Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide (Siegel et al.
2015). Although a variety of therapies such as surgical resection, liver transplantation, interventional treatment, and systemic therapies are employed, HCC patients have low 5-year survival rates and high recurrence rates due to invasion and metastasis (Au and Frenette
2015; Forner et al.
2012; Schlachterman et al.
2015). A better understanding of the molecular mechanisms that regulate HCC invasion and metastasis is essential for developing novel prognostic and of therapeutic strategies for HCC.
Radiofrequency ablation (RFA) is a local therapy that is an alternative to surgery for HCC < 3 cm and is widely used due to its simplicity, safety, minimal invasiveness, repeatability, and short hospitalization (Lee et al.
2017; Tsukamoto et al.
2018). However, cases of rapidly progressing residual HCC after insufficient RFA have been reported to be on the rise, with post-RFA recurrent rates ranging from 3.2 to 26% (Lam et al.
2008; Shiina et al.
2012). Moreover, local recurrences after RFA show more invasive growth and vascular invasion, and less differentiation compared with tumors from patients without RFA. Until now, the underlying mechanisms behind this phenomenon were still less unknown.
Lipid rafts function as physical platforms for various molecules that are involved in a variety of biological processes by serving as organizing centers for assembling signaling molecules into functional complexes (Babuke and Tikkanen
2007). Flotillins are key components of lipid rafts and include two members: flotillin-1 (FLOT1) and flotillin-2 (FLOT2). Previous studies have shown that flotillins are closely associated with tumor development, invasion, and metastasis (Bodin et al.
2014; Deng et al.
2018; Liu et al.
2018). Moreover, FLOT1 and FLOT2 were recently reported to be overexpressed and associated with progression and poor survival in HCC, suggesting the possibility of their use as prognostic markers or therapeutic targets in HCC (Wang et al.
2017; Zhang et al.
2013). Nevertheless, the biological role and molecular mechanism through which flotillins alter the biology of residual HCC after insufficient RFA remains unclear.
In this study, we investigated FLOT1 and FLOT2 expression in insufficient RFA tumor tissues in vivo and in heat-treated HCC cells in vitro, and further explored the roles of flotillins in altering the metastatic potential of residual HCC cells.
Materials and methods
Cell culture
The human HCC cell lines HCCLM3 (a highly metastatic HCC line established at the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China) and HepG2 cells (a modestly metastatic HCC line obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Rockville, MD, USA) with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA) and 100 U/mL penicillin at 37 °C in a humidified atmosphere containing 5% CO
2 (Li et al.
2004).
Vectors, retroviral infections, and transfection
The lentiviral vectors pGCSIL-GFP-shRNA-FLOT1, pGCSIL-GFP-shRNA-FLOT2 and pGC-FU-GFP-CTNNB1 were purchased from GeneChem Co., Ltd. (Shanghai, China). pGC-FU-GFP-CTNNB1 was transfected into HCCLM3 cells to overexpress β-catenin, and pGCSIL-GFP-shRNA-FLOT1 and pGCSIL-GFP-shRNA-FLOT2 were transfected into HCCLM3 cells to silence FLOT1 and FLOT2, respectively. pGCSIL-GFP and pGC-FU-GFP were used as controls.
Soft agar colony formation was performed using the CytoSelect 96-Well Cell Transformation Assay (Cell Biolabs Inc., San Diego, CA, USA) according the manufacturer’s protocol. Briefly, a bottom layer of 1.2% agar solution was plated and solidified, and then a top layer of equal volumes 1.2% agar solution, culture medium, and cell suspension (1 × 103 cells/well) was added. Culture medium (100 µL) was added to the top layer of the soft agar and replaced with fresh medium every 3 d. The cells were incubated for 6–8 days at 37 °C. Numbers of microscopically visible colonies (> 0.1 mm) were counted.
In vitro heat treatment
HCCLM3 and HepG2 cells were seeded into 6-well plates at 5 × 104 cells/well. After 24 h, the plates were sealed with Parafilm and submerged in a water bath set to the target temperature for 10 min. The target temperatures for HCCLM3 and HepG2 cells were 39 °C, 42 °C, 45 °C and 41 °C, 44 °C, 47 °C, respectively; the control temperature was 37 °C. The soft agar colony formation, and cell migration and invasion assays were performed 48 h after heat treatments.
Cell migration and invasion assays
Cell migration and invasion were assessed by transwell assays (Corning Inc., Corning, NY, USA). Briefly, 8 × 104 cells in serum-free DMEM were seeded into the upper chamber of each well of 24-well plates containing 8.0-µm pore size membranes. DMEM containing 10% FBS was added to the lower chamber of each well. After 48 h, cells that had reached the underside of the membrane were stained with Giemsa (Sigma-Aldrich, St. Louis, MO, USA), and then five randomly selected areas (200× magnification) per well were counted. Cell invasion assays were performed similarly, except 80 µL of Matrigel (0.8 mg/mL, BD Biosciences, Franklin Lakes, NJ, USA) was added to each well 6 h before cells were seeded onto the membranes.
