The Potential of Isoprenoids in Adjuvant Cancer Therapy to Reduce Adverse Effects of Statins

The mevalonate pathway provides sterols for membrane structure and nonsterol intermediates for the post-translational modification and membrane anchorage of growth-related proteins, including the Ras, Rac, and Rho GTPase family. Mevalonate-derived products are also essential for the Hedgehog pathway, steroid hormone signaling, and the nuclear localization of Yes-associated protein and transcriptional co-activator with PDZ-binding motif, all of which playing roles in tumorigenesis and cancer stem cell function. The phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT-mammalian target of rapamycin complex 1 pathway, p53 with gain-of-function mutation, and oncoprotein MYC upregulate the mevalonate pathway, whereas adenosine monophosphate-activated protein kinase and tumor suppressor protein RB are the downregulators. The rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is under a multivalent regulation. Sterol regulatory element binding protein 2 mediates the sterol-controlled transcriptional downregulation of HMGCR. UbiA prenyltransferase domain-containing protein-1 regulates the ubiquitination and proteasome-mediated degradation of HMGCR, which is accelerated by 24, 25-dihydrolanosterol and the diterpene geranylgeraniol. Statins, competitive inhibitors of HMGCR, deplete cells of mevalonate-derived intermediates and consequently inhibit cell proliferation and induce apoptosis. Clinical application of statins is marred by dose-limiting toxicities and mixed outcomes on cancer risk, survival and mortality, partially resulting from the statin-mediated compensatory upregulation of HMGCR and indiscriminate inhibition of HMGCR in normal and tumor cells. Tumor HMGCR is resistant to the sterol-mediated transcriptional control; consequently, HMGCR is upregulated in cancers derived from adrenal gland, blood and lymph, brain, breast, colon, connective tissue, embryo, esophagus, liver, lung, ovary, pancreas, prostate, skin, and stomach. Nevertheless, tumor HMGCR remains sensitive to isoprenoid-mediated degradation. Isoprenoids including monoterpenes (carvacrol, L-carvone, geraniol, perillyl alcohol), sesquiterpenes (cacalol, farnesol, β-ionone), diterpene (geranylgeranyl acetone), “mixed” isoprenoids (tocotrienols), and their derivatives suppress the growth of tumor cells with little impact on non-malignant cells. In cancer cells derived from breast, colon, liver, mesothelium, prostate, pancreas, and skin, statins and isoprenoids, including tocotrienols, geraniol, limonene, β-ionone and perillyl alcohol, synergistically suppress cell proliferation and associated signaling pathways. A blend of dietary lovastatin and δ-tocotrienol, each at no-effect doses, suppress the growth of implanted murine B16 melanomas in C57BL6 mice. Isoprenoids have potential as adjuvant agents to reduce the toxicities of statins in cancer prevention or therapy.

The mevalonate pathway provides sterols for membrane structure and nonsterol intermediates for the post-translational modification and membrane anchorage of growth-related proteins, including the Ras, Rac, and Rho GTPase family. Mevalonate-derived products are also essential for the Hedgehog pathway, steroid hormone signaling, and the nuclear localization of Yes-associated protein and transcriptional co-activator with PDZ-binding motif, all of which playing roles in tumorigenesis and cancer stem cell function. The phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT-mammalian target of rapamycin complex 1 pathway, p53 with gain-of-function mutation, and oncoprotein MYC upregulate the mevalonate pathway, whereas adenosine monophosphate-activated protein kinase and tumor suppressor protein RB are the downregulators. The rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is under a multivalent regulation. Sterol regulatory element binding protein 2 mediates the sterol-controlled transcriptional downregulation of HMGCR. UbiA prenyltransferase domain-containing protein-1 regulates the ubiquitination and proteasome-mediated degradation of HMGCR, which is accelerated by 24, 25-dihydrolanosterol and the diterpene geranylgeraniol. Statins, competitive inhibitors of HMGCR, deplete cells of mevalonate-derived intermediates and consequently inhibit cell proliferation and induce apoptosis. Clinical application of statins is marred by dose-limiting toxicities and mixed outcomes on cancer risk, survival and mortality, partially resulting from the statin-mediated compensatory upregulation of HMGCR and indiscriminate inhibition of HMGCR in normal and tumor cells. Tumor HMGCR is resistant to the sterol-mediated transcriptional control; consequently, HMGCR is upregulated in cancers derived from adrenal gland, blood and lymph, brain, breast, colon, connective tissue, embryo, esophagus, liver, lung, ovary, pancreas, prostate, skin, and stomach. Nevertheless, tumor HMGCR remains sensitive to isoprenoid-mediated degradation. Isoprenoids including monoterpenes (carvacrol, L-carvone, geraniol, perillyl alcohol), sesquiterpenes (cacalol, farnesol, β-ionone), diterpene (geranylgeranyl acetone), "mixed" isoprenoids (tocotrienols), and their derivatives suppress the growth of tumor cells with little impact on non-malignant cells. In cancer cells derived from breast, colon, liver, mesothelium, prostate, pancreas, and skin, statins and isoprenoids, including

INTRODUCTION
Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway (Goldstein and Brown, 1990). Widely prescribed as hypocholesterolemic agents, statins have been shown to inhibit cell proliferation and induce apoptosis in preclinical studies (Clendening and Penn, 2012). Clinical data suggest inverse association of statin use and risk of some, but not all, cancers. Dose-limiting toxicities of statins attributable to statin-induced compensatory upregulation of HMGCR and indiscriminate inhibition of HMGCR in normal and tumor cells pose constraints on the application of statins in cancer and call for novel approaches in reducing their side effects.
