6‐Gingerol suppresses tumor cell metastasis by increasing YAPser127 phosphorylation in renal cell carcinoma

According to the World Health Organization, the incidence and mortality rates of renal cell carcinoma (RCC) are rapidly increasing worldwide. Serious side effects caused by immune therapy and resistance to targeted drug therapy are urgent clinical problems facing kidney treatment. There is increasing global interest in developing natural products with a reduced number of side effects as adjunctive therapeutic options for RCC. Ginger is a spice and herbal remedy used worldwide, and 6‐gingerol is a major pharmacologically active ingredient in ginger. In our study, we found that 6‐gingerol suppressed RCC cell migration and metastasis in vitro and in vivo. Moreover, reduction in MMP2, Slug, and Vimentin protein levels was observed following 6‐gingerol treatment of 786‐O and ACHN cells. Furthermore, we revealed the mechanisms underlying the ability of 6‐gingerol to inhibit RCC cell migration and metastasis. 6‐Gingerol increased yes‐associated protein (YAP)ser127 phosphorylation and reduced YAP levels in cell nuclei. We also used a series of loss‐of‐function and gain‐of‐function experiments to support our results. Western blot results showed that MMP2, Slug, and Vimentin protein expression was downregulated in YAP‐silenced cells and upregulated in YAP‐overexpressing cells. Transwell data demonstrated that YAP suppressed RCC migration ability. Immunofluorescence images showed that 6‐gingerol decreased YAP levels, leading to disordered F‐actin and a reduction in cell lamellipodia. Overall, our results indicated that 6‐gingerol is a potential antimetastatic compound for use in kidney therapy.

sunitinib) have been used alone or in combination for the treatment of advanced or metastatic renal cancer. [3][4][5] The combination of nivolumab plus ipilimumab (anti-PD-1 plus anti-CTLA-4), avelumab plus axitinib (anti-PD-L1 plus VEGFR inhibitor), or atezolizumab plus bevacizumab (anti-PD-L1 plus VEGF inhibitor) improves the clinical outcome compared with sunitinib treatment in advanced or metastatic renal cancer. [6][7][8] However, serious side effects are still an urgent problem in clinical immunotherapy. Chemotherapy is still often used for kidney cancer after targeted drugs and/or immunotherapy have been attempted (http://www.cancer.org/cancer/kidney-cancer/ treating.html). Natural products are of increasing interest for investigating adjunctive therapeutic options in clinical therapy due to their nontoxic and wide antitumor effects.

| Western blot assay
The protocol was performed as described previously. [36] Antibodies

| Statistics
The data in this study, including standard deviations, medians, and ranges, were analyzed by GraphPad Prism 5 (GraphPad Software, La Jolla, CA). The significant differences between two groups were analyzed with unpaired the Student t tests. P < .05 is considered significant.

| 6-Gingerol inhibits renal cancer cell migration in vitro
To respectively). The IC50 of 6-gingerol was 30 μM in our previous data, [22] so 30 μM 6-gingerol was chosen for the following experiments. Furthermore, the inhibitory effect of 6-gingerol on RCC cell migration was confirmed by wound healing assays. As shown in 3.2 | 6-Gingerol suppresses renal cancer cell migration by decreasing YAP protein levels To explore the molecular mechanisms underlying the inhibition of cell migration by 6-gingerol in RCC, we first tested the effect of 6-gingerol on the levels of the Hippo pathway effector YAP; YAP plays a key role in tumor migration. The results showed that treatment with 10 to 50 μM 6-gingerol did not affect YAP messenger RNA levels ( Figure S1A). However, 6-gingerol increased YAP phosphorylation at Ser 127 , resulting in a dose-responsive decrease in the YAP amount in 786-O and ACHN cells from 10 μM 6-gingerol to 50 μM 6gingerol ( Figure 2A). To find the mechanism of 6-gingerol phosphorylates YAP, Lats1, the upstream of YAP, was detected in our study. As shown in Figure 2A Figure 2D). In sum, our results revealed that 6-gingerol inhibits cell migration by decreasing YAP protein levels in RCC.

| The inhibition ability of 6-gingerol is not reversed by ectopic expression of YAP in RCC cells
To further biochemically validate the effect of YAP on kidney cancer cell migration, ectopic expression of YAP was introduced in 769-P cells. As shown in Figure 3A, overexpression of YAP in 769-

