs contributed to sorafenib resistance by decreasing mitophagy mediated by the WNT7B-activated canonical WNT signal pathway. WNT7B is a potentially valuable molecular candidate for HBV-related HCC therapy. It is also a target for predicting sorafenib resistance in HBV-related HCC. HBV activity, including not only HBV DNA but also HBV structural proteins such L-HBs, plays an anti-chemotherapy effect in HCC. As a consequence, for advanced HCC patients with chronic HBV infection, a comprehensive reduction in HBV replication, including the formation of HBV structural proteins, may be beneficial.

2. Materials and Methods 2.1. Bioinformatics AnalysisThe Gene Expression Omnibus (GEO) dataset GSE87630 [21], which contains 64 cases of HCC specimens with HBV infection and 30 cases of nontumoral surrounding tissue specimens, was searched for genes that were significantly dysregulated in HBV-related HCC. The platform for this work was GPL6947 (Illumina Human HT-12 V3.0 expression beadchip). The Limma package [22] of R software was used for all bioinformatics analyses, with the standard of p-value The survival analysis was carried out using an online Kaplan–Meier plotter [23]. STRING bioinformatics tool was used to predict interacting proteins [24]. 2.2. Clinical Tissues

A total of 169 HCC samples with chronic HBV infection and 17 normal liver tissues were collected. All tissue samples were obtained from the Renmin Hospital of Wuhan University (Wuhan, China). The sample collection was carried out in accordance with consensus agreements and was authorized by the ethics committee of Wuhan University, School of Medicine (Ethics No. 14011). The study adhered to the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS).

2.3. Immunohistochemistry (IHC)

The clinical samples were fixed with 4% paraformaldehyde. The samples were then embedded in paraffin and sectioned. WNT7B was stained with the primary rabbit polyclonal antibody (PA5-103480, Thermo Fisher Scientific, Sunnyvale, CA, USA), followed by HRP conjugated goat anti-rabbit IgG (31460, Thermo Fisher Scientific). Gene expression was graded on a scale of 0–3 as follows: a score of 0 indicates that there is no membranous staining in any of the tumor cells; a score of 1 indicates that there is less than 10% of the tumor cells with any intensity or less than 30% of the tumor cells with weak intensity; a score of 2 indicates that there is staining in 10–30% of the tumor cells with moderate-to-strong intensity or staining in 30–50% of the tumor cells with weak-to-moderate intensity; a score of 3 indicates that there is staining in more than 50%. Positive samples had a score of 2 or above.

2.4. Plasmids Construction

Full-length cDNAs of HBV structural proteins (L-HBs, S-HBs, HBc, HBe, HBp) and human WNT7B were PCR-amplified from HepG2.2.15 cells and cloned into the pcDNA3.1(-) vector as directed by the manufacturer (V795-20, Invitrogen, Burlington, ON, USA).

WNT7B and FZD4 were knocked down using particular short hairpin RNAs (shRNA). WNT7B was targeted by the sequences 5′-CCCGAUGCCA UCAUUGUGAUU-3′ (WNT7B#1) and 5′-CAACAAGAUUCCUGGCCUA-3′ (WNT7B#2). The target sequence for FZD4 was 5′-GGUGAUGAAGAGGU GCCCUU-3′. The shRNA-coding oligonucleotides were annealed and ligated into the pSilencer 2.1-U6 neo following the manufacturer’s instructions (AM5764, Ambion, Austin, TX, USA).

2.5. Cell Culture and Transient Transfection

The American Type Culture Collection (ATCC, Manassas, VA, USA) provided the HepG2, HepG2.2.15, Huh7, and HCCLM3 cells used in this work. HL-7702 was gifted by Professor Wenjie Huang at Huazhong University of Science and Technology. Cells were cultured in Dulbecco’s modified Eagle medium (2347432, Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate at 37 °C with 5% CO2. When cells reached 70–90% confluence, Lipofectamine 2000 was used for transient transfection (11668019, Invitrogen).

2.6. RNA Extraction and Real-Time Quantitative Reverse Transcriptase-PCR (qPCR)

Total RNA was isolated from cells using TRIzol Reagent (15596026, Invitrogen). After removing genomic DNA contaminations from total RNA using DNase I (EN0521, Thermo Fisher Scientific), 1 μg of RNA was converted into cDNA using ReverTra Ace (FSK-101, TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.

qPCR was carried out in a final volume of 20 µL on the iCycler system (C1000, Bio-Rad, Hercules, CA, USA) using the SYBR Green master mixture (04913914001, Roche, Basle, Switzerland). To standardize the gene expression data, β-actin was employed as an endogenous control. Supplementary Table S1 lists the primer sequences. The reactions were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 10 s, 72 °C for 10 s, and 60 °C for 1 min. The 2(−ΔΔCt) method for the relative quantification of gene expression was used to determine the gene expression levels. 2.7. Protein and Mitochondria Isolation

After cleaning the cells with 1× PBS buffer, they were lysed using M-PER mammalian protein extraction reagent (78501, Thermo Fisher Scientific) supplemented with a Cocktail (11873580001, Roche).