In this review we first summarize the role of the mevalonate pathway in cell proliferation and cancer by highlighting the intertwined relations between the mevalonate pathway and several key signaling molecules in growth regulation (Mullen et al., 2016). The multivalent regulation of HMGCR, including sterol-mediated transcriptional downregulation and nonsterol-induced enhancement of degradation, in normal cells contrasts with the sterol-resistant, dysregulated tumor HMGCR that remains uniquely sensitive to isoprenoid-mediated downregulation (Mo and Elson, 2004). The mechanisms underlying the isoprenoid-mediated tumor suppression is elucidated, followed by studies showing the synergistic effect of statins and isoprenoids and suggesting the potential of isoprenoids in adjuvant therapy to reduce the toxicities of statins.

ROLE OF THE MEVALONATE PATHWAY IN CELL PROLIFERATION AND CANCER
The mevalonate pathway provides the bulk end product, cholesterol, and nonsterol isoprenoids such as heme-A, ubiquinone (or coenzyme Q 10 ), dolichol, isopentenyl adenine, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) (Goldstein and Brown, 1990). Cholesterol is important for membrane structure, whereas the mevalonate-derived nonsterol compounds play vital roles in iron-containing cofactors of hemoproteins (e.g., hemoglobin, myoglobin, catalase, endothelial nitric oxide synthase, and cytochrome), mitochondrial electron transport and cellular respiration, N-glycosylation of proteins, transfer RNA (Buhaescu and Izzedine, 2007), and post-translational prenylation and membrane anchorage of growth-related proteins, including the Ras, Rac, and Rho GTPase family (Hentschel et al., 2016;Wang and Casey, 2016). The prenylated proteins collectively support cell proliferation and cancer growth (Mullen et al., 2016) (Figure 1).
The mevalonate pathway is also intertwined with several signaling pathways with regulatory roles in cancer. The most frequently altered signaling pathway in cancer, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-AKT pathway, regulates cell survival and proliferation (Mullen et al., 2016). Through the downstream mammalian target of rapamycin (mTOR) complex 1 signaling (Duvel et al., 2010), PI3K-AKT pathway activates the mevalonate pathway (Ricoult et al., 2016) and concomitantly stimulates glucose uptake and glycolysis that provide acetyl-CoA and NADPH to support the mevalonate pathway. Gain-of-function mutations of p53 (Freed-Pastor et al., 2012), another frequently mutated gene in cancer, and the oncoprotein MYC (Wu et al., 2016), upregulate the transcription of mevalonate pathway genes. In contrast, adenosine monophosphate-activated protein kinase (AMPK), an energy sensor and central regulator of metabolism, downregulates the mevalonate pathway via phosphorylation (Beg et al., 1973(Beg et al., , 1978 and transcriptional control (Li et al., 2011) of HMGCR. The tumor suppressor protein RB also downregulates the mevalonate pathway (Shamma et al., 2009). Conversely, the mevalonate pathway provides sterols and nonsterol products for Hedgehog pathway (Eaton, 2008), steroid hormone signaling (Nguyen et al., 2015), and the nuclear localization of Yesassociated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) (Sorrentino et al., 2014;Koo and Guan, 2018), all of which have important roles in tumorigenesis and the cancer stem cell function (Mancini et al., 2018). FIGURE 1 | The regulation of the mevalonate pathway and the role of mevalonate-derived metabolites in cell proliferation. When cellular level of sterol is low, Insig dissociates from the SREBP-SCAP complex, allowing the latter to move to the Golgi apparatus. Following proteolytic cleavage of SREBP, its basic helix-loop-helix fragment enters the nucleus, binds to the SRE domain of target