Overexpression of BACH1 mediated by IGF2 facilitates hepatocellular carcinoma growth and metastasis via IGF1R and PTK2

Background: Accumulating studies manifest that BTB and CNC homology 1 (BACH1) facilitates multiple malignancies progression and metastasis, and targeting the BACH1 pathway enhances antitumor efficacy. Nevertheless, the exact mechanism of BACH1 promoting growth and metastasis and its therapeutic significance in hepatocellular carcinoma (HCC) remain unclear. Methods: The expression of BACH1 in human HCC specimens and HCC cell lines was analyzed by quantitative RT-PCR (RT-qPCR), western blot, and immunohistochemistry (IHC). The invasiveness and metastasis of HCC cells in vitro and in vivo were evaluated using transwell assays and orthotopic xenograft models. The luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays were performed to explore the transcriptional regulation of insulin-like growth factor 1 receptor (IGF1R) and protein tyrosine kinase 2 (PTK2) by BACH1. Results: BACH1 was prominently upregulated in human HCC samples and elevated BACH1 expression was associated with poor overall survival (OS) and high recurrence rates of HCC patients. BACH1 facilitated growth and metastasis of HCC by upregulating cell motility-related genes IGF1R and PTK2. Notably, insulin-like growth factor 2 (IGF2), the ligand of IGF1R, in turn upregulated BACH1 expression through the IGF1R-ERK1/2-ETS1 cascades, thus forming a positive feedback loop to provoke HCC growth and metastasis. Moreover, combining IGF1R inhibitor linsitinib with PTK2 inhibitor defactinib prominently suppressed BACH1-mediated HCC growth and metastasis. Conclusions: These results demonstrated the tumorigenic and pro-metastatic role of BACH1 in HCC, which could be a promising biomarker for predicting poor prognosis and selecting patients who could benefit from combination therapy of IGF1R-targeted and PTK2-directed.


In Vitro Migration and Invasion Assays
The migratory and invasive ability of HCC cells were evaluated using transwell inserts with an 8-µm pore size (Corning, NY, USA). DMEM supplemented with 10% FBS was added to the bottom chamber. Matrigel (50 µl, diluted 1:8 with DMEM, Corning, New York, USA) was coated on the top chambers and dried for invasion assay. 5 × 10 4 (migration assay) and 1 × 10 5 (invasion assay) cells were seeded in the top chamber in serum-free medium and were cultured in 5% CO2 at 37 °C for 24 h and 72 h, respectively. Cells that migrated or invaded to the lower surface of the membrane were fixed, stained, and imaged. The cell numbers from five fields per membrane of three inserts were used for statistical analysis. All experiments were performed in triplicate.

Construction of lentivirus and stable cell lines
Lentiviral vectors encoding shRNAs were generated using PLKO.1-TRC (Addgene) and designated as shBACH1, shIGF1R, shPTK2, shETS1 and shControl. 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 Supplementary Table S8. Lentiviral vectors encoding human BACH1, IGF1R, PTK2 and IGF2 were constructed in pLV-puro or pLV-neo (Addgene) and designated as LV-BACH1, LV-IGF1R, LV-PTK2 and LV-IGF2. 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, MA, USA). The lentiviruses were harvested twice on days 4 and 5. Viruses were filtered with a 0.45-μm filter and stored at -80 °C. For stable cell lines construction, HCC cells were transfected with lentivirus at a multiplicity of infection (MOI) of 10-30 for 12-24 h. 72 h after infection, HCC cells were selected with 2.5 μg/ml puromycin (OriGene) for 2 weeks. The stable cell lines were confirmed by qRT-PCR and western blotting.

Luciferase reporter assays
The Dual-Luciferase Reporter Assay (Promega, CA, USA) was used to detect luciferase activity according to the manufacturer's instructions. In brief, the cells transfected with plasmids were lysed and the lysates were centrifuged at maximum speed for 1 min. Relative luciferase activity was determined using a ModulusTM TD20/20 Luminometer (Turner Biosystems, CA, USA) and was normalized to Renilla luciferase activity.

