Basic Research Studies
The clinical studies discussed above have been complemented by basic research studies that focus on the mechanisms by which statins exert anti-tumor effects. Statins may affect tumor cells directly in four main ways: 1) growth suppression, 2) apoptosis induction, 3) anti-invasive and anti-metastatic effects, and 4) anti-angiogenic effects. These would be in addition to the indirect effects of lowering available cholesterol. However, the findings that most beneficial effects of statins accrue to those on lipophilic statins suggest direct effects on the tumor cells. Interestingly, the two exceptions wherein even hydrophilic statins present benefit, liver and prostate cancers, further support the direct effect hypothesis as these cancers possess the transporters that allow cellular entry of the hydrophilic statins.
Statin Suppression of Cancer Cell Growth
Previous in vitro studies have demonstrated that statins can halt cancer cell proliferation by inducing G0/G1 or G2/M arrest. Many pathways have been described that contribute to this anti-proliferative effect. First, simvastatin has been shown to induce G1 cell cycle arrest through reduction of CDK4/6 and Cyclin D1. Additionally, BRCA1 overexpression in breast cancer cells has been shown to sensitize cells to lovastatin treatment through regulation of CDK4/Cyclin D1. Second, simvastatin, fluvastatin, and lovastatin have been shown to block the CDK2/Cyclin E-mediated G1/S transition. Third, statins block the DNA-binding activity of NF-ĸB and Activator protein-1 (AP-1), which results in decreased transcription of genes that regulate cell proliferation. Fourth, statins have been shown to inhibit proliferation of breast cancer cells by suppressing FPP and GGPP modification and activation of Ras, Rac, and Rho small GTPases. Rho GTPases normally play an important role in p27 degradation, meaning inactivity results in the accumulation of p27 and cell cycle arrest. Similarly, statin-mediated decreases in Ras activity result in decreased downstream signaling through Akt and diminished cell proliferation. Finally, statins have been shown to inhibit DNA methyltransferases, altering gene transcription and reducing cancer cell proliferation. In summary, statins act to suppress cancer proliferation by inducing cell cycle arrest, blocking proliferation signals, and influencing gene transcription.
Statin Induction of Cancer Cell Apoptosis
In addition to suppressing growth of tumor cells, some literature reports that statins can induce apoptosis. Several pathways have been described that contribute to induction of apoptosis. First, statins have been shown to decrease the protein levels of anti-apoptotic proteins such as Bcl-2 and Bcl-xL.[63,64] Second, statins have also been shown to upregulate the activation of pro-apoptotic molecules such as Bax, Bad, and Caspases 3, 8, and 9.[63–66] Additionally, statins have been shown to decrease phosphorylation and degradation of Bim in breast cancer cells, promoting apoptosis. Third, statins can induce reactive oxygen species (ROS) generation, resulting in an increase in p38 MAPK and activation of apoptotic pathways. Similarly, iNOS-mediated generation of nitric oxide was shown to contribute to simvastatin- and fluvastatin-induced apoptosis in breast cancer cells. Fourth, statins can activate JNK-mediated apoptotic pathways that are independent of p53 activation. Finally, statins can induce calcium-dependent apoptosis through increased mitochondrial uptake of calcium through L-type calcium channels, resulting in the release of cytochrome C. It is suggested that these pro-apoptotic effects are mainly mediated by a reduction in GGPP- rather than FPP-modified proteins.
However, other studies demonstrate no influence of statins on apoptosis. For example, simvastatin was shown to decrease proliferation of bladder cancer cells without inducing apoptosis. Moreover, simvastatin at physiologically relevant concentrations was shown to protect osteosarcoma cells from oxidative stress-induced apoptosis by upregulation of Bcl-2. While atorvastatin treatment could reduce Bcl-2 expression in MDA-MB-231 breast cancer cells, two weeks of neoadjuvant atorvastatin at 80 mg/kg in human breast cancer patients was not found to alter Bax or Bcl-2 expression. Moreover, other studies report minimal effects of statins on inducing cancer apoptosis or a significant influence on apoptosis at concentrations much higher than currently used clinically. Thus, the precise mechanism and magnitude of statin-induced apoptosis of cancer cells remains unclear.