genes, and initiates the transcription and synthesis of a number of enzymes in the mevalonate pathway, including its rate-limiting enzyme, HMGCR. The activated mevalonate pathway produces sterols and nonsterols, including FPP and GGPP for protein prenylation and cell proliferation. Sterols block the transport of the SREBP-SCAP complex to the Golgi apparatus and induce the binding of HMGCR to Insig, leading to the ubiquitination and degradation of HMGCR. Exogenous geranylgeraniol is phosphorylated and converted to GGPP, which induces the dissociation of UBIAD1 from HMGCR and facilitates the degradation of HMGCR. The statins competitively inhibit HMGCR. Consequently, the lack of sterols and nonsterols results in a compensatory upregulation of HMGCR. Tocotrienols and potentially other isoprenoids block the processing and nuclear localization of SREBP and enhance the ubiquitination and degradation of HMGCR. HMGCR is upregulated in tumors but remains sensitive to isoprenoid-mediated downregulation. Synergistic impact of isoprenoids and statins on tumor HMGCR may offer a novel approach in enhancing the efficacy of statins with reduced toxicity.

REGULATION OF THE MEVALONATE PATHWAY IN NORMAL CELLS
In non-malignant cells, HMGCR is highly regulated by a multivalent feedback mechanism mediated by sterols and nonsterol isoprenoid end products of mevalonate metabolism. This tight regulatory feedback system, which operates at transcriptional and post-transcriptional levels, ensures proper signaling for sufficient sterol and isoprenoid syntheses and optimal cell growth (Goldstein and Brown, 1990). Transcription regulation, exerted by sterols and mediated by sterol regulatory element (SRE), is the dominant feedback mechanism. The SRE is a promoter sequence in the 5 ′ flanking region of more than 30 genes involved in lipid biosynthesis and uptake, including those of HMG CoA synthase, HMGCR, and low density lipoprotein (LDL) receptor (Goldstein and Brown, 1990;Brown and Goldstein, 1997;Horton et al., 2002;Goldstein et al., 2006). By providing a binding site for the membrane-bound transcriptional factors called SRE binding proteins (SREBPs), the SRE domain allows for SREBP-mediated transcriptional control of all these SRE-containing genes. Another ER membraneembedded protein, known as SREBP cleavage activating protein (SCAP), is coupled with SREBP and plays a critical role in the intracellular escort and proteolytic processing of SREBP (Osborne et al., 1985;Gil et al., 1988;Goldstein et al., 2006). SCAP and HMGCR share an intramembrane sequence named sterol-sensing domain that detects the concentration of membrane and intracellular sterols and mediates the interactions of SCAP and HMGCR with insulin-induced gene (Insig) proteins during transcriptional and post-transcriptional regulations Johnson and DeBose-Boyd, 2018).
When sterol levels diminish in cells, Insig dissociates from the SCAP-SREBP-Insig complex and undergoes ubiquitinmediated degradation (Gong et al., 2006). The liberated SCAP-SREBP complex is then taken into vesicles coated by COPII proteins (Sec23, Sec24, and Sar1-GTP) and transported to the Golgi apparatus (Brown and Goldstein, 1999;Goldstein et al., 2006;Espenshade and Hughes, 2007). The SREBP is cleaved on the cytosolic surface of Golgi membrane in a two-step proteolytic reaction by site-1 serine protease and site-2 Zn 2+ metalloproteinase, producing the transcriptionally active, basic helix-loop-helix fragment of SREBP. This active motif of SREBP enters nucleus, binds to the SRE domain, and initiates transcription of target genes that produce proteins taking part in cholesterol uptake and biosynthesis, including LDL receptor, HMG CoA synthase, HMGCR, Insig-1, and FPP synthase (Goldstein and Brown, 1990;Brown and Goldstein, 1999;Horton et al., 2002).
