c-Myc Oncoprotein: A Dual Pathogenic Role in Neoplasia and Cardiovascular Diseases?

Abstract

A growing body of evidence indicates that c-Myc can play a pivotal role both in neoplasia and cardiovascular diseases. Indeed, alterations of the basal machinery of the cell and perturbations of c-Myc-dependent signaling network are involved in the pathogenesis of certain cardiovascular disorders. Down-regulation of c-Myc induced by intervention with antioxidants or by antisense technology may protect the integrity of the arterial wall as well as neoplastic tissues. Further intervention studies are necessary to investigate the effects of tissue-specific block of c-Myc overexpression in the development of cardiovascular diseases.

Introduction

A great deal of work has focused on how oncogenes regulate cell cycle during normal development and in cancer, yet their roles in regulating cell growth have been largely unexplored [1]. Recent studies in several experimental models have demonstrated that homologues of some oncogenes regulate cell growth and suggested that certain effects of oncogenes on cell cycle may be a result of growth promotion [1]. These studies have also suggested how growth and cell cycle progression may be coupled.

The transcriptional regulatory proteins encoded by the c-Myc proto-oncogene family have been linked to multiple aspects of eukaryotic cell functions including cell cycle progression, differentiation, neoplasia, and apoptosis [2–8]. c-Myc is characterized by the presence of basic-helix-loop-helix (HLH) leucine zipper motif and is a transcriptional regulator involved in carcinogenesis through its role in growth control, cell cycle progression, and stimulation of the G1-S transition. Examples of both positive and negative regulation involving E-box and promoter elements have been reported. In both cases, c-Myc is thought to induce changes in transcription initiation. The HLH and zipper motif participate in protein dimerization, a prerequisite for DNA binding. The complete mechanism is still not well understood and c-Myc can also control cell proliferation, survival, and transformation regulating negatively p27 cyclin-dependent kinase inhibitor in smooth muscle cells, breast cancer, and leukemia cells [9]. In general, c-Myc regulates mammalian body size by controlling cell number but not cell size [10]. Biological evidence suggests the hypothesis that cancer and atherosclerosis and/or vasculoproliferative or other cardiovascular diseases may share pathologic mechanisms, emphasizing the need to perform studies investigating the involvement of somatic mutations in these pathologic conditions.

c-Myc in Vascular Proliferative Disorders

Vasculoproliferative disorders and neointimal formation occur during several pathophysiologic conditions. Together with c-myb, c-Myc has been reported to be essential for vascular smooth muscle cell (VSMC) proliferation [11]. For example, restenosis following arterial injury consists mainly of vascular cellular proliferation, which can ultimately lead to arterial occlusion [12]. In addition, cell migration, matrix deposition, and vascular remodeling are involved in restenosis after arterial injury [13]. Following balloon injury, c-Myc stimulates mitogenesis of quiescent VSMCs, and tissue-selective expression of dominant-negative proteins for c-Myc in combination with c-Myb blocks VSMC proliferation in rat as well as human transfected VSMCs, inducing apoptosis [14]. In another experimental setting, molecular and histologic data demonstrate that c-Myc mRNA was increased with a maximum at 4 hours after surgical treatment [15]. A recent model of vascular stenosis utilizes surgical injury for induction and development of stenosis in the rat common carotid artery, which mimics the injury occurring during implantation of arterial grafts, endarterectomy, or organ transplantation [16]. Histologic and histochemical analysis of carotid sections showed that morphologic changes, which occurred 30 days after surgical injury in the arterial wall, were associated with c-Myc overexpression. A number of data suggest that angiotensin II-dependent activation of the proto-oncogene c-Myc participates in the proliferative response of VSMCs of rats with spontaneous hypertension (SHR) [17,18]. Compared to normotensive Wistar-Kyoto rats, untreated SHR exhibited increased percentage of cells expressing c-Myc (P<0.05) and cyclin A (P<0.001) [17]. In quinapril-treated SHR compared with untreated SHR, there was decreased expression of c-Myc (P<0.005) and cyclin A (P<0.001). Thus, an enhanced expression of c-Myc may be involved in the increased proliferation seen in VSMCs from SHR. Quinapril administration normalizes proliferation in the VSMCs of SHR, possibly by inhibiting the expression of the oncoprotein c-Myc and its effects on the cell cycle. It was also demonstrated that ANG II receptors, c-Myc, and c-Jun in myocytes are overexpressed after myocardial infarction and ventricular remodeling [19]. However, nitric oxide-induced apoptosis was associated with c-Myc and CPP32 caspase activation [20] and the cytotoxic effect of sodium nitroprusside on cancer cells involves the suppression of c-Myc and c-Myb proto-oncogene expression [21].

c-Myc antisense oligomers reduces neointimal formation after arterial injury in pigs [22] as well as a phosphorothioated oligonucleotides in rat carotid arteries [23]. Moreover, local delivery of c-Myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon-injury model [24]. These interventional approaches require further investigation.

