Background
Ovarian cancer is the leading cause of death from gynecological malignancies in women [
1]. The survival rate of affected patients is comparably poor, especially due to the diagnosis at an advanced stage in the vast majority of the cases [
2‐
4]. A consistent number of patients present few clinical symptoms, which are also characteristic of various gastrointestinal, abdominal and urinary conditions [
5,
6]. In many cases, the late diagnosis leads to a clinical picture that includes intraperitoneal dissemination of cancer cells [
6]. Here, increased permeability of the peritoneal membrane and its associated vasculature leads to massive fluid accumulation (i.e. ascites) within the peritoneal cavity [
7,
8]. This process is sustained and accelerated by inflammatory cytokines, chemokines and growth factors secreted by cancer cells and additional cellular components of the tumor microenvironment, such as lymphocytes and tumor-associated macrophages (TAMs) [
4,
8,
9]. The resulting pro-inflammatory microenvironment supports the malignant invasive growth of the tumor and drives morbidity and mortality of affected patients [
4]. Furthermore, ascites is associated with chemoresistance, recurrence, and an overall poor prognosis [
4,
8]. Current therapeutic options in ovarian cancer management depend on the diagnosed stage and include radical cytoreductive surgery, which improves remission rates and results in a longer duration of tumor-free survival in over 90% of early-diagnosed cases [
10]. Adjuvant chemotherapy is more commonly administered in advanced ovarian cancer using carboplatinum and paclitaxel therapy which precedes and follows surgical cytoreduction [
11,
12]. The anti-angiogenic drug bevacizumab was recently approved as an additional first-line therapy [
13,
14]. Bevacizumab is administered in combination with the platinum-based chemotherapy and afterwards as maintenance monotherapy for a total of 15 months as used in the GOG-0218 trial [
13‐
15]). In addition, poly (ADP-ribose) polymerase (PARP) inhibitors have been approved in ovarian cancer [
16]. Here, olaparib is approved as maintenance therapy after initial platinum-based chemotherapy for
BRCA1/2 mutation carriers [
17]. In so-called platinum-sensitive ovarian cancer relapse PARP inhibitors are approved as maintenance therapy after response to platinum-based chemotherapy independent on the
BRCA mutation status [
18‐
21]. Major challenges in the management of ovarian malignancies are chemo-resistance to platinum-based therapy, metastases and disease recurrence [
22]. Although patients with resistant tumors eventually respond to second-line therapies, a large proportion of them experience short disease-free survival [
23]. Therefore, novel therapeutic options, especially for patients with chemo-resistant ovarian cancer, are urgently needed.
The mevalonate pathway is responsible for the biosynthesis of sterol and non-sterol isoprenoids, thereby playing a central role in cellular metabolism [
24,
25]. Moreover, it is important for the post-translational modifications of proteins, specifically by providing farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for farnesylation and geranylgeranylation, referred to as protein prenylation [
26]. Due to their central role in regulating cellular signaling processes, Rho-GTPases such as the Ras superfamily are among the best-studied prenylated proteins [
27,
28]. The dysregulation and involvement of mevalonate pathway enzymes and products such as cholesterol in human malignancies has been shown for several tumor entities including ovarian cancer [
25,
29‐
33]. Cholesterol can also be converted to 27-hydroxycholesterol by cytochrome P450 oxidase CYP27A1, which is expressed by both tumor cells and TAMs. CYP27A1 expression and 27-hydroxycholesterol promote tumor growth and are associated with reduced progression-free survival in breast and ovarian cancer [
34,
35]. In addition, Rho-GTPases are implicated in ovarian cancer tumorigenesis and platinum resistance [
36,
37]. Two major classes of drugs inhibit the mevalonate pathway at different levels: statins and amino-bisphosphonates (N-BP). Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway. They reduce cholesterol production and mediate the increased uptake of extracellular cholesterol by low density lipoprotein receptors [
38,
39]. Statins have evolved as a standard care for treating high cholesterol levels in patients [
39,
40]. N-BP are inhibitors of the farnesyl diphosphate synthase and induce apoptosis in bone-resorbing osteoclasts [
41,
42]. Therefore, N-BP are clinically used in osteoporosis or osteolytic bone metastases secondary to breast and prostate cancer [
41,
42]. Both classes of mevalonate pathway inhibitors show a multitude of pleiotropic antitumor effects, ranging from induction of apoptosis, inhibition of migration, metastasis and invasion, as well as modulation of tumor-promoting signaling molecules or the immune system [
25,
43‐
46]. In this study, we aimed at investigating the in vitro anti-tumor effects of several statins and the N-BP zoledronic acid in different human ovarian cancer cell lines.