Once intracellular sterol levels are restored by increased biosynthesis or uptake due to aforementioned SREBPupregulated events, a convergent feedback inhibition terminates the SREBP-directed transcriptional operation. Two of the SREBP-induced molecules, newly synthesized Insig-1 and newly biosynthesized or collected exogenous cholesterol, bind SCAP simultaneously and induce its conformational changes. As a result, the SCAP-SREBP complex is stabilized and retained in the ER membrane and prevented from undergoing any further transcriptional processing (Yang et al., 2002;Goldstein et al., 2006;Gong et al., 2006). This coordinated process ensures that cells attain sufficient amounts of both cholesterol and nonsterol isoprenoids for their growth and metabolic requirements before the SREBP transcriptional operation is fully halted. Furthermore, this feedback mechanism precludes overaccumulation of potentially toxic sterols.
As one of the genes in the mevalonate pathway under the transcriptional control of SREBP, HMGCR has a secondary line of regulation on its synthesis and degradation. While an earlier study postulated that an unknown mevalonatederived isoprenoid regulates HMGCR translation via a 5 ′untranslated region of HMGCR mRNA (Nakanishi et al., 1988), the mechanisms underlying the degradation of HMGCR have been more clearly elucidated. This post-transcriptional finetuning of HMGCR regulation is mediated by two mevalonatederived compounds, a reduced tetracyclic triterpenoid, 24, 25dihydrolanosterol, and a nonsterol diterpene, geranylgeraniol (Sever et al., 2003). 24, 25-Dihydrolanosterol and a less effective cholesterol induce binding of Insig to the sterol sensing domain of HMGCR, a process that requires a tetrapeptide sequence YIYF located in the second transmembrane segment of HMGCR (Roitelman and Simoni, 1992;McGee et al., 1996;Ravid et al., 2000;DeBose-Boyd, 2008). This binding is followed by the ubiquitination and proteasome-mediated degradation of HMGCR (Sever et al., 2004;Song et al., 2005;Lange et al., 2008;Nguyen et al., 2009). Addition of HMGCR inhibitors to cultured cells containing adequate amounts of sterols enhanced HMGCR protein expression and reduced its degradation, which was reverted with supplemental nonsterol metabolites (Goldstein and Brown, 1990), suggesting a nonsterol metabolite also regulates the expression and degradation of HMGCR. Using SV589 human fibroblasts that lack the monocarboxylate transporter and consequently the ability for mevalonate uptake, studies showed that geranylgeraniol is more potent than the 15-carbon isoprenoid farnesol in accelerating the sterol-induced and Insigdependent ubiquitination and degradation of HMGCR (Sever et al., 2003).
More recent studies have shown that geranylgeraniol might be phosphorylated and converted to GGPP, which serves as a potent regulator of HMGCR stability and degradation (Garza et al., 2009;Schumacher et al., 2016Schumacher et al., , 2018. Buildup of GGPP in ER membranes triggers dissociation of UbiA prenyltransferase domain-containing protein-1 (UBIAD1) from HMGCR, permitting ER-to-Golgi transport and maximal degradation of HMGCR (Schumacher et al., 2018). Additionally, pulse-chase experiments confirmed the synergistic effects of mevalonate or geranylgeraniol, but not farnesol, with sterols in enhancing complete HMGCR degradation (Sever et al., 2003).
The E2 conjugating enzyme Ubc7, E3 ubiquitin ligase glycoprotein 78 (gp78), and ATPase VCP/p97 facilitate delivery of ubiquitinated HMGCR from the ER membrane to the proteasomes for proteolysis and degradation (Espenshade and Hughes, 2007). A recent study (Hwang et al., 2016) confirmed that liver employs the same mechanism regulating the accelerated degradation of HMGCR and cholesterol homeostasis initially found in cultured cells. This operation safeguards HMGCR synthesis and stability until the cellular requirements of sterols and isoprenoids are met (Goldstein and Brown, 1990;Peffley and Gayen, 2003;Sever et al., 2003;Song et al., 2005;Goldstein et al., 2006;Lange et al., 2008;Nguyen et al., 2009). It was recently reported that the monoterpene linalool prevents the binding of SREBP-2 to HMGCR promoter and enhances the ubiquitinmediated degradation of HMGCR (Cho et al., 2011). A second monoterpene geraniol suppresses the protein level and specific activity of HMGCR in mouse liver (Galle et al., 2015) and A549 cells (Galle et al., 2014). These findings may add another layer of complexity to the regulation of HMGCR. It remains unknown whether GGPP mediates the effect of these monoterpenes on HMGCR.