Mitochondria were isolated using a commercial mitochondrial extraction kit, as directed by the manufacturer (SM0020, Solarbio, Beijing, China). 5 × 107 cultivated cells were homogenized for mitochondria isolation. For mitochondria isolation, 100 μg fresh minced tissues from subcutaneous xenografts in nude mice were homogenized.

Protein concentrations were determined using the BCA protein assay kit (23250, Thermo Fisher Scientific) as directed by the manufacturer.

2.8. Western Blotting Analysis

Protein extracts (30 μg) were separated in 4–12% SDS-polyacrylamide gels (80 V, 2.5 h). The proteins were electrophoretically transferred from gels to nitrocellulose membranes (10600002, GE Healthcare Life Sciences, Amersham, UK) using the TRANS-BLOT SD SEMI DRY TRANSFER CELL (BIO-RAD, 22v, 24 min). The membranes were treated with the appropriate primary antibodies and fluorescent-conjugated secondary antibodies after blocking in 5% skim milk for 60 min. The specific primary antibodies were as follows WNT7B (A7746, 1:1000, ABclonal, Wuhan, China), CTNNB1 (A19657, 1:1000, ABclonal), FZD4 (A8161, 1:1000, ABclonal), c-MYC (A1309, 1:1000, ABclonal), CCND1 (A19038, 1:1000, ABclonal), PINK1 (A7131, 1:1000, ABclonal), PARKIN (A11172, 1:1000, ABclonal), LC3B (A19665, 1:1000, ABclonal), and VDAC1 (A15735, 1:1000, ABclonal). To normalize the expression levels of different proteins, an anti-β-actin-peroxidase monoclonal antibody (A3854, Sigma-Aldrich, St. Louis, MO, USA) was utilized as an internal control. ECL reagents (34080, Millipore, Burlington, MA, USA) were used to view the bands in a Tanon 5200 MultiImage System (Tanon Science & Technology, Shanghai, China). Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.9. TOPFlash Reporter Assay

The relevant plasmids were cotransfected with either the WNT signaling reporter TOPFlash (TCF reporter plasmid) or the negative control FOPFlash (mutant, inactive TCF binding site) (17-285, Millipore) according to the manufacturer’s procedure to assess the transcriptional activity of the canonical WNT pathway.

The indicated plasmids were transiently co-transfected in Huh7 or HCCLM3 cells with either 2 µg TOPFlash or FOPFlash, and 0.5 µg pSV40-Renilla plasmid (E6911, Promega, Madison, WI, USA) as an internal control for 48 h. The firefly and Renilla luciferase activity ratio was determined using the Dual-Luciferase reporter assay system (E1960, Promega).

2.10. Cell Counting Kit 8 (CCK-8) Assay

Cells were plated at a density of 5 × 103 cells per well in 96-well plates. Cell viability was assessed using the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) at the given time after transient transfection and (or) drug treatment. In brief, 10 μL CCK-8 reagents were supplied to the cells and incubated for 40 min–1.5 h at 37 °C. A Multiskan FC plate reader was used to measure absorbance at 450 nm (Thermo Scientific, USA).

2.11. Colony Formation Assay

Soft agar dishes were made with a 0.6% agar under-layer in DMEM supplemented by 10% FCS. In the same mixture containing 0.35% agar, 2 × 103 cells were plated in the up-layer. After 2–3 weeks of incubation at 37 °C with 5% CO2, the colonies were counted using a microscope (Olympus CH-40; Olympus, Tokyo, Japan).

2.12. Wound Healing Assay

In 6-well plates, cells were grown until 80% confluent. Cells were subjected to serum deprivation (DEME supplemented with 1% FBS) for a further 24 h following transfection with the relevant plasmids. The monolayer was then scratched with a 200 μL pipette tip to create the linear wounds. An Olympus CH-40 microscope was used to examine cell migration into the wound area 24 h after the wound was created. For each plate, four distinct equidistant spots were measured and averaged. The migration rate was computed for each plate as the ratio of the mean distance between both borderlines after closure to the mean distance observed at 0 h.

