HMGB1-mediated elevation of KLF7 facilitates hepatocellular carcinoma progression and metastasis through upregulating TLR4 and PTK2

Background: Metastasis is a major cause of HCC-related deaths with no effective pharmacotherapies. Chronic inflammation promotes HCC dissemination, however, its underlying mechanisms are not fully understood. Here, we investigated the role of Krüppel-like factor 7 (KLF7) in inflammation-provoked HCC metastasis and proposed therapeutic strategies for KLF7-positive patients. Methods: The expression of KLF7 in human HCC specimens were examined by immunohistochemistry and quantitative real-time PCR. The luciferase reporter assays and chromatin immunoprecipitation assays were conducted to explore the transcriptional regulation related to KLF7. Orthotopic xenograft models and DEN/CCl4-induced HCC models were established to evaluate HCC progression and metastasis. Results: KLF7 overexpression promotes HCC metastasis through transactivating toll-like receptor 4 (TLR4) and protein tyrosine kinase 2 (PTK2) expression. High mobility group box 1 (HMGB1) upregulates KLF7 expression through the TLR4/advanced glycosylation end-product specific receptor (RAGE)-PI3K-AKT-NF-κB pathway, forming an HMGB1-KLF7-TLR4 positive feedback loop. The HMGB1-KLF7-TLR4/PTK2 axis is gradually activated during the progression of inflammation-HCC transition. Genetic depletion of KLF7 impedes HMGB1-mediated HCC progression and metastasis. The combined application of TLR4 inhibitor TAK-242 and PTK2 inhibitor defactinib alleviates HCC progression and metastasis induced by the HMGB1-KLF7 axis. In human HCCs, KLF7 expression is positively correlated with cytoplasmic HMGB1, p-p65, TLR4, and PTK2 levels, and patients positively co-expressing HMGB1/KLF7, p-p65/KLF7, KLF7/TLR4 or KLF7/PTK2 exhibit the worst prognosis. Conclusions: HMGB1-induced KLF7 overexpression facilitates HCC progression and metastasis by upregulating TLR4 and PTK2. Genetic ablation of KLF7 via AAV gene therapy and combined blockade of TLR4 and PTK2 represents promising therapy strategies for KLF7-positive HCC patients.

In addition, 10 normal liver tissues and 25 pairs of adjacent nontumor and HCC tissues preserved in liquid nitrogen were used for examining the mRNA levels of KLF family genes ( Figure S1); 20 normal liver tissues and 50 pairs of HCC tissues and adjacent nontumor tissue samples preserved in liquid nitrogen were used to detect the mRNA expression of KLF7 ( Figure 1C); 16 pairs of fresh adjacent nontumor tissues, primary HCC tissues and matched metastatic HCC tissues were collected after surgical resection and were used for KLF7 IHC staining ( Figure 1E); 20 pairs of fresh adjacent nontumor tissues, primary HCC tissues and matched metastatic HCC tissues were collected after surgical resection and were used to detect the expression profiles of HMGB1, KLF7, TLR4, and PTK2 ( Figure 6G, Figure S8).

Plasmid construction
Plasmid construction was performed according to standard procedures. The primers were shown in Table S8. For example, the KLF7 gene complete CDS construct, pCMV-KLF7, was generated by using cDNA from human PBMCs. It was generated with forward and reverse primers incorporating EcoRI and HindIII sites at the 5' and 3'-ends, respectively. The polymerase chain reaction (PCR) product was cloned into the EcoRI and HindIII sites of the pCMV-Tag2B vector. The TLR4 promoter construct, (-1918/+473) TLR4, was generated from human genomic DNA. This construct corresponds to sequence from -1918 to +473 (relative to the transcriptional start site) of the 5'-flanking regions of human TLR4 gene. It was generated with forward and reverse primers incorporating SacI and NheI sites at the 5' and 3'-ends, respectively.
Other promoter constructs were cloned in the same manner.

Construction of lentivirus and stable cell lines
Lentiviral vectors encoding shRNAs were generated using PLKO.1-TRC (Addgene) and designated as LV-shKLF7, LV-shTLR4, LV-shPTK2, LV-shRAGE, LV-shp65, and LV-shcontrol. "LV-shcontrol" is a non-target shRNA control. The vector "pLKO.1-puro Non-Target shRNA Control Plasmid DNA" (purchased from Sigma, SHC016) contains an shRNA insert that does not target any known genes from any species. The shRNA sequences can be found in Table S9. Lentiviral vectors encoding human KLF7, TLR4, PTK2, and HMGB1 genes were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-KLF7, LV-TLR4, LV-PTK2 and LV-HMGB1. An empty vector was used as the negative control and was designated as LV-control. The lentivirus and cell infection were produced according to the lentiviral vector protocol recommended by Addgene. Briefly, the lentiviral plasmid and packaging plasmids pMD2.G and psPAX2 (Addgene plasmid #12259 and #12260) were transfected into HEK-293T cells with transfection reagent (Lipofectamine®3000, Thermo Fisher Scientific) and OPTI-MEM media (Invitrogen, Waltham, MA, USA). The lentiviruses were harvested twice on days 4 and 5. Viruses were filtered with a 0.45μm filter and stored at -80 °C. Lentiviral infection of target cells were performed in cell culture media with 5 μg/ mL polybrene (Sigma H9268). Seventy-two hours after infection, cells were selected for 2 weeks using 2.5 μg/mL puromycin (OriGene). Selected pools of cells were used for the following experiments.