UPREGULATION OF HMGCR IN TUMORS
Tumorigenesis has been associated with alterations and reprograming of energy, carbohydrate and lipid metabolisms with intertwined relations (Hanahan and Weinberg, 2011; Pavlova and Thompson, 2016). Activated oncogenes including ras and myc, for example, have been associated with alterations in glucose metabolism termed "aerobic glycolysis, " one of the hallmarks of cancer cells that is also known as the "Warburg effect" (Warburg, 1956;Hanahan and Weinberg, 2011). Hypoxia and the Ras oncoprotein that requires mevalonate-derived FPP for its post-translational modification can independently stimulate hypoxia-inducible factor 1α (HIF1α) and HIF2α transcriptional factors, which in turn upregulate glucose transporters and enzymes of the glycolytic pathway (Hanahan and Weinberg, 2011).
Consistent with these observations is the finding that exogenous mevalonate or artificially overexpressed HMGCR promotes growth. Supplemental mevalonate promoted growth of metastatic human breast cancer MDA-MB-435 cells in xenograftbearing mice (Duncan et al., 2004a). Supplemental mevalonate increased the proliferation of cancer cells by upregulating cyclindependent kinase 2 (CDK2) activity and accelerating entry of cells into the S phase of cell division cycle (Duncan et al., 2004a). Ectopic expression of full-length or splice variant of HMGCR promotes transformation (Clendening et al., 2010). Finally, overexpression of mevalonate pathway genes is correlated with poor prognosis of recurrence-free and overall survival in breast cancer patients (Kimbung et al., 2016).
A recent study found that mutant p53-mediated upregulation of the mevalonate pathway is both necessary and sufficient for architectural phenotypes in breast cancer (Freed-Pastor et al., 2012), offering a new perspective for the role of the mevalonate pathway in cancer. The correlation between p53 mutation and highly expressed mevalonate pathway genes likely involves SREBP-2 and to a lesser extent, SREBP-1, which may assist the binding of p53 to the promoter of HMGCR gene. The mutant p53 and the mevalonate pathway also form a feedforward loop in promoting cell proliferation. Mevalonate kinase upregulates DNAJ heat shock protein family (Hsp40) member A1 (DNAJA1), a type-I Hsp40, which inhibits the ubiquitination and degradation of mutant p53 mediated by the C-terminus of Hsc70-interacting protein (CHIP) E3 ubiquitin ligase (Parrales et al., 2016(Parrales et al., , 2018. Further implicating the role of the mevalonate pathway in tumorigenesis is the finding that mevalonate pathway inhibitors have cytostatic and cytotoxic effects in von Hippel-Lindau (VHL)-deficient clear cell renal cell carcinoma (CC-RCC) through an HIF-dependent mechanism (Thompson et al., 2018). Overall, these findings demonstrate the cancer promoting effects of excessive mevalonate and HMGCR activity.

Species Tissues Upregulation in tumors References
Human Adrenal gland Adrenal tumors had >70-fold higher HMGCR activity Lehoux et al., 1984 Blood and lymph Mononuclear blood cells and leukocytes from leukemia patients had several-fold higher HMGCR activity than those from healthy subjects; HMGCR was upregulated by up to 50-fold in stimulated lymphocytes; upregulation of mevalonate pathway was correlated with lower survival of patients with acute myeloid leukemia Yachnin and Mannickarottu, 1984;Harwood et al., 1991;Vitols et al., 1994;Hentosh et al., 2001;Kuzu et al., 2016 Brain HMGCR activity in metastatic brain tumors was higher than that in primary tumors; glioblastoma multiforme cell line U343 had higher HMGCR mRNA than normal human astrocytes; FPP synthase was overexpressed in glioblastoma compared to tumor-free peripheral brain and normal human astrocytes Maltese, 1983;Laezza et al., 2015;Abate et al., 2017 Breast Upregulation of the mevalonate pathway enzymes including HMGCR was found in breast apocrine cysts, DMBA-and MNU-initiated mammary tumors, breast cancer stem cell-derived basal/mesenchymal tumorspheres, and Tamoxifen-resistant breast cancer El-Sohemy and Archer, 2000; Celis et al., 2006;Ginestier et al., 2012 Colon HMGCR mRNA and activity were up to 8-fold higher in