RNA extraction and real-time quantitative (RT-q)PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to cDNA using the PrimeScript RT reagent kit (Takara, Shiga, Japan). SYBR Premix Ex Taq (Takara) was used for RT-qPCR according to the manufacturer’s instructions (Livak and Schmittgen
2001). Relative mRNA expression levels were calculated by the 2
− ΔΔCt method after normalizing to β-actin as an internal control. (16) Sequences of the RT-qPCR primers were as follows: FLOT1, forward: 5′-CCCATCTCAGTCACTGGCATT-3′ and reverse: 5′-CCGCCAACATCTCCTTGTTC-3′; FLOT2, forward: 5′-CCCCAGATTGCTGCCAAA-3′ and reverse: 5′-TCCACTGAGGACCACAATCTCA-3′; E-cadherin, forward: 5′-CCCGGGACAACGTTTATTACTATG-3′ and reverse: 5′-TCAGCCGCTTTCAGATTTTCA-3′;
N-cadherin, forward: 5′-CACGCCGAGCCCCAGTAT-3′ and reverse: 5′-GCCCCCAGTCGTTCAGGTAA-3′; Vimentin, forward: 5′-CTCTCAAAGATGCCCAGGAG-3′ and reverse: 5′-GCACGATCCAACTCTTCCTC-3′; Snail, forward: 5′-TGCAGGACTCTAATCCAAGTTTACC-3′ and reverse: 5′-GTGGGATGGCTGCCAGC-3′; β-actin, forward: 5′-GCTCCTCCTGAGCGCAAG-3′ and reverse: 5′-CATCTGCTGGAAGGTGGACA-3′.
Western blotting
Western blotting was performed as in previous studies using anti-FLOT1, anti-FLOT2 (Sigma-Aldrich), anti-E-cadherin, anti-
N-cadherin, anti-Vimentin, anti-Snail, anti-β-catenin and anti-Cyclin-D1 antibodies (Abcam, Cambridge, UK) (Zhang et al.
2014a). Membranes were stripped and re-probed with anti-β-actin (Sigma-Aldrich) as a loading control.
Xenograft mouse model
Male BALB/c nude mice (5-week-old, 18–20 g) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under specific pathogen-free conditions. The experimental protocol was approved by the Ethical Committee on Animal Experiments of the Animal Care Committee of Fudan University. Xenograft HCC models were established by orthotopic implantation of histologically intact tumor tissue into the nude mouse liver according to previous protocols (Sun et al.
1996). Two weeks after orthotopic implantation, the insufficient RFA operation was performed as follows: insufficient RFA mice underwent laparotomy to expose the tumor, then, considering the weight and volume of nude mice, RFA was performed at a lower energy protocol, in which the out power was 5 W and the duration was 30 s. This ensured the presence of residual cancer. Mice in the control group were sham-operated by inserting a needle electrode into the tumor without performing ablation. 5 weeks later, all mice were euthanized and tumor volumes were measured. Tumor volume was calculated according to the formula: volume (mm
3) = (largest diameter × shortest diameter
2)/2 (Zhang et al.
2014a).Then, tumors and lungs were placed in a 4% paraformaldehyde solution. Lung tissues were serially sectioned and stained with hematoxylin and eosin.
Immunohistochemistry
Tumor tissues were fixed, embedded, and sliced into 5-µm thick sections. FLOT1, FLOT2, E-cadherin,
N-cadherin, Vimentin and β-catenin immunohistochemical staining were performed as previously described. Staining results were viewed under a light microscope (Olympus, Tokyo, Japan) (Zhang et al.
2014a). Stained slides were scored by two investigators according to values obtained from IRS systems.
Statistical analysis
Experimental data are presented as mean ± SD. First, the Kolmogorov–Smirnov test was used to determine the normality of the data in each group. Differences between two normal distribution groups were estimated using Student’s t test. Differences among three or more normal distribution groups were analyzed using ANOVA. Differences between non-normal distribution groups were analyzed using nonparametric analyses of Chi-square tests. All analyses were performed using SPSS v20.0 software (IBM. Armonk, NY, USA); a two-tailed P value < 0.05 was considered statistically significant.
Discussion
In this study, we first found that FLOT1 and FLOT2 were overexpressed in HCC tissues after insufficient RFA and in HCC cell lines after heat treatment. Next, we explored the role of FLOT1 and FLOT2 in HCC growth and metastasis in vivo and in vitro. In accordance with studies in other cancers (Berger et al.
2013; Song et al.
2012; Wang et al.
2013), knocking down FLOT1 and/or FLOT2 inhibited the invasion, migration, and anchorage-independent growth of HCCLM3 cells in vitro and decreased their tumor growth and metastasis in vivo.
Flotillins encode caveolae-associated integral membrane proteins that belong to the lipid raft family, and are involved in vesicular trafficking and signal transduction(Bickel et al.
