Statins as Repurposed Drugs in Gynecological Cancer: A Review

Discovering new drugs is an expensive and time-consuming process, including target identification, bioavailability, pharmacokinetic (PK) tests, pharmacodynamic (PD) tests, toxicity profiles, recommended dosage test, and observation of the side effects, etc. Repurposed drugs could bypass some steps, starting from phase II trials, and shorten the processes. Statins, also known as HMG-CoA inhibitors (HMGCR), are commonly used to manage and prevent various cardiovascular diseases and have been shown to improve the morbidity and mortality of patients. In addition to the inhibitory effects on the production of cholesterol, the beneficial effects of statins on the prognosis and risk of various cancers are also shown. Statins not only inhibited cell proliferation, metastasis, and chemoresistance but affected the tumor microenvironment (TME). Thus, statins have great potential to be repurposed in oncology. Hence, we review the meta-analysis, cohort, and case-control studies of statins in gynecological cancers, and elucidate how statins regulate cell proliferation, apoptosis, tumor growth, and metastasis. Although the results in gynecological cancers remain controversial and the effects of different statins in different histotypes of gynecological cancers and TME are needed to elucidate further, statins are excellent candidates and worthy of being repurposed drugs in treating gynecological cancers.


Introduction
Gynecological cancer is any cancer that starts in a woman's reproductive organs, including cervical, endometrial, ovarian, vaginal, and vulvar cancer. The treatments generally include surgery, radiation therapy, chemotherapy, target therapy, and immunotherapy. Combination therapy is a trend worldwide. However, discovering new drugs or targets is always the mission against cancers. There is an established setting for new drug discovery from pre-clinical results, in vitro and in vivo, to human studies, phase I and II trials, and a phase III randomized controlled trial (RCT). It is expensive and takes over 10 years in all processes [1]. Thus, if the existing drugs could be repurposed, it can dramatically reduce costs and save time, benefiting patients who suffer from these malignant and lethal diseases.
Statins, as 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase competitive inhibitors (HMGCR), are commonly used as lipid-lowering drugs, preventing cardiovascular diseases. However, the anti-cancer properties of statins have been investigated in recent decades, showing better prognoses in various types of cancer through various mechanisms [2,3]. The evidence of the anti-cancer effects of statins in gynecological cancers is sparse and controversial, thus, we review and assess the potential of statins as repurposed drugs in gynecological cancers.

Endometrial Cancer and Its Risk Factors
Endometrial cancer (EC) is the sixth most common cancer in females and the second most commonly diagnosed cancer of female reproductive organs. Around 417,000 new cases were detected, and 97,000 women died worldwide from the disease in 2020 [10] [11]. There are two main types of ECs that were characterized. Type I ECs, around~80%, are mostly well differentiated with endometrioid histology and show a high level of estrogen receptor (ER). Type II ECs are poorly differentiated with serous or clear cell histology and show a high recurrence rate (80%~90%) within 3 years, representing a poor prognosis [12]. In addition, ECs can be low-grade (grades 1 and 2) tumors which are generally associated with a better prognosis, or high-grade carcinomas (grade 3) carrying an intermediate prognosis [13]. The risk factors for EC include high body mass index (BMI: kg/m 2 ), often with other components of metabolic syndrome (e.g., hypertension, diabetes), nulliparity or infertility, early menarche, and late menopause.
The relative risk (RR) for developing EC with metabolic syndrome was 1.89 [95% confidence interval (CI) 1.34 to 2.67, p < 0.001] among the components of metabolic syndrome. Obesity (BMI > 30) was associated with the greatest increase in RR of 2.21 (p < 0.001). Other components of the metabolic syndrome linked to endometrial cancer include hypertension, with a RR of 1.81 (p < 0.05). [14] Type II Diabetes mellitus (DM) showed an independent risk factor for EC, with an approximate doubling of risk [Odds ratio (OR) was 2.18, 95% CI 1.40 to 3.41] [15]. Among the causes of infertility, polycystic ovarian syndrome (PCOS) showed an increase in risk (OR = 2.79, 95% CI 1.31 to 5.95) [16]. Both early menarche (RR was 2.4 for women <12 vs. ≥15 years) [17], and late menopause (RR = 1.8 for women ≥55 versus <50 years) [18] are associated with increased risk for EC.