Statin Suppression of Angiogenesis
Statins exert biphasic effects on angiogenesis—pro-angiogenic effects at low (nanomolar) concentrations, anti-angiogenic effects at higher (micromolar) concentrations. At low concentrations, statins can induce mobilization and differentiation of endothelial progenitor cells (EPCs) through stimulation of the PI3K-Akt pathway. Similarly, statin treatment can induce proliferation in EPCs through upregulation of cyclin genes and downregulation of the CDK2 inhibitor p27. Moreover, low statin concentrations can enhance endothelial cell migration and tube formation in a PI3K-Akt- and endothelial nitric oxide synthase (eNOS)-dependent manner. The PI3K-Akt pathways seem to be required for statin-mediated angiogenesis, as blocking PI3K eliminates pro-angiogenic effects of statins.
In contrast, at higher concentrations, statins negatively impact angiogenesis through multiple mechanisms. First, statins have been shown to reduce membrane localization of RhoA by blocking its geranylgeranylation, which reduced the tube-forming ability of human endothelial cells in vitro and was reversed with GGPP supplementation. This inhibition of RhoA subsequently suppresses signaling through pathways that require RhoA activity, such as VEGFR, FAK, and Akt. Second, statins decrease the abundance of caveolin-1 in endothelial cells, which reduces VEGFR2-mediated angiogenic signaling in a cholesterol-independent manner. Third, endothelial cells treated with relatively higher doses of statins show a decrease in hypoxia-stimulated secretion of VEGF and expression of VEGFR2. Finally, simvastatin has been shown to upregulate vascular epithelial cadherin (VE-cadherin), which limits endothelial cell proliferation, migration, and tube forming ability.
In the context of cancer, statins can reduce tumor angiogenesis through several different mechanisms. First, statins can directly affect the endothelial cells to reduce tumor angiogenesis. For example, simvastatin treatment was shown to decrease tumor vascularization in mice at high statin doses. Second, statins can act indirectly on the endothelium by reducing pro-angiogenic or increasing anti-angiogenic protein secretion by tumor cells. Atorvastatin has been shown to reduce circulating VEGF concentrations in human patients with coronary artery disease. Finally, simvastatin and atorvastatin can reduce matrix metalloproteinase (MMP)-9 expression in endothelial cells, which reduces their invasive capability. Moreover, MMP-9 has been shown to be important for releasing the VEGF that is sequestered in breast cancer extracellular matrix (ECM). Thus, a reduction in MMP-9 impacts both the stimulation for and competency of endothelial cells to initiate angiogenesis.
Statin Suppression of Cancer Cell Invasion and Metastasis
Many in vitro studies have suggested statins reduce the invasiveness and metastatic potential of cancer cells through multiple different mechanisms. First, statin treatment destabilizes the cytoskeletal structure of tumor cells in a RhoA/RhoC-dependent manner. Upon statin treatment, Rho delocalizes from the membrane, which causes breakdown of the actin cytoskeleton, loss of actin stress fibers and focal adhesion sites, and cell rounding.[84,85] Moreover, statin treatment is sufficient to block EGF-mediated RhoA membrane localization and formation of actin stress fibers. Second, statins block adhesion of cancer cells to both ECM proteins by reducing integrin binding activity. Similarly, statins downregulate E-selectin on tumor endothelial cells, which reduces tumor cell adhesion and invasion through an endothelial barrier. Third, statins reduce expression and activity of the pro-migratory proteases MMP-2, MMP-9, and urokinase by inhibiting Ras and Rho activity. Fourth, statin treatment can downregulate the cancer stem cell marker CD44 in breast cancer cells, which reduces cell migration and invasion. Fifth, statins can reduce expression of the transferrin receptor in breast cancer cells, which causes iron starvation and a reduction in tumor invasiveness. Finally, we have shown that E-cadherin can induce tumor cell resistance to statins. This suggests that statins specifically target the cells primed to undergo the epithelial–mesenchymal transition (EMT), invade, and metastasize.