colorectal cancer cells than those in colon epithelial cells, fibroblasts and mucosa; farnesyltransferase and FPP synthase activities and cholesterol synthesis were also upregulated Cerda et al., 1995;Hentosh et al., 2001;Notarnicola et al., 2004;Caruso and Notarnicola, 2005;Tyagi, 2005 Connective tissue Upregulation of mevalonate pathway was correlated with lower survival of sarcoma patients Kuzu et al., 2016 Embryo HMGCR mRNA in embryonic tumors was higher than that in fetal tissues Engstrom and Schofield, 1987 Esophagus HMGCR in esophageal squamous cell carcinoma was higher than that in esophageal tissue Shi et al., 2013 Liver HMGCR activity and cholesterol biosynthesis were several-fold higher in hepatocellular carcinoma and human HepG2 and Hep3B hepatoma cells than those in liver, hepatocytes and fibroblasts Kawata et al., 1990;Tam et al., 1991;Sohda et al., 2013 Lung HMGCR activity in A549 lung carcinoma cells was 2 to 4-fold higher than that in fibroblasts; overexpressed GGPP synthase was found in lung adenocarcinoma tissues and correlated with large tumors, high TNM stage, lymph node metastasis and poor prognosis Rat Breast DMBA-and MNU-induced mammary tumor had 2 to 4-fold higher HMGCR that was resistant to dietary cholesterol; total cholesterol in neoplastic tissue was 2 to 3-fold higher; neoplastic cholesterol synthesis was 5 to 6-fold higher Rao et al., 1988;El-Sohemy and Archer, 2000 Liver HMGCR activities in hepatoma, carcinogen-induced hepatic nodules and preneoplastic foci were up to 14-fold higher than that in liver; HMGCR in hepatoma and preneoplastic foci was less responsive to cholesterol feedback Siperstein et al., 1971;Mitchell et al., 1978;Feingold et al., 1983;Gregg et al., 1986;Erickson et al., 1988;Azrolan and Coleman, 1989;Coni et al., 1992;Olsson et al., 1995 Pancreas Tumor and fetal pancreas had higher HMGCR activity; fast-growing AT3A tumor had higher HMGCR activity than the slow-growing AT3B tumor Rao et al., 1983 (Continued) Frontiers in Pharmacology | www.frontiersin.org

Mouse Liver and bone marrow
Baseline HMGCR activity in liver tumors was 2 to 8-fold higher than that in liver; 1% dietary cholesterol led to >90% reduction in liver HMGCR but had much less response in hepatoma; ectopic expression of HMGCR in normal bone marrow or fetal liver cells increased myeloid colony formation Kandutsch and Hancock, 1971;Clendening et al., 2010 Guinea pig Lymph L2C leukemic lymphocytes had >30 times higher cholesterol biosynthesis, 8 times higher HMGCR, and 25 times higher fatty acid biosynthesis than normal lymphocytes Philippot et al., 1977 hinder the ubiquitination and degradation of mutant p53 protein, statins also suppress the growth of mutant p53-expressing cancer cells (Freed-Pastor et al., 2012;Freed-Pastor and Prives, 2016;Parrales et al., 2016). A recent study suggest that the anticancer effect of statins is associated with the epithelial-to-mesenchymal transition phenotype (Yu et al., 2018). Clinical efficacy of statins in cancer reduction may be tissue specific. Statin use was found to be associated with lower risks of primary liver cancer (McGlynn et al., 2015), hepatocellular carcinoma (Kim et al., 2018), HPV-negative squamous cell carcinoma (SCC) of the larynx, hypopharynx, and nasopharynx (Lebo et al., 2018), and subtypes of non-Hodgkin lymphomas including diffuse large B-cell lymphomas and plasma cell lymphomas (Ye et al., 2018), reduced aggressiveness (Allott et al., 2016) and mortality (Yu et al., 2014) of prostate cancer, and lower cancer specific and all-cause mortalities in esophageal cancer . However, statins do not affect survival after colorectal cancer (Hoffmeister et al., 2015) and small-cell lung cancer (Seckl et al., 2017), the risk of pancreatic cancer (Hamada et al., 2018), or the progression of prostate cancer in certain minority-enriched subpopulations (Allott et al., 2018). The type and hydrophilicity of statins, length of statin use, and ethnicity, lifestyle, and preexisting health condition of subjects may have contributed to the diverse outcome in statin and cancer studieswith some but not all studies showing the anticancer effect of statins (Gong et al., 2017).