Ovarian Cancer and Its Risk Factors
Ovarian cancer is the leading disease of death in females diagnosed with gynecological cancers. In the meantime, it is women's fifth most frequent cause of death. There are approximately 21,750 new ovarian cancer cases in the US, comprising 1.2% of all cancer cases. The estimated number of deaths related to ovarian cancer was 13,940 in 2020 [19]. Among the ovarian cancer subtypes, type II high-grade serous carcinoma (HGSC) is the most prevalent and lethal, representing more than 70% of ovarian cancer. Type I tumor includes low-grade serous, endometrioid, clear-cell, and mucinous carcinomas, presenting at an early stage and carrying a good prognosis except for clear-cell [20]. HGSCs arise from serous tubal intraepithelial carcinoma (STIC) in the fimbriae of the tube, undergoing malignant transformation and metastasizing to the nearby ovaries and peritoneum [21,22].
The risks of ovarian cancer were increasing in postmenopausal women and those with a family history of breast or ovarian cancer. At the same time, pregnancy, lactation, and oral contraceptive pills reduced the risks [23]. Moreover, obesity was an independent prognostic factor in addition to advanced tumor staging and positive ascites cytology. The hazard ratio (HR) of overall survival (OS) was 1.871, 95% CI 1.005 to 3.486 in all ovarian cancer patients, and the HR was elevated to 2.405, 95% CI 1.335 to 4.333 in pT3 stage patients [24].

Statins, Lipid-Lowering Drugs
Statins are traditionally applied in cardiovascular diseases to reduce cholesterol [25] and could be divided into two groups: type-I derivatives (from fermentation, including mevastatin, lovastatin, pravastatin, and simvastatin), and type-II drugs (from the synthetic origin, including fluvastatin, atorvastatin, cerivastatin, pitavastatin, and rosuvastatin) [26,27]. The main role of statin in the mevalonate pathway is inhibiting HMG-CoA reductase (HMGCR), resulting in the depletion of LDL cholesterol [28] (Figure 1). The statins were used between 10-80 mg, and the metabolic pathway of statins was major through CYP3A4 [29] (Table 1). However, recent studies suggested that statins could have anti-tumor effects (Figures 1 and 2), from meta-analysis and bench, in vitro and in vivo.

Statins in Cancer
The most investigated statin in cancer is simvastatin. In general, the role of statins was tumor suppressor. Statins could induce cancer cell apoptosis through traditional Figure 2. The effects of statins on anti-tumor progression and tumor microenvironment (TME). TME includes immune cells and MSCs. Green arrows represent inhibitory effects, and red arrows represent promoting effects.

Statins in Cancer
The most investigated statin in cancer is simvastatin. In general, the role of statins was tumor suppressor. Statins could induce cancer cell apoptosis through traditional caspases cascade and inhibit cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and chemoresistances in various types of cancer ( Figure 2), including breast, lung, pancreas, and liver cancer [30]. Statins induced apoptosis of cancer cells through NFκB and the canonical caspase pathway and reduced proliferation through MEK1/2, ERK1/2, and JNK pathways [31]. Statins also induced cell cycle arrest of cancer cells by activating AMPK and increasing p21 and p27 expression [32]. Simvastatin suppresses the invasion of cancer cells by decreasing Pituitary Tumor-Transforming Gene 1 (PTTG1) [33]. Furthermore, statins could also regulate epigenetic machinery resulting in cell cycle arrest. DNMTs could be the targets of statins and the downstream p16 protein [34] and p21 [35]. In conclusion, statins showed anti-tumor progression in various cancers ( Figure 2).

