Metformin inhibits the growth of ovarian cancer cells by promoting the Parkin-induced p53 ubiquitination

Ovarian cancer is the most lethal diseases among women. The chemo-resistance has been a big challenge for the cancer treatment. It has been reported that metformin may inhibit ovarian cancer and is able to impede the development of drug resistance, but the molecular mechanisms remain elusive. In this study, we explored the molecular roles of metformin in Parkin expression and p53 ubiquitination in chemo-resistant ovarian cancer cells. Firstly, ovarian cancer and chemo-resistant ovarian cancer cells were selected for determining the expression of Parkin, p53, and p53 signaling pathway-related factors. Then the cell proliferation and viability after loss- and gain-of-function assays were measured. Besides, immunoprecipitation (IP) was used to determine the interactions between Parkin and p53, and the ubiquitination level of p53 was measured using in vitro ubiquitination assay. Finally, the degradation of p53 proteasome regulated by Parkin was monitored using the MG132 proteasome inhibitor. We found that metformin significantly inhibited the growth of ovarian cancer parental cells and chemo-resistant cells, and metformin promoted Parkin expression in chemo-resistant cells. Further, up-regulated Parkin expression promoted the ubiquitination and degradation of p53, and metformin inhibited the expression of p53 to suppress the proliferation of chemo-resistant ovarian cancer cells. Mechanistically, metformin could inhibit the growth of ovarian cancer cells by promoting the Parkin-induced p53 ubiquitination. Altogether, our study demonstrated an inhibitory role of metformin in the growth of chemo-resistant cancer cells through promoting the Parkin-induced p53 ubiquitination, which provides a novel mechanism of metformin for treating ovarian cancer.


Introduction
Ovarian cancer is one of the most prevalent gynecological malignant tumors [1]. The incidence of ovarian cancer differs among countries: the highest incidence rates are shown in the developed regions, while the lowest rates observed in African and Asian parts [2]. Due to the limitations of therapeutic treatments, there's rapid development and frequent cancer recurrence for this disease, making the outcome of ovarian cancer treatment comparatively poor [3]. Moreover, increasing number of patients with relapse in one to two years under different circumstances gradually develop resistance to the chemotherapies [4]. Therefore, it is urgent to explore the underlying molecular mechanisms of progression of chemo-resistance of ovarian cancer and to identify novel targets for the therapy of ovarian cancer. Recently, a study has indicated that metformin may have an anti-cancer effect on ovarian cancer through inhibition of the ovarian cancer cell proliferation [5].
Metformin (1,1-dimethylbiguanide) is one of the most effective treatment to patients with type 2 diabetes mellitus (T2DM), making itself the most commonly prescribed therapy for T2DM [6].
Many studies have reported a survival benefit with metformin use in many cancers. For example, a study found that those patients with non-small cell lung cancer (stage IV) who received metformin showed notably higher survival rate [7]. Additionally, the previous study has demonstrated that metformin suppresses the expression of ovarian cancer-related genes, cell migration and proliferation of ovarian cancer cells, which suggests that it can be a therapeutic target for ovarian cancer [8]. However, the role of metformin mechanism in the chemo-resistance in ovarian cancer is rarely discussed and needs to be further explored. Among the various ubiquitin ligase, the impacts of Parkin on different diseases have been recently revealed. For instance, as an E3 ubiquitin ligase, Parkin is involved in the cell biological function of keloids [9]. In Parkinson's disease, Parkin is a tumor suppressor that affects necroptosis and inflammation [10]. Moreover, the chemo-resistance is Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200679/892162/bsr-2020-0679.pdf by guest on 18 October 2020 Bioscience Reports. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BSR20200679 controlled by Parkin in mitophagy [11]. However, there are few studies on the relationship between Parkin and ovarian cancer. Furthermore, as one of the most studied oncogenes, p53 mutations are commonly found and its expression is altered in nearly a half of human malignant tumors [12]. It is reported that the aggregation of p53 exerts crucial function in tumor progression and cell survival, which is associated with the chemo-resistance reversal among ovarian cancer patients [13], which makes it a critical therapeutic target for cancers.
From all above, we hypothesized that metformin could affect the chemo-resistant ovarian cancer cells by regulating the expression of Parkin as well as the p53 ubiquitination. Therefore, in our study, we explored the correlations among metformin treatment, Parkin and p53, aiming to provide a novel treatment and therapeutic targets for the ovarian cancer.
A2780-cis and IGROV1-cis cells were cultured in a 6-well plate, with the concentration of 2 × 10 5 cells per well. When the cell confluence reached 80%, the cells were transfected according to Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200679/892162/bsr-2020-0679.pdf by guest on 18 October 2020 Bioscience Reports. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BSR20200679 Lipofectamine 2000 instructions (11668-019, Invitrogen, Carlsbad, California, USA). The cells were treated with the plasmids of siRNA against Parkin or p53 and their corresponding non-sense RNA negative control (NC). All the plasmids above were purchased from GenePharma Co., Ltd (Shanghai, China). The cells after treatment were cultured at 37℃ and 5% CO 2 for 48 h, which was used for subsequent experiments.