2.13. Transwell Assay

Cell invasion was measured using a 24-well Transwell with a pore membrane diameter of 8 μm (Costar, Rochester, NY, USA). Matrigel (BD Biosciences, San Diego, CA, USA) diluted to 200 μg/mL was applied to the chambers and incubated for 24 h at 37 °C. Transwell chambers were seeded with 2−6 × 104 cells in the serum-free medium. The bottom chamber contained 20% FBS medium. After 48 h of incubation, the cells that had migrated through the membrane were fixed, stained with crystal violet, and counted under an Olympus CH-40 microscope. Four fields in each membrane’s four quadrants were counted and averaged.

2.14. Sorafenib Treatment

Sorafenib (IS0220, Solarbio) was diluted in dimethyl sulfoxide (DMSO) to a concentration of 20 mmol/L. Sorafenib was added to the culture media in serial dilutions or at the stated concentrations either directly or after plasmid transfection. The cells were cultured for 1–3 days before being tested for IC50 using the CCK-8 assay. The cells were cultured for 24 h before being tested for additional detection.

2.15. Flow Cytometry

After the floating and detached cells were collected and combined, cells were treated by an Annexin V-FITC/propidium iodide apoptosis detection kit (ZP327-1, Beijing Zoman Biotechnology, Beijing, China) according to the manufacturer’s protocol. A total of 10,000 cells per sample were analyzed via Epics Altra II cytometer (Beckman Coulter, Miami, FL, USA). A Beckman Coulter Epics Altra with Expo32 software (Beckman Coulter) was used to measure cell apoptosis.

2.16. Subcutaneous Xenograft Experiment

The subcutaneous xenograft experiment was utilized to assess L-HBs and WNT7B’s anti-sorafenib role in vivo. Cells were transfected with the indicated plasmids. The indicated plasmids were transfected into the cells. Sorafenib was added to the culture medium at a final concentration of 20 μmol/L after 24 h of transfection. After another 24 h, 2 × 107 cells were collected and resuspended in 200 μL 0.9% sodium chloride solution before being subcutaneously injected into the flank of each 4–5-week old BALB/c-nu (nude) mouse. At regular intervals (3 days), tumor formation in mice was seen. Anesthesia was used to sacrifice mice, and tumors were dissected over a 4–6 week period. The National Institutes of Health’s Guide for the Care and Use of Laboratory Animals was followed for all animal care and handling procedures. The Animal Ethics Committee of Wuhan University, as well as the Wuhan University Center for Animal Experiment/A3 Laboratory, approved the animal experiments (Animal Using Protocol number: WP202220233).

2.17. Confocal Imaging Mitophagy was visually assessed using the colocalization of mitochondria and lysosomes [25]. To observe mitochondria, HCCLM3 cells were dyed with 200 nmol/L MitoTracker Red CMXRos (M9940, Solarbio) for 30 min at 37 °C. The cells were then stained for 60 min with 100 nmol/L LysoTracker Green DND-26 (L7400, Solarbio) to detect lysosomes. After experimenting with new cultural mediums. Images were captured using an Olympus FV3000 confocal microscope (Olympus). Fluorescences were stimulated at 578 nm (Red) and 488 nm (Green) (Green). Images were analyzed using Fluoview FV31S-SW software (Olympus) to determine MitoTracker and LysoTracker colocalization. 2.18. Statistical Analysis

The experiments were based on at least three separate trials. To compare qualitative variables, the Chi-squared or Fisher exact tests were used. The mean ± standard deviation (SD) or standard error of the mean (SEM) of at least three independent experiments was used to express numerical data. An unpaired Student’s t-test, one-way ANOVA followed by Fisher’s post hoc comparison test, or two-way ANOVA with multiple post hoc comparisons were used to compare significant differences between treatments. GraphPad Prism software was used to create the graphical representations (GraphPad, San Diego, CA, USA). p < 0.05 (*), p < 0.005 (**), p < 0.001 (***), and p < 0.0001 (****) were used to represent statistical significance.