Statins in Immune Cells
Mostly, statins showed anti-inflammatory effects and enhanced the number of regulatory T cells (Treg) [36], which may result in the suppression of the Th1 immune response [37]. In addition, statin treatment reduced the Th17 population [38]. Treg obtained immunosuppressive effects on immunotherapy. However, a high dose of Atorvastatin could reduce the in vitro function of conventional T and regulatory T (Treg) cells [39]. Furthermore, statins were associated with better clinical outcomes in patients treated with PD-1 inhibitors [40]. Statins plus Th1 cytokines or dendritic cells (DC)-based immunotherapy could suppress breast tumor growth [41]. Statins could stimulate immunogenicity and promote an antimelanoma immune response [42]. These data showed the conflicting roles of statins in immunotherapy. Thus, the roles of statins in immune cells and immunotherapy are needed to be elucidated.

Statins in MSCs
Statins had several effects on mesenchymal stem cells (MSCs). Statins could enhance the osteogenic differentiation, angiogenic potential, migration, homing, survival, and proliferation of MSCs [43], which may have improved therapeutic outcomes in regenerative medicine. The evidence of statins in regulating cancer-associated MSCs (CaMSCs) is limited. Simvastatin could decrease CCL3 expression from cancer cells and ICAM-1, VCAM1, IL-6, and CCL2 expression from CaMSCs, disrupting the crosstalk of the cancer cells and tumor microenvironments (TME) and inhibiting tumor progression [44]. Therefore, the roles of statins in the TME-not only in immune cells but also MSCs, especially CaMSCs-need further investigation.
In summary, the effects of statins in treating EC are still controversial. However, large results suggested that statins may be potent drugs to decrease the risks and mortalities of EC, and are worth performing clinical trials.
Because ovarian cancer has different histotypes, statin use did not show significant differences in risks in serous and clear cell types [59], but the mortality decreased [75,76]. The results of statin use in ovarian cancer patients remained controversial. Thus, additional studies are needed to elucidate the effects of different statins on different histotypes of ovarian cancer.

Cohort Studies in Other Gynecological Cancers
The HR association between the risk of cervical cancer and statin use was 0.83, 95% CI of 0.67 to 0.99. Statin use was associated with decreased total gynecological cancer mortality, (HR = 0.70, 95% CI 0.50 to 0.98) [77]. The statin use group had a better prognosis compared with the non-user (progression-free survival: HR = 0.062, 95% CI 0.008 to 0.517; overall survival: HR = 0.098, 95% CI 0.041-0.459) in cervical cancer patients [78] ( Table 4). The effects of statin use against cervical cancer and vulvar cancer are not conclusive due to too few studies and case numbers [61]. In conclusion, statin use may obtain protective effects on cervical cancer, but the evidence is too few. CI: confidence interval. HR: hazard ratio.