P cells resulted in a significantly higher number of migrating cells than what was observed in the vector-transfected cells (P = .013).
In addition, 6-gingerol treatment was assessed in migration assays, and we found that 6-gingerol significantly decreased the number of cells that passed through the transwell membrane compared to that of the control (P = .002). However, following the ectopic ex-

| 6-Gingerol decreases YAP nuclear localization
YAP is the major mediator of the Hippo-YAP pathway, and YAP translocates to the cell nucleus to activate downstream gene transcription.
Our results show that 6-gingerol phosphorylated YAP (Figure 2A). We thus speculate that 6-gingerol inhibits RCC cell migration by decreasing YAP nuclear localization. We first tested cytoplasmic and nuclear protein  Taken together, the molecular mechanism underlying 6-gingerol inhibition of RCC cell migration was 6-gingerol-mediated phosphorylation of YAP, resulting in a decrease in the nuclear localization of YAP.

| 6-Gingerol inhibits tumor migration in nude mice
The migration inhibition capacity of 6-gingerol was validated in an ACHN xenograft model. A total of 2 × 10 6 cultured ACHN cells were injected into nude mice by tail vein injection (n = 10), and the mice were randomly separated into two groups (five mice in one group).
Control group mice (n = 5) were treated with 100 μM corn oil, and the 6-gingerol treatment group (n = 5) was treated with 2.5 mg/kg 6gingerol every 3 days ( Figure 5A). Meanwhile, mice were weighed every 3 days, and the physical state of the mice was monitored throughout all experiments. As previously reported, there was no significant body weight loss between the 6-gingerol treatment group and the control group ( Figure 5B). We observed that tumor metastasis was strongly inhibited in the 6-gingerol group compared with that of the control group by measuring the value of maximum and minimum luminescence signal ( Figure 5C). As shown in Figure 5C, there were multiple tumor metastases in the control group (n = 5), including leg metastases, spinal metastases, and lung metastases.
However, in the 6-gingerol treatment group, tumor cell numbers at metastatic sites were reduced compared with those in the control group. Our results suggested that 6-gingerol inhibits RCC cell migration in vivo.