Clonogenic assay
Cell proliferation was detected using the clonogenic assay. First, 1.2 % agar was heated, dissolved and then placed in water at 46℃. The prepared ovarian cancer cells were counted and suspended in preheated RPMI1640 medium (40℃) containing 40% fetal calf serum (FCS). A total of 325 μL of the cell suspension (including 1 × 10 5 cells) was added to each well of the 24-well culture plate, and 50 μL of the sample to be tested was added. A suitable amount of 125 μL preheated agar (1.2%) was mixed with the cell suspension and the sample to be tested in a 24-well plate. The mixture was incubated at 37℃ and 5% CO 2 for 8-10 days after coagulation, and then observed under an inverted microscope. Each experiment was repeated three times.
(Cambridge, UK). The gray value of each band was analyzed by gel image analysis software Image J.

Immunoprecipitation (IP)
The cells were lysed in the EBC lysis buffer. The soluble supernatant was mixed with polyclonal anti-p53 antibody and incubated overnight at 4℃. Protein A/G beads were then added to the reaction mixture. After washing the immune complexes, the protein was re-suspended in SDS sample buffer and separated by SDS-PAGE. The monoclonal rabbit antibody against Parkin (ab77924, 1 : 100, Abcam Inc., Cambridge, UK) was analyzed by western blot analysis. The experiment details were referred to the methods established before [15].

In vitro ubiquitination assay
In vitro ubiquitination assay was carried out using the commercially available kit (Boston Biochem, Inc., Cambridge, Ma, USA) according to the manufacturer's instructions. The specific operation methods were referred to previous study [15]. Briefly, the HEK293 cell was transfected with the plasmids Ha-ubiquitin (2 μg), Flag-p53 (1 μg) and myc-Parkin (1 μg) using the Lipofectamine 3000.
After 48 h, the cells were collected for western blot analysis and ubiquitination detection. The protein was analyzed and eluted by western blot analysis with p53 monoclonal antibody.

Statistical analysis
Statistical analysis was conducted using SPSS 21.0 statistical software (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. The unpaired data conforming to normal distribution and homogeneous variance between two groups were analyzed by unpaired t-test. Comparisons among multiple groups were conducted using the one-way analysis of variance (ANOVA) with Tukey's post hoc test used. Data comparison among groups were analyzed by the repeated measures ANOVA followed by Bonferroni's post hoc test. A value of p < 0.05 was considered as statistically significant.

Metformin inhibits the growth of ovarian cancer parental cells and chemo-resistant cells
In order to explore the effect of metformin on the chemo-resistance of ovarian cancer cells, the ovarian cancer parental cells A2780 and IGROV1 and chemo-resistant cell A2780-cis, IGROV1-cis were treated with different concentrations (0, 5, 10, 20 mM) of metformin, respectively. The proliferation of the aforementioned cells treated with different concentrations of metformin was measured by the clonogenic assay. The results showed that the growth of cells was inhibited by metformin in a dose-dependent manner ( Figure 1A and 1B). Subsequently, MTT assay was used to measure the viability of cells treated with different concentrations of metformin. The results depicted that the increase of metformin concentration led to decreased cell viability, and the viability of chemo-sensitive cells was significantly lower than that of chemo-resistant cells ( Figure   1C). The aforementioned findings revealed that metformin significantly inhibited the growth of both chemo-sensitive and chemo-resistant ovarian cancer cells.

Metformin promotes the Parkin-mediated ubiquitination of p53 in chemo-resistant ovarian cancer cells
Given that Parkin is an important E2 ubiquitin ligase, we aimed to determine whether metformin promoted the ubiquitination and degradation of p53 by upregulating the expression of Parkin.
Therefore we detected the binding of Parkin to p53 in A2780-cis and IGROV1-cis cells by IP assay.
The results demonstrated the interaction between Parkin and p53 in A2780-cis and IGROV1-cis cells ( Figure 3A). Following after, the ubiquitination of p53 in cells treated with siRNA against  Figure 3E). These results suggested that the ubiquitination of p53 was mediated by Parkin in ovarian cancer chemo-resistant cells, and the degradation of p53 was promoted by metformin.