Transient transfection
The cells were plated at a density of 1×10 5 cells/well in a 24-well plate. After 12-24 hours, the cells were co-transfected with 0.6 μg of expression vector plasmids, 0.18μg of promoter reporter plasmids, and 0.02 μg of pRL-TK plasmids using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. After 6h of transfection, the cells were washed and allowed to recover overnight in fresh medium supplemented with 1% FBS for 48 h. Serum-starved cells were used for the assay.

Luciferase reporter assay
Luciferase activity was detected using the Dual Luciferase Assay (Promega, USA) according to the manufacturer's instructions. The transfected cells were lysed in culture dishes containing a lysis buffer and the resulting lysates were centrifuged at maximum speed for 1 min in a microcentrifuge. Relative luciferase activity was determined using a ModulusTM TD20/20 Luminometer (Turner Biosystems, USA), and the transfection efficiencies were normalized according to the Renilla activity.

Western blot analysis
Proteins from lysed cells were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 5% BSA in TBST (120 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20) for 2 hours at room temperature. Blots were incubated with a specific antibody overnight at 4 °C.
Western blotting of β-actin on the same membrane was used as a loading control. The membranes were then washed with TBST 3 times and incubated with an HRPconjugated secondary antibody. Proteins were visualized using an ImmobilonTM Western Chemiluminescent HRP substrate (Millipore, USA).
The primary antibodies used are listed below. 1h. At the end of the hour, we filtered the dissociated cells through 100-μm-pore filters rinsed with fresh media. The 1×red cell lysis was added to the tissues and incubated for 5 minutes to lysis the red blood cell, followed by another rinse. The dissociated cells were crosslinked using 1% formaldehyde for 10 minutes at 37℃. After cell lysis, the DNA was fragmented by sonication. ChIP grade antibody or IgG (negative control) was used to immunoprecipitated the fragment DNA. Then, qRT-PCR was used to amplify the corresponding binding site on the promoters.

Cell treatment
Established HCC cell lines were seeded in 6-well plates and allowed to attach overnight. Reagents used to treat these cells for 24 hours.

In vitro migration and invasion assays
Transwell inserts were placed inside 24-well cell culture plates (Corning, USA). The upper chamber was coated with 60µL of Matrigel (Corning, 200 mg/mL) and dried overnight for invasion assays. For the migration and invasion assays, 5×10 4 and 1×10 5 cells, respectively, were plated in the top chamber, and the lower chamber was filled with 600µL of complete medium. After incubation for 24h, the cells on the upper surface of the membranes were removed by swabbing, and the cells on the lower surface were fixed with methanol for 15 min and stained with 0.1% crystal violet for 15min.
The average number of cells in five fields per membrane was counted on three inserts.

Colony formation assay
Transfected cells (1000 per well) were cultured in 6-well plates. After 10-14 days of culture, the cells formed stable colonies. The cell colonies were fixed with 70% ethanol and then stained with a crystal violet solution for 10 minutes. Colonies containing more than 50 cells were counted and each group included three replicates. Reaction product was visualized with diaminobenzidine for 2 min. Images were obtained under a light microscope (Olympus, Japan) equipped with a DP70 digital camera.

The Human Liver cancer RT 2 profiler PCR array
The Human Liver cancer PCR array was applied to PLC/PRF/5-KLF7 cells and

Hepatocytes and non-parenchymal cells isolation
Hepatocytes and non-parenchymal cells were isolated after liver perfusion according to previously published protocol [2]. Briefly, the liver was perfused at the speed of 7 mL/min for 4 minutes through the portal vein with perfusion buffer containing collagenase Ⅳ. The resulting suspension was centrifuged at 50g and washed 3 times with sterile Hank's solution, and the hepatocytes were in pellet and the nonparenchymal cells were in supernatant. Cells were then cultured and used for further analysis.

Cytoplasmic protein extraction
Cytoplasmic protein extraction of indicated HCC cells was performed using a was added, and the cells were resuspended and homogenized on ice for 1 min. The homogenate was centrifuged at 12000g for 5 min at 4 °C. The resulting supernatant was the cytoplasmic protein fraction.

Figure S1
Figure S1 The mRNA levels of KLF family genes in normal livers (n = 10) and 25 pairs of adjacent nontumor and HCC tissues were detected by RT-PCR. KLF7 is the most upregulated KLF gene in HCC tissues. *P < 0.05.

Figure S10
Figure S10 The sequence of the KLF7 promoter. The NF-κB binding sites are highlighted in yellow, the AP-1 binding sites are highlighted in blue, the STAT3 binding sites are highlighted in green, and the transcription start site is marked in red.   Primers for TLR4 promoter construct: