Sophoridine suppresses lenvatinib‐resistant hepatocellular carcinoma growth by inhibiting RAS/MEK/ERK axis via decreasing VEGFR2 expression

Abstract Hepatocellular carcinoma (HCC) is one of the most lethal cancer types with insufficient approved therapies, among which lenvatinib is a newly approved multi‐targeted tyrosine kinase inhibitor for frontline advanced HCC treatment. However, resistance to lenvatinib has been reported in HCC treatment recently, which limits the clinical benefits of lenvatinib. This study aims to investigate the underlying mechanism of lenvatinib resistance and explore the potential drug to improve the treatment for lenvatinib‐resistant (LR) HCC. Here, we developed two human LR HCC cell lines by culturing with long‐term exposure to lenvatinib. Results showed that the vascular endothelial growth factor receptors (VEGFR)2 expression and its downstream RAS/MEK/ERK signalling were obviously up‐regulated in LR HCC cells, whereas the expression of VEGFR1, VEGFR3, FGFR1‐4 and PDGFRα/β showed no difference. Furthermore, ETS‐1 was identified to be responsible for VEGFR2 mediated lenvatinib resistance. The cell models were further used to explore the potential strategies for restoration of sensitivity of lenvatinib. Sophoridine, an alkaloid extraction, inhibited the proliferation, colony formation, cell migration and increased apoptosis of LR HCC cells. In vivo and in vitro results showed Sophoridine could further sensitize the therapeutic of lenvatinib against LR HCC. Mechanism studies revealed that Sophoridine decreased ETS‐1 expression to down‐regulate VEGFR2 expression along with downstream RAS/MEK/ERK axis in LR HCC cells. Hence, our study revealed that up‐regulated VEGFR2 expression could be a predicator of the resistance of lenvatinib treatment against HCC and provided a potential candidate to restore the sensitivity of lenvatinib for HCC treatment.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the most common malignancy of primary liver cancer, which accounts for nearly 90% of all cases. 1 There are multiple aetiologies responsible for HCC, including hepatitis B virus or hepatitis C virus infection, aflatoxin-contained food consumption, alcohol abuse, obesity, type 2 diabetes and smoking. 2,3 China has a heavy burden of million-level people with chronic hepatitis infection, leading to the incidence and mortality of HCC in China, respectively, ranks as the fourth and the third place. 4 Globally, HCC has a population of more than 800 000 new cases in 2018, and at the same time, death cases were of more than 780 000. 5 Moreover, men have both higher incidence and mortality rate of HCC compared to women. 5 These data are a proxy that HCC has already become a heavy health burden, more medical investments in both preclinical and clinical practice are still in urgent need.
Currently, the curative treatment for early-stage HCC patients includes liver resection, liver transplantation and local ablation. 6 It is noted that most HCC patients are diagnosed with advanced stage which means they cannot undertake those treatments because of dysfunction of liver. 7 Alternatively, only two targeted therapies sorafenib and lenvatinib have been approved by Food and Drug Administration as the standard frontline treatments for advanced HCC patients. 8 Similar to sorafenib, lenvatinib is an orally multi-targeted tyrosine kinase inhibitor that selectively inhibits vascular endothelial growth factor receptors (VEGFR), fibroblast growth factor receptors (FGFR), platelet-derived growth factor receptor α (PDGFRα), KIT and RET. 9,10 It is highlighted that the resistance of targeted therapy still exists because of the primary resistance or adaptive resistance, which has hindered the treatment of advanced HCC. 11,12 Therefore, exploring the potential underlying mechanisms of lenvatinib resistance is necessary with clinical significance.
Besides the investigation of resistance mechanisms, another solution could be searching for potential combined therapy to overcome or ameliorate resistance. We have noted that Sophoridine is a natural bioactive alkaloid extracted from the seeds of Sophora alopecuroides L with multiple pharmacological functions, 13 including anti-tumour, 14 anti-inflammation, 15 anti-osteoporosis 16 and anti-virus. 17,18 For its anti-tumour function, previous studies demonstrated that Sophoridine could suppress the tumour growth of gastric cancer, 13 lung cancer, 19 medulloblastoma, 20 pancreatic cancer, 21 glioma, 22 colorectal cancer 23 and HCC. 24 However, the therapeutic effect of Sophoridine on lenvatinib-resistant (LR) HCC and whether Sophoridine can sensitize HCC to lenvatinib are still unknown.
Here, we revealed that up-regulated VEGFR2 expression and its downstream RAS/MEK/ERK signalling mediated the lenvatinib resistance of HCC. Transcription factor E26 transformation specific sequence 1 (ETS-1) was responsible for VEGFR2 mediated lenvatinib resistance. In vivo and in vitro studies revelated Sophoridine distinctly suppressed LR HCC and sensitized the therapeutic of lenvatinib.
These data provided potential evidence for the underlying mechanism of lenvatinib resistance and approved that Sophoridine could be a novel combined therapy with lenvatinib for HCC treatment.

| Cell culture
HepG2 and Huh7 cell lines were cultured in DMEM medium supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin. Then, the cell lines were maintained in cell incubator in a humidified atmosphere containing 5% CO 2 at 37°C. For each experiment, cell lines were harvested by 0.25% trypsin.

| Cell viability
Cell viability was measured by Cell counting kit-8 (CCK-8; Yeasen, cat# 40203ES60). According to the standard protocol, 5 × 10 3 cells were seeded into 96-well palates with three replicates. Then, cells were treated with lenvatinib or Sophoridine for 24-96 hours at 37°C in 5% CO 2 . Last, 10 μL CCK-8 was added into each well and incubated for another 4 hours. OD value of each well was detected by Microplate Reader at 450 nm.

| Development of lenvatinib resistance cell lines
First, the IC 50 of HepG2 and Huh7 cell lines to lenvatinib were detected. HepG2 or Huh7 cells were seeded into 96-well plates and treated with various doses of lenvatinib. After incubation for 72 hours, the cell viability was determined by CCK-8. Then, 1 × 10 4 HepG2 or Huh7 cells were seeded into 6-well palates and incubated with lenvatinib concentrations just below their IC 50 . During the following weeks, the dosages of lenvatinib were slowly increased at 0.25 μmol/L per time. Over 6-7 months, we established HepG2 and Huh7 cell lines resistant to lenvatinib (HepG2-LR and Huh7-LR).
After establishment, these resistant cell lines were continuously cultured with the presence of lenvatinib.

| Colony formation assay
First, HepG2, HepG2-LR, Huh7 cell and Huh7-LR cells were seeded into 6-well palates at a density of 500 cells/per well and then treated with lenvatinib or Sophoridine for 24 hours. Then, the drug-contained medium was discarded, and the fresh medium was added into plates. Cells were incubated for another 2 weeks under 37°C in 5% CO 2 . Last, the colonies were fixed with 4% paraformaldehyde and stained with crystal violet.

| Cell apoptosis assay
The apoptosis of HepG2-LR and Huh7-LR cells was determined by the FITC/Annexin V apoptosis detection Kit (BD Pharmingen, cat#556547). In brief, HepG2-LR and Huh7-LR cells were previously seeded in 6-well plate (1 × 10 6 cells/well) for 6 hours and then treated with different concentration of Sophoridine (0, 20, 40 and 80 µmol/L) for another 24 hours. HepG2-LR and Huh7-LR cells were harvested and re-suspended in binding buffer. Next, 100 μL cell solution was transferred into 1.5 mL centrifuge tube and 5 μL FITC Annexin V and 5 μL PI was added into each tube. Finally, these tubes were incubated for 15 minutes at room temperature in the dark and 400 μL binding buffer was added into each tube before analysing by flow cytometry.

| Quantitative real-time PCR analysis
Following the manufacturer's instructions, total RNA from HepG2, HepG2-LR, Huh7 and Huh7-LR cells were collected by TRIzol reagent (Invitrogen) and RNeasy kit (Qiangen). Then, the RNA was reverse transcribed into cDNA by a FastKing One Step RT-PCR Kit (Tiangen). qRT-PCR was performed on 7500 real-time PCR system

| Western blotting
The HepG2, HepG2-LR, Huh7 and Huh7-LR cells were lysed in RIPA buffer. Protein concentration of each sample was detected by using

| In vivo study
For in vivo study, a total of 1 × 10 6 HepG2-LR cells were injected subcutaneously into the left flank of BALB/c nude mice (4 weeks old, male). When the tumour volumes reached around 100 mm 3 , the tumour-bearing mice were randomized into four groups, including control group (saline solution, daily, intraperitoneally), Sophoridine group (50 mg/kg, daily, intraperitoneally), lenvatinib group (30 mg/kg, daily, intragastrically) or Sophoridine combined with lenvatinib group. Tumour volume was measured by caliper and calculated with the formula tumour volume = 1/2(length × width 2 ). After 16 days, the mice were killed, and the tumour weight was also weighted and recorded.

| Development of lenvatinib-resistant cell lines
In order to explore the underlying mechanism of lenvatinib resistance in HCC, we developed two LR HCC cell lines by culturing HCC cells with long-term exposure to lenvatinib in the culture medium. LR cells were acquired by gradually increasing the dosages of lenvatinib over repeated cell passages (6-7 months). LR cell lines were successfully constructed when HCC cell lines can tolerate higher doses of lenvatinib compared to parental cell lines. Then, two LR HepG2 and Huh7 cell lines were established ( Figure 1A). Those two cell lines were characterized by higher cell viability than the parental cells (WT) with lenvatinib treatment ( Figure 1A). The IC 50 of the parental and LR cells were also determined ( Figure 1B). In the resistant cells, the IC 50 of lenvatinib showed a higher concentration compared to parental cells ( Figure 1B). Accordingly, lenvatinib treatment had no influence on the growth of individual clones of resistant cells compared to their parental cells ( Figure 1C,D).

| Up-regulated VEGFR2 expression mediated lenvatinib resistance by activating RAS/MEK/ ERK signalling
As lenvatinib is multi-targeted tyrosine kinase inhibitor including VEGFR1-3, FGFR1-4 and PDGFRα/β, 10 (Figure 2A and S1). Thus, we further measured the VEGFR2 protein levels, which also presented higher expression in LR cells ( Figure 2B). RAS/MEK/ERK axis is recognized as the downstream pathway of VEGFR2. 25 Consistently, the protein level of RAS was increased and the p-MEK, p-ERK levels were also up-regulated in LR cells compared to parental cells ( Figure 2C).
These results suggested that up-regulated VEGFR2 expression may mediate lenvatinib resistance by activating RAS/MEK/ERK signalling.

| ETS-1 was responsible for VEGFR2 mediated lenvatinib resistance
Previous studies have pointed out that activation of ETS-1 promotes the FOX:ETS motif bind to the first intron enhancer of VEGFR2 to enhance VEGFR2 expression. 26  HepG2-WT or Huh7-WT cells ( Figure 3F). Herein, we concluded that ETS-1 was responsible for VEGFR2 mediated lenvatinib resistance.

| Sophoridine inhibited the proliferation, colony formation and increased apoptosis of lenvatinibresistant HCC cells
To

| Sophoridine decreased ETS-1 expression to down-regulate VEGFR2 expression along with downstream RAS/MEK/ERK axis in lenvatinibresistant HCC cells
We have demonstrated that up-regulated VEGFR2 expression mediated lenvatinib resistance by activating downstream RAS/MEK/ERK signalling in vitro. We also would like to know whether Sophoridine could influence the VEGFR2 expression and subsequently inhibited the RAS/MEK/ ERK axis activation to play an anti-tumour effect against LR HCC cells.
First, we measured the expression of VEGFR2 and p-ERK in tumours after indicated treatment by immunohistochemistry ( Figure 6I). Results

| D ISCUSS I ON
Hepatocellular carcinoma is hard to discern at the early stage based on regular body check, leading to the high occurrence of advanced HCC. 27 With the lack of effective therapies, 5-year survival of HCC patients is only 18%. 3,28 Sorafenib was the first approved targeted therapy; however, the clinical benefits of sorafenib were modest and the 5-year relative survival remains low. 8,29 The clinical trials of lenvatinib showed lenvatinib had a significant clinical improvement in objective response rate, time to progression and progressionfree survival compared to sorafenib. 30 Hence, the discovery and  inducing cell cycle arrest in G0/G1 phase or S phase arrest. 13,[19][20][21][22] However, the effect and underlying mechanism of Sophoridine against LR HCC is unknown. In this research, we found Sophoridine inhibited the proliferation, colony formation, increased apoptosis and sup-

| CON CLUS ION
In summary, we identified the responsible role of ETS-1 induced increased VEGFR2 expression and its downstream RAS/MEK/ERK axis activation in LR HCC for the first time. In addition, we found a novel function and mechanism of Sophoridine against LR HCC.
Sophoridine had the capacity to restore the sensitivity of lenvatinib against LR HCC via suppressing ETS-1 mediated up-regulated VEGFR2 expression. Hence, we uncovered the underlying mechanism of lenvatinib resistance in HCC and provided an alternative candidate for sensitizing the therapeutic effect of lenvatinib against LR HCC.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.