Plasmid construction
Plasmid construction was performed according to standard procedures. The primers were shown in Supplementary Table S7. For example, the BACH1 gene complete CDS construct, pCMV-BACH1, was generated by using cDNA from human PBMCs.
It was generated with forward and reversed primers incorporating EcoRI and BamHI sites at the 5' and 3'-ends, respectively. The polymerase chain reaction (PCR) product was cloned into the EcoRI and BamHI sites of the pCMV-Tag2B vector. The IGF1R promoter construct, (-2128/+70) IGF1R, was generated from human genomic DNA.
This construct corresponds to sequence from -2128 to +70 (relative to the transcriptional start site) of the 5'-flanking region of human IGF1R gene. It was generated with forward and reverse primers incorporating KpnI and MluI sites at the 5' and 3'-ends, respectively. The polymerase chain reaction (PCR) product was cloned into the KpnI-and MluI sites of the pGL3-Basic vector (Promega, CA, USA). The 5'flanking deletion constructs of the IGF1R promoter, (-1720/+70) IGF1R, (-572/+70) IGF1R were similarly generated using the (-2128/+70) IGF1R construct as the template. The BACH1 binding sites in the IGF1R promoter were mutated using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, CA, USA). The constructs were confirmed by DNA sequencing. Other promoter constructs were cloned in the same manner.

Transient transfection
The cells were plated at a density of 1 × 10 5 cells/well in a 24-well plate. After 12-24 h, the cells were co-transfected with 0.6 μg of expression vector plasmids, 0.18 μg of promoter reporter plasmids, and 0.02 μg of pRL-TK plasmids using Lipofectamine 2000 (Invitrogen, MA, USA) according to the manufacturer's instructions. After 6 h 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.

Chromatin immunoprecipitation Assay (ChIP)
Cells were immersed in 1% formaldehyde for 10 min at 37 °C to stimulate crosslinking. Then, glycine was used to quench the formaldehyde after cross-linking to stop formaldehyde fixation. After washing with PBS, the cells were resuspended in lysis buffer (1 mM PMSF, 1% SDS, 10 mM EDTA and 50 mM Tris (pH 8.1) -total volume 300 μl). Sonication was then performed to produce fragmented DNA. A slurry of protein G-Sepharose and herring sperm DNA (Sigma-Aldrich) was used to clear the supernatant. The recovered supernatant was then subjected to a 2-hour incubation period with specific antibodies or an isotype control IgG in the presence of protein G-Sepharose beads and herring sperm DNA, followed by antibody denaturation with 1% SDS in lysis buffer. Precipitated DNA was extracted from the beads by immersing them in a 1.1 M NaHCO 3 solution and 1% SDS solution at 65 °C for 6 h.
Immunoprecipitated DNA was retrieved from the beads by immersion in 1% SDS and a 1.1 M NaHCO 3 solution at 65 °C for 6 h. The DNA was then purified using a PCR Purification Kit (Qiagen, Germany). The primers were shown in Supplementary Table   S7.
For ChIP assays of tissues, cells were first separated from six pairs of fresh frozen HCC tissues and normal liver tissues collected after surgical resection. In detail, surgically extracted tumor tissues were first washed by 1 × cold PBS, 5 min, for three times and added to medium supplemented with antibiotics and antifungal agents. Use a clean razor blade to cut a pie of tissue (around 5 mm 3 ) into small piece (typical 1 mm 3 or smaller). Then, digestion the tissues with DNase I (20 mg/mL; Sigma-Aldrich) and collagenase (1.5 mg/mL; Sigma-Aldrich) and placed on a table concentrator, 37 ℃, for 1 h. At the end of the hour, we filtered the dissociated cells through 70 μmpore filters rinsed with fresh media. The 1 × red cell lysis was added to the tissues and incubated for 5 min to lysis the red blood cell, followed by another rinse. The dissociated cells were crosslinked using 1% formaldehyde for 10 min at 37 ℃. After cell lysis, the DNA was fragmented by sonication. ChIP grade antibody or IgG (negative control) was used to immunoprecipitate the fragment DNA. Then, qRT-PCR was used to amplify the corresponding binding site on the promoters.
The antibodies used in ChIP were listed below. For cell proliferation studies, HCC cells were seeded into 96-well plates (5000 cells/well). Six wells of each group were detected every day. The cells were incubated into 100 μl of fresh medium containing 10 μl CCK8 at 37 °C for 2 h, and then the medium was replaced by 100 μl of DMSO and shaken at room temperature for 10 min.
The absorbance was measured at 450 nm.

Colony formation assay
For colony formation assays, HCC cells were seeded into 35 mm dishes (500 cells/dish). Then the cells were incubated at 37 ℃ in 5% CO2 for 2 weeks.
Subsequently, the medium was removed. The cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and imaged with light microscope (Olympus, Japan). Only positive colonies (diameter > 40 um) in the dishes were counted and compared.    The sequences highlighted in yellow represent the two binding sites of BACH1 on the IGF1R promoter, and the arrow represents the transcription initiation sites. The sequences highlighted in yellow represent the three binding sites of BACH1 on the PTK2 promoter, and the arrow represents the transcription initiation sites.