Discussion
Among human gynecologic malignancies, ovarian cancer is the most lethal one [
56]. The vast majority of patients are diagnosed at an advanced stage, when the long term survival rates are found to be as low as 20–30% [
5]. Current therapeutic options are limited, particularly for advanced ovarian cancer, because of recurrence, metastasis and chemo-resistance to primary treatments or secondarily acquired resistance [
10]. Statins and N-BP as inhibitors of the mevalonate pathway have been widely studied for their pleiotropic anti-tumor properties [
24].
The rationale for targeting the mevalonate pathway in ovarian cancer is underpinned by the evidence of a role of the mevalonate pathway in ovarian cancer tumorigenesis. The HMGCR enzyme is overexpressed in several human ovarian cancer cell lines, including IGROV-1 and A2780 [
29]. Moreover, late-stage metastatic ovarian cancer cells show a marked molecular reprogramming with upregulation of several mevalonate pathway genes including HMGCR [
57]. High levels of cholesterol have been linked to an increased risk of developing ovarian and other cancer entities [
33]. A number of retrospective and case-control studies have shown a significant association between pre- and/or post-diagnostic use of statins and a reduced overall as well as ovarian cancer-specific mortality have been described [
58,
59]. A reduced risk of developing ovarian or endometrial cancers was seen in patients prescribed with bisphosphonates for more than 1 year before the diagnosis [
60].
In our study, we demonstrate that statins and the N-BP zoledronic acid reduce cell vitality and induce apoptosis in the ovarian cancer cell lines A2780 and IGROV-1. These observations match with studies describing similar effects in vitro and in vivo in a number of human malignancies [
57,
61‐
72]. In the present study, the lipophilic statins atorvastatin and simvastatin were more potent than the hydrophilic rosuvastatin which is in line with studies in breast and additional ovarian cancer cell lines, including Hey 1B and OVCAR-3 [
52,
63,
73‐
75]. The differences in the chemical structure, the transport into cells via passive membrane diffusion or via transporters and the varying potential to alter the sterol metabolism within tumor cells may account for variances in the anti-tumor effects exerted by different statins [
41,
63].
Not only did the individual effects among the used statins differ, but also the sensitivity of the used cell lines, which is a known phenomenon in human cancer cell lines [
52,
76,
77]. This could be due to differences in the molecular profile of A2780 and IGROV-1 cells. Although both of them are estrogen receptor negative, A2780 cells carry few mutations, whereas IGROV-1 are considered as hyper-mutated and carry both familial and sporadic mutations [
78]. Moreover, simvastatin exerted significant effects on apoptosis in cisplatin-resistant A2780cis cells, although higher concentrations compared to parental A2780 cells were necessary, indicating a relative cross-resistance. Future studies might also investigate potential statin resistance mechanisms and biomarkers of statin or N-BP sensitivity and whether these may cross-interfere with mechanisms of cisplatin resistance. We and others have demonstrated a restorative feedback loop by induction of the statin targeting enzyme, the HMGCR, as a mediator of statin resistance in human breast and prostate cancer as well as multiple myeloma cells [
52,
79,
80]. Similar mechanisms might occur in ovarian cancer cells, especially as cisplatin and simvastatin induce a strong accumulation of HMGCR in ovarian cancer cells [
81,
82]. In times of personalized medicine, it would be greatly relevant to identify patients with those molecular subtypes, that would mostly benefit from a statin or N-BP therapy, individually or in combination with chemotherapy, as well as to define the optimal therapeutic window.
We demonstrated that the cytotoxic effects by statins and zoledronic acid in IGROV-1 and A2780 cells were mediated by an inhibited geranylgeranylation, rather than by an inhibited farnesylation. These observations were made in breast cancer cells using the same agents [
51]. In mice bearing Ovcar-4 ovarian cancer xenografts, geranylgeraniol limits the anti-tumor effects of pitavastatin [
29]. The underlying mechanisms are potentially mediated by a disrupted function of geranylgeranylated Rho proteins. Alendronate inhibits ovarian cancer cell migration through an inhibited geranylgeranylation and Rho protein activation [
83]. In addition, pitavastatin alters the subcellular localization of RhoA, CDC42 and Ras in several ovarian cancer cell lines [
77].