| DISCUSSION
Because RCC is considered to be an incurable disease that commonly develops drug resistance, [37] prior drugs targeting VEGF and mTOR signaling caused by VHL mutations and immunotherapeutic treatments have shown limited clinical benefit in RCC clinical treatment. [23] 6-Gingerol is one of the most consumed dietary components worldwide. [9,11] Our study conducted a series of experiments to expand the understanding of 6-gingerol antitumor activity in RCC. In our study, we found that 6-gingerol suppresses kidney cancer metastasis in vivo and in vitro. Furthermore, we demonstrated that 6-gingerol decreased cell migration primarily by decreasing Vimentin, Slug, and MMP2 protein levels. Meanwhile, F-actin staining assays showed that cell pseudopods and F-actin were reduced in 6-gingerol-treated cells. The mechanism underlying the 6-gingerol inhibition of RCC migration was that the amount of YAP in the nucleus was decreased by 6-gingerol.
Ginger is a food product and has been used in medicine and daily life. [11] In our study, a significant decrease in organ metastasis F I G U R E 3 YAP could not reverse the effect of 6-gingerol on tumor cell migration. A, Transwell assays were performed to test the migration ability of 769-P cells overexpressing YAP. 769-P cells were seeded in 6 cm dishes and were transfected with a plasmid ectopically expressing either YAP or NC. Then, the cells were harvested, and 4 × 10 4 cells were seeded in the upper chamber and incubated for 24 hours. The cells were stained with crystal violet and were visualized under a microscope. The cell numbers were determined using ImageJ. Statistical analysis of the data between two groups was performed using the Student t tests, and error bars indicate ±SD. B, Immunoblot analysis of Vimentin, Slug, and MMP2 was performed on 769-P cells ectopically expressing YAP. 769-P cells were transfected with plasmids ectopically expressing either YAP or NC for 48 hours. Then, the cells were lysed, and the protein levels of Vimentin, Slug, MMP2, YAP, and p-YAP were analyzed by Western blot assay. SD, standard deviation; YAP, yes-associated protein and tumor cell numbers in metastatic sites were observed in the 6-gingerol treatment group ( Figure 5). In addition, our results confirmed that 6-gingerol had no effect on mouse body weight and lifespan when we compared the 6-gingerol treatment group with the control group ( Figure 5B). These findings were verified and are consistent with previous reports in other cancers. 6-Gingerol has been previously shown to suppress tumor cell growth by inhibiting the AKT-GSK 3β pathway and cyclins in RCC, [22] pancreatic cancer, [13] cervical adenocarcinoma cells, [12] and colorectal cancer cells. [16] Previous research results showed that 6-gingerol induced tumor cell apoptosis by activating caspases 3 and 9 and by modulating mitochondrial functions in colorectal cancer cells, [15] oral tumor cells, [19] and cervical tumor cells. [18] 6-Gingerol has also been shown to suppress breast cancer cell invasion by decreasing MMP2 and (A) F I G U R E 4 6-Gingerol decreases YAP localization in the nucleus. A, YAP in nuclear and cytoplasmic protein fractions was measured after 6-gingerol treatment. 786-O cells were seeded in 10 cm dishes and treated with 30 μM 6-Gingerol for 48 hours. Cells were lysed, and the amount of YAP protein in the nucleus and cytoplasm was analyzed by Western blot assay. B, YAP and F-actin were detected using an immunofluorescence assay. 786-O and ACHN cells were treated with 6-gingerol for 48 hours. YAP expression and distribution were detected using immunofluorescence, F-actin was stained by Phalloidin-iFluor 594, yellow arrow represents lamellipodia, and nuclei were labeled with DAPI. DAPI, 4′,6-diamidino-2-phenylindole; YAP, yes-associated protein MMP9 levels. [21] However, the mechanism behind these decreases is unclear. In our experiments, we confirmed that the activities of YAP were decreased by 6-gingerol treatment and found that the levels of phosphorylated YAP were increased in 786-O and ACHN cells that were treated with 6-gingerol ( Figure 2B). Meanwhile, 6-gingerol enhanced phosphorylated AMPKα amount in a dose-dependent manner (Figure 2A), and AMPKα was reported to activate Lats1. [26] In our results, Lats1 was also increased after 6-gingerol treatment ( Figure 2A). In addition, 6-gingerol was found to decrease the amount of YAP protein in the nucleus of 786-O and ACHN cells after treating them with 6-gingerol at IC50 levels (30 μM) (Figure 4). 6-Gingerol inhibited RCC cell migration through the AMPK/Last1/YAP pathway in our study.
YAP is the key downstream target of the Hippo signaling pathway, [38] it has been reported to be highly expressed in human kidney cancer and other types of cancers. [33,39] Previous studies confirmed that YAP is an important regulator of organ growth, stem cell self-renewal, differentiation, and tumor cell growth/migration. [38] MMP2, Vimentin, and Slug significantly contribute to the migration and metastasis of tumor cells. In our study, we confirmed that 6-gingerol suppressed MMP2, Vimentin, and Slug protein levels by decreasing the nuclear levels of YAP. YAP has been reported to act as a promoter in tumor metastasis in many cancers. Bin You et al [40] demonstrated that ERK1/2 inhibitors inhibited the migratory and invasive activity of NSCLC by promoting YAP degradation. Haskins et al [41] reported that members of the epidermal growth factor (A) (B) (C) F I G U R E 5 6-Gingerol inhibits tumor metastasis in vivo. A, The protocol of the animal experiment. Ten BALB/c nude mice were injected with 2 × 10 6 ACHN cells by tail vein and then were randomly separated into two groups. One group of mice (n = 5) was treated with corn oil, and one group of mice (n = 5) was treated with 2.5 mg/kg 6-gingerol every 3 days thereafter via tail vein injection of tumor cells until the end of the experiment. B, Mice were weighed every 3 days, and the weights were compared between the two groups. Statistical analysis of the data between two groups was performed using the Student t tests; error bars indicate ±SD, n = 5. C, Luminescent images of the metastases were captured in the control and 2.5 mg/kg 6-gingerol groups. Mice were injected intraperitoneally with 300 mg/kg D-luciferin potassium salt 10 minutes before imaging, and representative bioluminescence imaging of the metastases was acquired with IVIS Lumina. The value of bioluminescence was compared between the two groups. Statistical analysis of the data between two groups was performed using the Student t tests, and error bars indicate ±SD. max represents maximum; min represents minimum. SD, standard deviation receptor family ERBB4 promote breast cancer cell migration by activating YAP to regulate its target genes. Feng et al [42] showed that thromboxane A2 receptors stimulate vascular smooth muscle cell migration by upregulating YAP/TAZ. Furthermore, F-actin homeostasis and tumor cell lamellipodia were all disordered in 6-gingeroltreated 786-O and ACHN cells ( Figure 2B). In addition, we demonstrated that F-actin was regulated by YAP in our study. These results were consistent with those reported by Kim et al [27] in SKMEL28 and WM3248 cells and by Feng et al [42] in vascular smooth muscle cells.
Therefore, we concluded that 6-gingerol induces RCC cell migration inhibition though the Hippo-YAP signaling pathway in vitro.
In conclusion, our results confirmed that 6-gingerol inhibits metastasis of the 786-O and ACHN kidney cancer cell lines in vivo and in vitro. The mechanism behind the anticancer effect of 6-gingerol was the phosphorylation of YAP, which led to a decrease in YAP nuclear localization. Considering that 6-gingerol is one chemical component of ginger, which is commonly ingested in the human diet (250 mg-1 g/day), our results suggested that 6-gingerol would be safe and useful for preventing or treating kidney cancer.