4. DiscussionHCC is a serious cancer with a slow beginning, invasive growth, a high recurrence rate, and a high mortality rate. The median survival of patients with advanced HCC is less than one year. Systemic chemotherapy is the only option for such patients [1,7]. Despite advances in chemotherapies, the median overall survival remained less than a year [30]. Chemoresistance mechanisms include dysregulated efflux and inflow transporters, metabolic reprogramming, an abnormal immunological microenvironment, extracellular vesicles, and others [31,32,33]. HBV is also linked to chemoresistance, according to recent limited data and our earlier studies [10,12,34]. Our data in this article show sorafenib resistance in response to L-HBs. We also discovered that L-HBs stimulated the WNT7B-mediated canonical WNT signaling pathway, which aided in the development of HCC and sorafenib resistance by suppressing mitophagy. Research on predictive and prognostic HCC biomarkers and their reaction to chemoresistance is required in order to improve patient outcomes and achieve therapeutic advances in systemic chemotherapy. Sadly, no biomarkers for either systemic treatment were reported to predict responses in HCC [30]. In this investigation, we discovered that HBV-associated HCC tissues and HBV-replicating cell lines both had high levels of WNT7B expression, indicating that WNT7B may be a critical factor in HBV-induced HCC. Patients with late-stage HCC who tested positive for WNT7B had a shorter overall survival. When combined with the finding that individuals with late-stage HCC can only get systemic treatment [7], it is possible that WNT7B is implicated in chemoresistance.WNT signaling pathways, particularly canonical WNT signaling, have been demonstrated to be regularly active in HBV-induced HCC [35,36]. The canonical WNT signaling pathway is activated by inhibiting the phosphorylation-dependent degradation of the transcriptional coactivator CTNNB1. CTNNB1 accumulates in the cytoplasm and then translocates to the nucleus, where it interacts with TCF/LEF family members, initiating a variety of intracellular signaling cascades [13]. The WNT/CTNNB1 signaling pathway can be activated by HBx, which increases the expression and stability of WNT pathway target genes [37,38]. S-HBs has also been shown to raise the levels of LEF-1, c-MYC, and CCND1, all of which are downstream from the WNT pathway [39]. Here, we discovered that the HBV whole genome and L-HBs can increase WNT7B and FZD4 expression. L-HBs can activate canonical WNT signaling in HCC cells via WNT7B, as evidenced by increased downstream genes c-MYC and CCND1. Our results suggest that L-HBs also activated the canonical WNT pathway mediated by WNT7B. A study [40] found that the T-complex protein-1 ring complex subunit (also known as TCP1) activates the WNT7B-mediated canonical WNT pathway in HCC, validating our findings that WNT7B can activate CTNNB1-dependent WNT signaling in HCC.WNT7B is involved in a number of cancers. Wnt7B knockdown inhibits pancreatic cancer stem cell proliferation [14]. In breast cancer cells, WNT7B stimulates cell proliferation and migration [28]. WNT7B mRNA from extracellular vesicles is transported to and modulates human umbilical vein endothelial cells toward higher proliferative and angiogenesis [29]. Our findings demonstrate that WNT7B overexpression can increase the proliferation, malignant transformation, and invasion of HCC cells, implying an oncogenic role for WNT7B in HCC. Because HBs has been linked to an HCC risk in patients with low viral loads [5,6], it is believed that L-HBs caused HCC development by hyperactivating WNT7B-mediated canonical WNT signaling.Sorafenib is an effective first-line therapy for late-stage HCC [41]. However, due to the early onset of sorafenib resistance, most patients did not benefit long-term [42]. Clinical trial data reveal that HBV infection is related to a decreased response to sorafenib treatment [10]. HBV-replicating hepatoma cells are also less responsive to sorafenib treatment [43,44]. Mechanism investigations revealed that HBx depletion reduces the effects of sorafenib resistance in HCC [37]. In this work, we discovered that L-HBs prevented sorafenib-induced apoptosis and increased the growth of xenografts in nude mice, showing that L-HBs play a role in sorafenib resistance in HCC.Canonical WNT signaling was also implicated in sorafenib resistance [45,46]. A fraction of HBV-replicating cells in HBV-associated HCC increases WNT signaling and is resistant to sorafenib [47]. One study found that cutting down GSK3, a critical component of the CTNNB1 destruction complex, eliminates the inhibitory effects of HBV X protein depletion on sorafenib resistance in HCC [37], showing that canonical WNT signaling is involved in sorafenib resistance in HBV-related HCC. Our findings demonstrated that L-HBs produced sorafenib resistance via WNT7B, revealing a novel mechanism whereby HBV induces sorafenib resistance by modulating the WNT signaling pathway.Mitophagy activation has a dual role in modulating sorafenib sensitivity [42]. In Chen et al.’s study, ketoconazole, which resulted in PARKIN mitochondrial translocation and excessive mitophagy, sensitized HCC cells to sorafenib treatment [48]. Another study [49] found hypoxia-induced sorafenib-resistant HCC cells with hyperactivated mitophagy mediated by PINK1/PARKIN. In sorafenib-treated HCC cells, PINK/PARKIN-mediated mitophagy was induced, according to our findings. PINK1 was also found to be downregulated in HBV-HCC tissues. In HCC, L-HBs induced sorafenib resistance by blocking PINK1/PARKIN-mediated mitophagy via WNT7B. As a result, we postulated that WNT7B/CTNNB1 signaling activation in HBV-associated HCC contributed to the development of HCC and that L-HBs generated sorafenib resistance by reducing mitophagy.

留言 (0)

沒有登入
gif