Reported dose-limiting toxicities of statins may further deter the use of statins-at least as single therapies-in cancer treatment. Observations in clinical practices note approximately 20% adverse reaction rates to statins (Bruckert et al., 2005;Maningat and Breslow, 2011;Zhang et al., 2013Zhang et al., , 2017. Possible adverse effects include diabetes mellitus, hemorrhagic stroke, cognition decline, tendon rupture, interstitial lung disease, and muscle problems . The Effect of Statins on Skeletal Muscle Function and Performance (STOMP) study suggested 5-10% myalgia incidence in statin users (Parker et al., 2013). Concerned about the potential adverse effects of statins on cognition, glycemic control, and incident diabetes as well as their wide range of interactions with other medications, the US Food and Drug Administration issued and implemented new safety labeling for statins in 2012.
The dose-limiting toxicities of statins may be at least partially attributed to statin-mediated compensatory upregulation of HMGCR. By mimicking and competing with HMG CoA, the substrate for HMGCR, statins render HMGCR unavailable for catalyzing the formation of mevalonate and downstream sterols and nonsterols including FPP and GGPP. Statins also change the conformation of HMGCR and prevent it from attaining a functional structure, attaining high efficacy and specificity in inhibiting HMGCR (Sirtori, 2014). By removing the sterol and nonsterol products that downregulate HMGCR, persistent exposure to statins induces compensatory overexpression of the HMGCR in vitro and in vivo (Roglans et al., 2002). Elevated HMGCR expression by approximately 6-to 10folds has been observed in liver microsomes and fibroblasts (Bensch et al., 1978;Sirtori, 2014). In our hands lovastatin also induced upregulation of HMGCR in human DU145 prostate carcinoma cells (Yeganehjoo et al., 2017). Exposure to rosuvastatin, lovastatin, and atorvastatin caused 6-, 11-, and 15fold greater expression of hepatic HMGCR protein, respectively, and enhanced SREBP-2 processing of some target genes in mice (Schonewille et al., 2016). Since majority of cholesterol synthesis occurs in the liver, the significant hepatic uptake of statins and the limited bioavailability of many statins in extra-hepatic tissues can cause compensatory induction of mevalonate pathway in these tissues. This can, in turn, stimulate overproduction of isoprenoid metabolites (FPP and GGPP) and downstream prenylated proteins (Duncan et al., 2005;Brown, 2007;Solomon and Freeman, 2008). A compensatory increase in HMGCR expression in extrahepatic tissues following cholesterol-lowering statin therapy may provide a mechanistic basis for the elevated risk of statin-related tumorigenesis observed in some studies (Sacks et al., 1996;Coogan et al., 2002;Shepherd et al., 2002;Beck et al., 2003).
In summary, preclinical and mechanistic studies support the anti-cancer property of statins. The indiscriminate inhibition of HMGCR in tumor and non-tumor cells and the resulted overexpression of HMGCR may have contributed to doselimiting toxicities of statins and mixed outcomes in clinical investigations. Efforts have been devoted to identifying agents capable of synergizing with statins in cancer growth inhibition.
Diverse isoprenoids of plant mevalonate metabolism trigger post-transcriptional events to regulate the HMGCR expression and activity. These isoprenoid-mediated modulations include blockade of HMGCR mRNA translation and induction of HMGCR degradation in proteasomes.

SYNERGISTIC EFFECT OF STATINS AND ISOPRENOIDS ON TUMOR GROWTH MAY REDUCE STATIN TOXICITY
Since long-term use of a single compound requires a relatively high dose, which tends to be associated with adverse effects, synergistic effects of therapeutic compounds can provide the same or enhanced efficacy with lower doses of each agent and plausibly fewer adverse effects to lesser degrees. Investigations with blends of isoprenoids have shown favorable outcomes. Studies using tumor cells of skin McAnally et al., 2007), prostate (Mo and Elson, 2004), and pancreas (Hussein and Mo, 2009) have delineated the synergistic or cumulative effects of HMGCR suppressors on mevalonate signaling and tumor cell growth. Geranylgeraniol and d-δtocotrienol synergistically suppressed growth of the murine b16 melanoma cells (Katuru et al., 2011). Dietary γ-tocotrienol (2 mmol/kg) and β-ionone (2 mmol/kg) administered individually or together also improved survival in mice bearing implanted melanomas (He et al., 1997).