Metformin suppresses the proliferation of chemo-resistant ovarian cancer cells through downregulation of the expression of p53
To further explore whether p53 is involved in the chemo-resistance of ovarian cancer cells, we treated A2780-cis and IGROV1-cis cells with different concentrations of metformin to measure the related protein expression of p53 and p53 pathway-related factors (phosphorylated p53, p21 and MDM2). The results showed that the expression of p53 and p53 pathway-related factors were decreased significantly by metformin treatment in a dose-dependent manner ( Figure 4A). Given the effects of metformin treatment on p53 signaling, we'd like to know whether metformin affected the chemo-resistance of ovarian cancer cells through Parkin/p53 signaling pathway. Thus we silenced p53 expression in A2780-cis and IGROV1-cis cells with siRNA with the silencing efficacy confirmed by RT-qPCR and western blot analysis that the mRNA and protein expression of p53 were notably reduced after siRNA against p53 treatment ( Figure 4B). Then with silenced p53 expression, we treated the cells with 20 mM metformin and measured the expression of p53.
Surprisingly, the expression of p53 was further reduced with metformin treatment ( Figure 4B). Then clonogenic assay was used to detect the proliferation of cells in different groups. Silencing p53 significantly inhibited the proliferation of cells which was further inhibited by metformin treatment ( Figure 4C). MTT assay was used to detect the cell viability in different groups. The results demonstrated that silencing p53 notably reduced the effect of metformin on cell viability ( Figure   4D). Additionally, Western blot analysis was performed to measure the protein expression of p53 pathway in different groups of cells. The results revealed that silencing p53 suppressed the expression of p53 pathway-related factors in cells. Besides, cells with siRNA against p53 after metformin treatment showed further inhibited expression of p53 pathway-related factors, and increased Parkin expression ( Figure 4E). These results indicated that through Parkin/p53 pathway, metformin inhibited the proliferation of ovarian cancer chemo-resistant cells.

Discussion
The etiology and molecular biology of the malignancy of ovarian cancer remain elusive [16]. With the progress of ovarian cancer, the cell biological properties of the cancer cells becomes more and more complicated, and most patients who previously positively respond to chemotherapy may die of recurrent chemo-resistant tumors. This indicates that the chemo-resistance is dynamically developed and hard for patients to overcome [13]. Therefore, to better understand the molecular mechanisms of chemo-resistance and discovery of therapeutic targets is critical for the treatment of ovarian cancer cells. As a FDA approved drug for treating T2DM, metformin has also been reported to play an inhibitory role in the development of cancer stem cells and epithelial ovarian cancer cell [17]. However, the mechanism remains unclear. Our study explored the role of metformin in ovarian cancer chemo-resistant cells and we found that metformin inhibits the growth of ovarian cancer parental cells and chemo-resistant cells through acting on Parkin and p53 ubiquitination. As we have shown, metformin significantly suppressed the growth of chemo-resistant ovarian cancer cells. What's more, many other studies have found that metformin is a potential treatment for diverse cancers. For example, a previous study reveals that metformin up-regulates miR-708-5p expression and promotes the cell apoptosis in prostate cancer [18]. The correlation between metformin and ovarian cancer has been explored. Metformin affects the ovarian cancer cell metabolism, prevents the growth of tumors and strengthens the sensitivity to chemotherapies both in vivo and in vitro, indicating that metformin could be a drug for the treatment of ovarian cancer [5]. Consistent with our findings, a previous study has demonstrated the inhibitory effect of metformin on ovarian cancer cell migration and proliferation as well as the expression of cancer-related genes of adipocyte-mediated cell [8].
The growth of cancer cells was restrained by the regulation of Parkin by metformin. As a tumor suppressor, Parkin also plays an inhibitory role in breast cancer metastasis [19]. A previous study shows that Parkin expression was downregulated in the cells and tissues of breast cancer, suggesting that Parkin is involved in the development of breast cancer [20]. Previous studies also revealed the correlation between metformin and Parkin. For example, metformin could significantly increase the expression of Parkin and promote the interaction between Parkin and hypoxia-inducible factor-1α, indicating that the cooperation of metformin and Parkin can be a therapeutic agent for keloids [9]. This finding is exactly consistent with our results that metformin promoted the expression of Parkin. Moreover, our study also showed that silencing Parkin by siRNA could increase the proliferation and viability of chemo-resistant A2780-cis and IGROV1-cis cells.
Correspondingly, previous study unraveled that the loss of Parkin accelerated the progression of cancers including lung cancer and breast cancer [21]. Taken together, Parkin plays critical roles in multiple cancers.
Additionally, we also found that metformin treatment decreased the expression of p53 and p53 pathway-related factors in a dose-dependent manner. Qiang et al. reported that metformin could inhibit the activation of MDM2 to inhibit malignant phenotypes of endometrial cancer cells [22]. Importantly, targeting p53-MDM2 pathway is demonstrated as the goal of preventing and treating cancers in the current stage [23]. Interestingly, our study further illustrated the relation between Parkin and p53 ubiquitination. It has been reported that Parkin interacts with p53, which leads to the loss of p53 function in rheumatoid arthritis [15]. A previous study has demonstrated that the binding of metformin and 2-deoxyglucose can promote the p53-dependent apoptosis in prostate cancer In conclusion, we find that metformin can upregulate the expression of Parkin and promote p53 ubiquitination to inhibit the growth of ovarian cancer parental cells and chemo-resistant cells ( Figure 5). These findings revealed that metformin could serve as a therapeutic agent for ovarian cancer. However, more studies remains needed to further explore the mechanisms and overcome the limitations of this study. For instance, the in vivo experiments are required in future study. Besides, the interactions among metformin, Parkin and p53 remain to be more identified in ovarian cancer.
We will further explore the underlying rules governing metformin-Parkin interaction in the future.