Autologous saphenous vein is one of the conduits of choice for the bypass of arterial occlusive disease in either the peripheral or coronary arterial circulation. This technique is limited by high primary graft failure rates (approaching 20% in the first year for peripheral arterial disease and 50% at 10 years for coronary artery bypass grafting), which is often consequent to intimal hyperplasia. A role for c-Myc in this process is suggested by its overexpression in vein graft intimal hyperplasia [25], and may be mediated by the c-Myc oncoprotein binding elongation 2 factor (E2F) that is involved in the basal machinery of the cell. Interestingly, the PREVENT trial describes a novel, relatively safe and effective means of ex vivo transfection of harvested vein grafts with an E2F decoy oligonucleotide, resulting in 70% to 74% decreases in the level of proliferating cell nuclear antigen (PCNA) and c-Myc mRNA expression within the vessel (reviewed in Ref. [26]). This translated into a statistically significant reduction in failure of primary grafts when these were subsequently used to bypass peripheral arterial occlusions in a high-risk human patient population.

Finally, local delivery of mithramycin (a cell cycle inhibitor) restores vascular reactivity and inhibits neointimal formation in injured arteries and vascular grafts [27]. These studies indicated that perivascular implantation of drug-loaded matrices led to 50% reduction of neointimal formation and reduced the c-Myc expression and VSMC proliferation in comparison to control implants. Moreover, local perivascular mithramycin treatment limits the functional alterations caused by the grafting of venous segments in high-pressure arterial environment and potently inhibits vascular stenosis secondary to grafting or angioplasty injury.

c-Myc and Atherogenesis

Accumulation of lipids and proliferation of VSMCs are the main histologic features of atherosclerotic plaque formation. The most prominent classical theory concerning the pathophysiologic mechanisms of atherosclerotic plaque formation is the “inflammatory response to injury” hypothesis, which describes VSMC proliferation as an inflammation-fibroproliferative reaction to different insults to the artery wall [28]. However, alterations at the DNA level may contribute significantly to the development of the disease, and form the basis for the “monoclonal” hypothesis of atherosclerosis [29,30]. According to this hypothesis, atherosclerosis could begin as a mutation or viral infection, transforming a single, isolated VSMC into the progenitor of a proliferative clone, as seen in carcinogenesis. Such a phenomenon may involve the enhanced expression of c-Myc demonstrated in human VSMCs cultured from aortic plaques [31] and in carotid atherosclerotic lesions [32]. Transforming genes and c-Myc overexpression were also found in coronary VSMCs when plaque DNA was transfected into fibroblast and the transformed cells were injected into nude mouse [33]. The mechanism by which c-Myc is activated is not well known but oxidative stimuli activate c-Myc (its dimerization with Max) E2F and AP-1, both in vitro and in vivo [34]. Indeed, c-Myc-dependent signaling was found activated in early coronary lesions of Watanabe Heritable Hyperlipidemic rabbits [34] and in early coronary lipidotic accumulation in coronary vascular wall of hypercholesterolemic pigs [35]. Abnormal expression and dysfunction of this apoptosis-regulated gene may either attenuate or accelerate vascular cell apoptosis and affect the integrity and stability of plaques [34–36]. More important, antioxidants downregulated c-Myc overexpression [34,35] in a similar fashion to that observed in tumor cells [37].