1997). The roles of flotillins in cancer progression have been studied in various cancers (Gao et al.
2015; Li et al.
2014; Zhang et al.
2014b). Flotillin overexpression promotes the proliferation, invasion, migration, and metastasis of cancer cells. Our previous studies had demonstrated that thermal intervention can enhance the metastasis of residual cancer by activating the FAK/PI3k signaling pathway (Zhang et al.
2017). A recent research pointed out that overexpression of flotillin can interact with α-actin and affect the activity of FAK, thereby enhancing the migration and metastasis of cancer(Banning et al.
2018). Whether thermal interference can affect the expression of flotillin is worth exploring. The present study confirmed for the first time that the expression of flotillin was up-regulated in residual cancer tissues after thermal interference.
In this study, we found that insufficient RFA increased both FLOT1 and FLOT2 in HCCLM3 cells but not in HepG2 cells, indicating these human HCC cell lines have different biological characteristics. Inversely, knocking down either FLOT1 or FLOT2 almost completely blocked the enhanced aggressiveness of heat-treated HCCLM3 cells in vitro and of residual cancer after insufficient RFA in vivo. These findings indicated a prominent role for flotillins in insufficient RFA-induced metastasis.
EMT is a biological process through which polarized epithelial cells undergo phenotypic changes into mesenchymal cells with enhanced invasiveness, survival, and extracellular matrix production(Kalluri and Weinberg
2009). Furthermore, EMT contributes to metastasis in a variety of human cancers (Hugo et al.
2007; Yeung and Yang
2017). Several studies have demonstrated that suboptimal RFA accelerated HCC growth and spread by transiently inducing an EMT-like and more aggressive cellular phenotype (Iwahashi et al.
2016; Yoshida et al.
2013). Meanwhile, the EMT-related transcript factors snail and twist were significantly higher in RFA group than those in the non-RFA group in clinical HCC cases (Iwahashi et al.
2016; Zeng et al.
2018). Herein, we showed that silencing FLOT1 and FLOT2 increased E-cadherin expression and decreased
N-cadherin, Vimentin and Snail in heat-treated HCCLM3 cells. Finally, these changes were also found in xenografts in nude mice after insufficient RFA. Thus, this study provides new insights into the mechanisms through which EMT is regulated in residual HCC after insufficient RFA and suggests FLOT1 and FLOT2 as potential targets for anti-metastatic therapy.
Multiple signaling pathways regulate EMT, but considering the central role of Wnt/β-catenin in HCC biology and the well-characterized association between Wnt/β-catenin signaling and EMT, we focused on the Wnt/β-catenin pathway (Dahmani et al.
2011; Monga
2015; Waisberg and Saba
2015). In this study, the effects of flotillins on β-catenin expression were examined, and elevated total and intra-nuclear β-catenin levels were verified in HCCLM3 cells in vitro. The key downstream gene of Wnt/β-catenin signaling, Cyclin-D1, was also upregulated. Moreover, we also detected pAKT levels because AKT phosphorylation can enhance β-catenin nuclear accumulation (Fang et al.
2007). Moreover, AKT signaling pathway is closely related to EMT in HCC, colorectal cancer, gastric cancer and breast cancer (Duan et al.
2018; Huang et al.
2018; Jiang et al.
2019; Zhang et al.
2018). Li et al. found that up- and downregulation of FLOT1 remarkably affected cervical cancer cell motility and invasion, respectively, through the EMT (via Wnt/β-catenin) and NF-κB pathways (Li et al.
2016). Liu et al. showed that FLOT2 promoted metastasis in nasopharyngeal carcinoma by activating the NF-κB and PI3K/Akt3 pathways (Liu et al.
2015). In bladder cancer, up-regulation of FLOT1 could also reverse the suppressed cell proliferation caused by miR-608 via activating AKT signaling (Liang et al.
2017). Additionally, as a direct target of miR-133, FLOT2 was regulated via Akt signaling and played pro-metastatic role in lung adenocarcinoma cell (Wei et al.
2018). Our results showed that pAKT was increased by heat treatment and decreased by silencing FLOT1 and/or FLOT2. Conversely, overexpressing β-catenin rescued the increase in motility and invasion of HCCLM3 cells following heat treatment that was lost when FLOT1 or FLOT2 were knocked out. Our findings demonstrate that insufficient RFA enhances the invasion and metastasis of residual cancer cells via Akt/Wnt/β-catenin-regulated EMT.
In summary, this study demonstrated that insufficient RFA enhanced the metastatic ability of HCC cells in vivo and in vitro, which was mediated by Akt/Wnt/β-catenin signaling. Furthermore, insufficient RFA increased FLOT1 and FLOT2 expression, suggesting these lipid raft protein promote these signaling pathways. Therefore, future studies will attempt to decrease the invasion and metastasis of residual cancer cells to improve the curative effects of RFA in HCC patients.
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