Direct Targeting of CREB1 with Imperatorin Inhibits TGFβ2‐ERK Signaling to Suppress Esophageal Cancer Metastasis

Abstract Metastasis accounts for 90% of cancer death worldwide, and effective therapeutic strategies are lacking. The aim of this work is to identify the key drivers in tumor metastasis and screen therapeutics for treatment of esophageal squamous cell carcinoma (ESCC). Gene Ontology analysis of The Cancer Genome Atlas (TCGA) gene expression datasets of ESCC patients with or without lympy metastasis identifies that TGFβ2 is highly enriched in the pathways essential for tumor metastasis and upregulates in the metastatic ESCC tumors. High TGFβ2 expression in ESCC correlates with metastasis and patient survival, and functionally contributes to tumor metastasis via activating extracellular signal‐regulated kinases (ERK) signaling. By screening of a library consisting of 429 bioactive compounds, imperatorin is verified as a novel TGFβ2 inhibitor, with robustly suppressive effect on tumor metastasis in multiple mice models. Mechanistically, direct binding of imperatorin and CREB1 inhibits phosphorylation, nuclear translocation of CREB1, and its interaction with TGFβ2 promoter, represses TGFβ2 expression and fibroblasts‐secreted CCL2, and then inactivates ERK signaling to block cancer invasion and abrogates the paracrine effects of fibroblasts on tumor angiogenesis and metastasis. Overall, the findings suggest the use of TGFβ2 as a diagnostic and prognostic biomarker and therapeutic target in ESCC, and supports the potential of imperatorin as a novel therapeutic strategy for cancer metastasis.

min. Next, 500 μL extraction buffer with 1% Triton-100 was added to the cell supernatant to solubilize plasma membrane and leave the nuclear membrane intact. To obtain the nuclear pellet, the cell supernatant was incubated on ice for 20 min, and 500 μL nuclear isolation buffer (10 mM HEPES-KCl [pH 7.6], 10 mM KCl, 5 mM MgCl 2 , sucrose) was added, and then the homogenates were centrifuged at 600 g for 10 min at 4 °C. The supernatant fraction is the cytosolic fraction, and the pellet fraction is the enriched nuclear fraction.

Western blot
Cells were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA) according to the manufacturer's instructions, and a BCA kit (Thermo Fisher Scientific) was used to determine the protein concentration [7]. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% nonfat milk for 1 h, the membrane was incubated with primary antibody at 4°C overnight, and washed with Tris-Buffered Saline Tween-20 (TBST) followed by a incubation with the corresponding HRP-conjugated secondary antibodies (Cell Signaling Technology) at room temperature for 1 h. The reaction was visualized using ECL (Bio-Rad, Hercules,

Enzyme-Linked Immunosorbent Assay (ELISA)
Human TGFβ2 and CCL2 ELISA Kit (RayBiotech, Norcross, GA) was used to determine the concentration of TGFβ2 and CCL2 in the conditioned medium or human serum samples, respectively, according to the manufacturer' instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using Trizol reagent according to the manufacturer's protocol (Life Technologies). RNA was converted to cDNA using PrimeScript II first Strand cDNA Synthesis Kit (Takara, Dalian, China) and the subsequent quantitative PCR was performed on a Bio-Rad Mini Opticon real-time PCR system using SYBR Premix Ex TaqII (Takara) according to the manufacturer's instructions. Actin was included as internal control. The primers were listed as Supplementary Table 2.

Molecular Docking and molecular dynamics analysis
The nuclear magnetic resonance (NMR) structure of CREB1 was downloaded from RCSB Protein Data Bank (PDB code: 2LXT), and the imperatorin structure was constructed using UCSF Chimera [8]. DOCK 6.7 program was then utilized to conduct semi-flexible docking where 10000 different orientations were generated [9, 10]. The clustering analysis was performed to obtain the best scored poses, with a RMSD threshold of 2.0 Å. All molecular dynamics simulations were performed using GROMACS version 2016. 4 [11] with AMBER ff99SB-ILDN force filed. CREB1-imperatorin complexes were centered in a cubic box of 10 Å solvated using TIP3P water model and SPC216 solvent configuration. MM/PBSA method was employed to calculate the binding free energies of ligand and protein as decribed previously [12].
Luciferase activity was measured by using Dual-luciferase Reporter Assay System (Promega) as previously described [14].

Chromatin immunoprecipitation (ChIP)-quantitative PCR
The ChIP assay was performed as previously described by using simple CHIP enzymatic chromatin IP kit (Cell Signaling) according to the manufacturer's manual [15]. In brief, in vivo protein and DNA crosslinking was performed using 37% formaldehyde, followed by sonication and chromatin digestion. The protein-DNA complexes were immunoprecipitated using CREB1 antibody or negative control IgG antibody, and the purified DNA was subjected to SYBR Green PCR analysis (Takara).
Relative expression was calculated using the comparative Ct method after normalization to GAPDH control.

Protein purification and surface plasmon resonance (SPR) assay
Protein was purified by using Glutathione S-transferase (GST) tag protein purification kit (Beyotime Biotechnology). The pGEX-6P-1 plasmid expressing GST-tagged wild-type or mutant CREB1 was transformed into E. coli BL21 star (DE3) cells, and isopropyl β-D-thiogalactopyranoside (IPTG) was added when bacteria culture were grown to a 600 nm (OD600) optical density of about 0.6. After obtaining CREB1-GST protein, the GST tag was cut with PreScission Protease (Beyotime Biotechnology) according to manufacture's protocol. SPR analysis was performed using the Biacore X100 system (GE Healthcare Life Sciences). Wide type CREB1 and mutant CREB1 protein was immobilized by amine coupling onto a CM7 chip (GE Healthcare Life Sciences) as described previously (8). Imperation in PBS buffer was added with a speed of 30 μL/min for 90 s, and dissociation was evaluated by passing HBS buffer alone over the chip at 30 μl/min for 10 min.

Tumor xenograft model
Female BALB/c nude mice aged 6-8 weeks were maintained under standard conditions and cared for according to the institutional guidelines for animal care. All the animal experiments were approved by the Committee on the Use of Live Animals in Jinan University. ESCC cells were subcutaneously injected into flanks of mice, and the mice were randomized into treatment and control groups when the tumors reached ~5 mm diameter. The treatment group received oral gavage of imperatorin (50 mg/kg dissolved in corn oil) thrice weekly, whereas the control group received the vehicle only. A sub-group of imperatorin-treated mice also received twice weekly intravenous injections of CCL2 recombinant protein (300 ng/mouse). At the end of the experiment, the tumors were collected for immunohistochemical analysis of CD31 as described previously [16].

Immunohistochemistry
Paraffin-embedded sections were deparaffinized and rehydrated, followed by antigen retrieval for 15 min in 10 mM citrate buffer (pH 6). After blocking with normal serum, the slides were incubated with primary antiobdy at 4°C overnight, and then incubated with corresponding biotinylated secondary antibody. After incubation with peroxidase-conjugated avidin-biotincomplex, immunostaining was visualized Supplementary