Isoprenoids have also shown synergistic effect with statins ( Table 3). Figure 2 lists the structures of representative isoprenoids that potentiate the anti-cancer efficacy of statins in vitro and in vivo. All three two-way blends of tocotrienols, lovastatin, and β-ionone synergistically suppressed the proliferation of murine B16 melanoma and human A549 lung carcinoma, A2058 melanoma, MIA PaCa-2 pancreatic carcinoma, and DU145 prostate carcinoma cells (He et al., 1997;Mo and Elson, 1999;McAnally et al., 2007;Fernandes et al., 2010;Katuru et al., 2011). In human MCF-7 mammary tumor cells resistant to doxorubicin and tamoxifen, simvastatin and FIGURE 2 | Structures of representative isoprenoids that impact tumors or potentiate the statin-mediated tumor suppression. Differing from the monoterpenes (carvacrol, carvone, perillyl alcohol, geraniol), sesquiterpenes (cacalol, farnesol, β-ionone), and diterpene (geranylgeranyl acetone), the tocotrienols are "mixed" isoprenoids with only part of their structure derived from the mevalonate pathway. The number and location of methyl group (R 1 and R 2 ) on the chromanol ring vary among the members (α, β, γ, and δ) of tocotrienols.
The effect of tocotrienols on cell viability correlates with that on SREBP-2 activity, consistent with the finding that downregulation of SREBP2 and subsequent lipid biosynthesis effectively suppresses the growth of colon cancer proliferation and tumor spheroid formation (Wen et al., 2018). SREBP-2 and HMGCR were overexpressed in cisplatin-resistant A2780 epithelial ovarian cancer cells (Zheng et al., 2018). SREBP-2 promotes the expression of Staphylococcal nuclease and tudor domain containing 1 gene that is associated with the progression and malignancy of colon, breast, prostate, lung, glioma, skin, and liver cancers (Armengol et al., 2017), providing potential new applications for tocotrienols in cisplatin-resistant cancers. Mevalonate rescues the effect of statin, but not that of tocotrienol, on prostate cancer cell viability, consistent with tocotrienol-mediated increase in the degradation of nuclear, mature SREBP-2, which maintains cholesterol homeostasis through transcriptional regulation of enzymes in cholesterol biosynthesis and uptake beyond HMGCR (Krycer et al., 2012). The tocotrienol-enhanced degradation of SREBP-2 suggests that tocotrienol may impact, in addition to HMGCR, multiple enzymes of the mevalonate pathway, offering another aspect of synergy with statins, which specifically inhibit HMGCR. Such suitable and optimal combinations of statins and tocotrienol could lead to favorable synergistic effects with lower effective doses of statins with minimized or no side effects. The pair's combined lipid-lowering effects have been shown in chickens (Qureshi and Peterson, 2001) and hypercholesterolemic humans .

CONCLUSION AND FUTURE DIRECTIONS
The widely prescribed statins, originally designed for hypercholesterolemia, hold promises for cancer therapy as the multiple roles of the mevalonate pathway in growth support and regulation unfold. The repurposing of this class of wellestablished drugs for cancer stems from their ability to deplete mevalonate-derived sterol and nonsterol products essential for the prenylation of proteins involved in growth regulation and signaling pathways for cell proliferation. Nevertheless, the indiscriminate inhibition of HMGCR in normal and tumor cells and compensatory upregulation of HMGCR induced by statins may have contributed to their dose-limiting toxicities and conflicting clinical outcomes. The upregulated and sterol-resistant tumor HMGCR remains responsive to the isoprenoid-mediated downregulation, offering isoprenoids tumor-targeted growth suppression with little toxicity to normal cells. Isoprenoids also attenuate statin-induced compensatory upregulation of HMGCR via augmented, proteasome-mediated degradation of HMGCR, providing a new approach in enhancing the efficacy of statins with reduced adverse effects associated with lower statin doses. Most studies on the combination of isoprenoids and statins, however, are limited to in vitro models. A major gap hence remains in the lack of clinical data on the bioavailability, pharmacokinetics, pharmacodynamics, efficacy and mechanisms of action of the isoprenoids. The activities of the vast majority of the estimated 22,000 isoprenoids (Bach, 1995) remain unexplored. New formulation including those employing nanoparticles may improve the bioavailability of isoprenoids. Isoprenoid derivatives (Jung et al., 1998;Mo et al., 2000) with improved potencies and lower toxicities may provide more opportunities for optimal formulations. The combinations of isoprenoids and statins with complementary mechanisms and synergistic actions hold promise for cancer prevention and therapy and warrant further clinical studies.