The treatments with statins and zoledronic acid also significantly reduced the expression of the anti-apoptotic genes
Bcl-2 and
Svv, even in chemo-resistant A2780cis cells, which is in line with studies in breast, colorectal, and prostate cancer [
84‐
87].
Bcl-2 as well as
Svv have been shown to be overexpressed in ovarian carcinomas and are associated with chemo-resistance and a worse prognosis [
88‐
92]. The suppression of both genes by statins and zoledronic acid offers a potential strategy to overcome such resistance. Platinum resistance is a major problem in the treatment of ovarian cancer, for which different mechanisms account such as increased drug efflux and inactivation, the ability to repair platinum-DNA adducts, or overexpression of anti-apoptotic regulators [
93]. Additional studies might investigate, as to whether or not statins and N-BP modulate anti-apoptotic genes in these cells and if a concomitant treatment with platinum would re-sensitize the cells and induce cytotoxicity as seen in cisplatin-resistant SKOV3 cells [
94]. The potential advantage of such a combinatory approach is the reduction of individual concentrations without the loss or even with the increase of therapeutic effects and reduced side effects such as nephro- and neurotoxicity by cisplatin [
11].
Our study also demonstrates, that especially statins significantly suppressed
TNF-α,
TFG-β1 IL-6,
IL-8 and
VEGF in IGROV-1 and A2780 cells. These factors play central roles in the accumulation of peritoneal fluid, inflammation, and angiogenesis, as well as in supporting tumor promotion and metastases [
54,
95‐
101]. Moreover, all of them are overexpressed and associated with chemo-resistance and a poor prognosis in affected patients with ovarian cancer [
54,
95‐
101]. The constitutive expression of
IL6 by ovarian cancer cells stimulates TAMs of the tumor microenvironment thereby promoting tumor growth [
102]. IL-6 also promotes the production of pro-angiogenic factors, like VEGF and IL-8 [
96] and the latter itself drives cell motility [
103]. Constitutive secretion of TNF-α leads to the further release of IL-6, VEGF, and other factors that concomitantly sustain the vicious cycle of malignant ascites [
9,
54]. Targeting these tumor-promoting soluble factors offers a therapeutic strategy in ovarian cancer and might modulate the response of chemo-resistant tumor cells to chemotherapy. VEGF suppression was seen to reduce tumor burden and ascites formation in several preclinical studies [
104]. Successful translation of these findings has led to the development and evaluation of the VEGF targeting antibody bevacizumab in clinical trials and its approval for the use in patients with ovarian cancer [
13,
14]. Pharmacological inhibition of IL-6 has also been positively evaluated in preclinical and clinical studies [
105]. The knockdown of IL-6 and IL-8 was shown to sensitize the tumor cells to cisplatin treatment in lung and ovarian cancer, respectively [
106,
107]. Additional in vivo models using chemo-sensitive and -resistant ovarian cancer cell lines are necessary to evaluate the effects of statins and zoledronic acid, individually and in combination with chemotherapy, on these pro-tumorigenic factors.
Our study has several limitations, including the use of in vitro cell models which do not fully reflect the heterogenous pattern of molecular subtypes of human ovarian cancer. Moreover, the levels of statins that are reached in the serum of patients range between 0.002 and 0.1 μM [
108] and the maximum concentration of zoledronic acid administered to patients is 2 μM [
109]. Hence, the clinically achievable concentrations of these agents within tumor tissues and ascites might be too low for direct antitumor effects. However, the clonogenic assay revealed that even low concentrations of simvastatin significantly impaired the number of newly formed IGROV-1 colonies as a marker of the clonogenic potential. These findings might translate into effective anti-tumor effects of statins or even N-BP at low levels in the stage of single tumor cell dissemination to secondary sites and needs to be comprehensively analyzed in additional studies. In breast cancer, disseminated tumor cells can be eliminated with N-BP therapy and this is associated with an improved survival [
110,
111]. For ovarian cancer, no trials exist that investigated adjuvant N-BP therapy in the context of single tumor cell dissemination.
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