Statins also impact tumor invasion and metastasis in ex vivo and in vivo models. Statins were shown to reduce prostate cancer migration towards human bone marrow stroma without impacting ECM adhesion. Also, lovastatin, atorvastatin, and simvastatin have been shown to reduce metastatic seeding of melanoma and breast cancer cells in the lung and bone.[85,89,93] In breast cancer patients receiving 40 mg simvastatin per day for 4–6 weeks before mastectomy, statin treatment was able to decrease post-explant tumor migration. Moreover, simvastatin treatment reduced the activity of Rho associated protein kinase (ROCK) and expression of RhoC, chemokine receptor type 4 (CXCR4), and CD44. In a sophisticated ex vivo microphysiological model of breast cancer metastasis to the liver, breast cancer outgrowth in response to an inflammatory lipopolysaccharide/EGF stimulus could be suppressed with atorvastatin treatment. Finally, we have shown in mouse models of spontaneous breast cancer metastasis to liver and lung that atorvastatin is able to suppress metastatic proliferation but not the proliferation of the primary tumor. These data suggest that statins preferentially target mesenchymal, metastatic cells.
Many basic research studies have shown that statins can reduce tumor cell growth and proliferation by inducing cell cycle arrest. Additionally, some, but not all, studies suggest that statins can induce apoptosis in cancer cells. In addition to affecting tumor cell growth and apoptosis, statins can negatively impact the tumor vasculature through suppression of angiogenesis. While statins exhibit a biphasic effect on angiogenesis, their anti-angiogenic effects are observed at similar doses to those used in studies that demonstrate anti-tumor effects. While statins have been shown to reduce cancer cell migration and invasion in vitro, few studies have demonstrated a reduction in metastasis with statin treatment in vivo. These previous studies motivate the investigation of statins in the context of breast cancer metastasis, in particular dormancy and emergence, which is the main cause of morbidity and mortality in this patient population. We propose a model in which statins block the secondary EMT and outgrowth of tumor cells by preventing Ras, Rac, and RhoA prenylation (Figure 2).
Proposed model for statin action in breast cancer. a The breast cancer metastatic cascade. Statins (red) block emergence of dormant breast cancer cells at the site of micrometastasis to prevent their emergence to form clinically evident metastases. b Statins (red) block HMG-CoA reductase (HMGCR) to decrease the number of prenylation groups (Pr), such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, available for prenylating small G proteins, such as Ras (shown), Rac, and RhoA. Decreased prenylation reduces membrane tethering of these G proteins, which reduces downstream proliferative and pro-EMT signaling. Drawing made using images from Servier Medical Art 
An important consideration for repurposing statin drugs for breast cancer treatment is toxicity. Breast cancer patients already receive chemotherapeutic compounds that exhibit varying degrees of toxicities. Fortunately, statins are typically well-tolerated drugs. The most common adverse events associated with statin usage involve muscle tissues, which range in severity from myalgia to fatal rhabdomyolysis. Muscle pain (myalgia) is relatively more common among statin users, with an incidence of approximately 5%. Myopathy, or myositis, only occurs in 0.1% of patients taking statins and involves pain, weakness, and mobility restrictions that often are accompanied by an elevation in serum creatine kinase (CK) to ten times the upper limit of normal. Rarely, myopathy can progress to rhabdomyolysis, which involves CK elevations above 50 times the upper limit of normal and myoglobin accumulation in the blood (myoglobinemia) and urine (myoglobinuria), which can induce renal failure. The incidence of rhabdomyolysis is extremely rare, with an incidence of 3.4 per 100,000 person years and mortality rate of approximately 10%. Rhabdomyolysis incidence is higher in patients taking atorvastatin, lovastatin, and simvastatin, which is due to their extensive metabolism by CYP3A4 and potential for drug–drug interactions. The less common adverse events observed with statin usage involve the liver, pancreas, kidney, and peripheral nerves.
The key concern for breast cancer patients is that the toxicity of statins leads to generalized inflammation. The initiation of inflammatory cascades can awaken dormant micrometastases to outgrow.[99,100] Thus, higher dose statins may be perversely contrary and promote cancer progression if they cause a cytokine storm or lead to fibrosis of chronic inflammation. As the clinical correlation studies did not account for toxicity of statins, the benefits may be understated.
Breast Cancer Res. 2018;20(1) © 2018 BioMed Central, Ltd.
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