The Mechanisms of the Anti-Tumor Effects of Statins on Gynecological Cancer
Simvastatin exhibits anti-metastatic and anti-tumorigenic effects in ECC-1 and Ishikawa EC cells through mitogen-activated protein kinase (MAPK) but not the Akt/mTOR pathway [79]. The drug for diabetes, metformin, combined with simvastatin, synergistically inhibited cell growth and induced Bim expression and apoptosis in RL95-2, HEC1B, and Ishikawa EC cells. The combination treatment of metformin and simvastatin upregulated phosphorylated AMPK (pAMPK) and downregulated downstream phosphorylated S6 (pS6), suggesting the mTOR pathway may regulate these anti-proliferative effects [80]. Lipophilic (lovastatin and simvastatin) but not hydrophilic (pravastatin) statins induced apoptosis in ovarian cancer cell lines A2780 and UCI 101; endometrial cancer cell line, Ishikawa; and cervical cancer cell line, HeLa, which all expressed high levels of HMG-CoA reductase [81] (Table 5). AMPK, Akt/mTOR. [92] Lovastatin and Pravastatin decreased metastasis through RhoA signaling in vitro and in vivo of SKOV3 ovarian cancer cells [82]. In addition, lovastatin and atorvastatin induced apoptosis in Hey 1B and Ovcar-3 ovarian cancer cells and suppressed anchorageindependent growth of these cells through the JNK/Rac1/Cdc42 pathway [83]. Lovastatin synergizes with doxorubicin to induce apoptosis by a novel mevalonate-independent mechanism [84]. In the mogp-TAg mice model, the promoter of oviduct glycoprotein-1 was used to drive the expression of SV40 T-antigen, and serous tubal intraepithelial carcinomas (STICs) were developed in gynecologic tissues. Lovastatin significantly reduced the development of STICs in mogp-TAg mice and inhibited ovarian tumor growth in the mouse xenograft model. Furthermore, lovastatin induced autophagy in ovarian cancer cells in vitro [85]. Simvastatin inhibited the proliferation of ovarian clear cell RMG-1 and TOV21-G in vitro and tumor growth in vivo [86]. All statins except pravastatin demonstrated single-agent activity against monolayers (IC50 = 1-35 µM) and spheroids (IC50 = 1-13 µM) of ovarian cancer cells. Furthermore, simvastatin could activate and block autophagy through the Rab7/p62/LC3-II pathway, and the lipophilic statins, simvastatin, and fluvastatin were more potent than hydrophilic statins [87]. In the ID8 syngeneic mice model, simvastatin induced apoptosis and inhibit tumor growth of ovarian cancer [88]. In a K18-gT121+/− p53fl/fl Brca1fl/fl (KpB) mouse model, a unique serous ovarian cancer mouse model specifically and somatically deletes Brca1 and p53 and inactivates the retinoblastoma (Rb) proteins; simvastatin reduced the orthotropic xenograft tumor. In vitro studies showed that simvastatin obtained anti-metastatic and anti-tumorigenic effects through MAPK and AKT/mTOR pathways [89] (Table 5).
Atorvastatin, fluvastatin, and simvastatin induced apoptosis in cervical cancer cells, CaSki, HeLa, and ViBo (established from a biopsy derived from a cervical tumor) [90]. Moreover, simvastatin reduced the phosphorylation of Raf, ERK1/2, Akt, and mTOR and prenylated Ras, resulting in the induction of apoptosis and inhibition of cervical cancer tumor growth. A combination of simvastatin and paclitaxel abolished tumor growth in vivo [91]. In addition, apoptosis and autophagy were induced by atorvastatin through the AMPK/Akt/mTOR pathway. The xenograft tumor was reduced when treated with atorvastatin [92] (Table 5).
In summary, statins showed great potential to reduce cell proliferation and tumor growth of gynecological cancer in vitro and in in vivo. Akt/mTOR is the most important pathway in regulating cell proliferation, and a combination of statins with chemo drugs could synergize the anti-tumorigenic effects. Based on this foundation, statins may be a candidate repurposed drug for gynecological cancers.

Conclusions and Perspective
The mevalonate pathway and lipid metabolism are linked to the key regulators, influencing gene expression, chromatin remodeling, cellular differentiation, stress response, and tumor microenvironment that collectively enhance tumor development [93]. Statins obtain pleiotropic roles to decrease tumor progression through mevalonate-dependent and independent pathways. Statins reduced the prenylated small GTPase and other signaling pathways, such as Akt/mTOR, to induce apoptosis and autophagy and inhibit cell proliferation and metastasis, resulting in anti-tumor development.
This review of statin use in gynecological cancers showed positive and negative results. Some studies cannot avoid confounders, including multiple comorbidities, lifestyle factors, health-related behaviors, stage and grade of disease, and other medications. The study or clinical trials of different statins (e.g., lipophilic or hydrophilic) on different histotypes of cancer (e.g., serous type or non-serous type; type I or type II) and in combination with chemo drugs are required to validate since there are only 3 trials on EC, 6 trials on ovarian cancer, compared to breast cancer which has 52 trials.
If statins are to be applied clinically to gynecological cancers, they may be used as a single agent. We advocate that using statins in combination with other drugs is more potent. In addition, the identification of the response prediction markers is just undergoing. In ovarian cancer cells, VDAC1 and LDLRAP1 were positively and negatively correlated with the response to statins, respectively [94].
The value of statins as therapeutic drugs against gynecological cancer is inestimable because the repurposing of inexpensive, commonly used, and FDA-approved medications to exploit their anti-cancer effects yields the development of cost-effective approaches to cancer therapy. Most important, it can directly benefit the patients, life-saving or prolonging.

Conflicts of Interest:
The authors declare no conflict of interest.