In addition, estrogen and progesterone may also affect VSMC gene expression of growth regulatory molecules such as platelet-derived growth factor (PDGF), interleukin-1 (IL-1), interleukin-6 (IL-6) and proto-oncogene c-Myc. In a recent study, VSMCs were exposed to estrone sulfate (E1-S) and medroxyprogesterone acetate (MPA) to induce differentiation [38]. E1-S inhibited the expression of PDGF-A chain, IL-1, IL-6, and c-Myc mRNA, whereas MPA had no effect. Inhibition by E1-S was not affected by treatment combined with MPA. These findings suggest that estrogen but not progesterone modulates growth regulatory molecules and c-Myc gene expression in VSMC. Thus, estrogen may interfere with signals in the arterial wall through inhibition of growth regulatory factors and this could affect vascular diseases and atherogenesis. However, the c-Myc protooncogene is an estrogen target gene in hormone-responsive breast cancer, and hormonal progression of breast cancer can be elicited by its enhanced expression through c-Myc gene amplification and enhanced mRNA stability [39], suggesting that its modulation by estrogen may depend on other coexisting factors.

c-Myc in Cardiac Hypertrophy

Cellular oncogenes like c-Myc may participate in both the normal developmental cell proliferation as well as in pressure overload or angiotensin II-induced cardiac hypertrophy [40–42]. Recently, c-Myc overexpression was also reported to correlate with left ventricular hypertrophy and dysfunction in patients with chronic aortic regurgitation [43]. The expression of c-Myc was induced when the myocardial hypertrophy was moderate. Nevertheless, when the myocardial hypertrophy was severe, cardiac myocytes failed to express the c-Myc [44]. Similarly, the expression of c-Myc during pressure-overloaded hypertrophy of the rat heart was transient [40]. Several growth factors interacting with c-Myc [38] influence cardiac hypertrophy, but cytokines do not seem to play a mayor role in subsequent heart failure [45]. Furthermore, c-Myc, c-fos, and c-Jun, have been reported to be implicated in the pathogenesis of myocardial remodeling [40–42]. More recently, to determine the effects of de novo c-Myc activity in adult postmitotic myocardium in vivo, a novel transgenic model was investigated in which c-Myc is expressed and inducibly activated specifically in cardiac myocytes [46]. Activation of c-Myc in adult myocardium was sufficient to reproduce the characteristic changes in myocyte size, protein synthesis, and cardiac-specific gene expression seen in cardiac hypertrophy. However, despite the increased cardiac mass, left ventricular function remained normal. Hence, future studies will be needed to define the role of c-Myc in cardiac hypertrophy and remodeling.

c-Myc in Cerebrovascular Ischemia and Reperfusion

Various stimuli also seem to activate c-Myc in the cerebrovascular system. For example, reactive oxygen species implicated in several pathologies act by activating several transcription pathways, such as NFκB as well as c-Myc. During reperfusion injury after focal cerebral ischemia in mice, reactive oxygen species regulate the activity of NFκB and c-Myc [47]. These factors were found increased in ischemic zone and colocalized in apoptotic neuron. Furthermore, overexpression of copper/zinc superoxide dismutase in transgenic mice prevented activation of NFκB, B, blocked the expression of c-Myc, and reduced the ischemic damage, suggesting a role for this pathway in cerebrovascular reperfusion injury [47].

c-Myc and Neoplasia

The quantity of c-Myc is normally carefully controlled, and its actions to induce and repress genes are modulated by interactions with other regulatory proteins [2–8,48]. It is well known that the c-Myc proto-oncogene plays a central role in proliferation and malignant transformation of both human and animal cells, and its amplification and/or overexpression have been reported in most types of human malignancy [7,8,49]. c-Myc regulation of growth affects both cellular size and tissue differentiation, and this gene participates in most aspects of cellular function, including replication, growth, metabolism, differentiation, and apoptosis. The recent availability of DNA chip microarray screen leads to the identification of genes differentially involved in c-Myc and n-Myc oncogenesis in human tumors [50]. c-Myc disrupts genetic control in the early G1 phase of the cell cycle, and its coexpression with Bcl-2 antagonizes the effects of the p53 tumor suppressor gene p53 on G1 arrest and apoptosis [7,8,48,49,51]. c-Myc integrates the cell cycle machinery with cell adhesion, cellular metabolism, and the apoptotic pathways [7,8,49,51,52], and deregulation of its expression is a common feature of multiple cancers [7,8], especially many breast cancers [53]. Interestingly, there is c-Myc overexpression in breast cancer and also in adjacent non-neoplastic tissue [54]. This suggests an important causal role of c-Myc-dependent downstream effects in breast cancer. Furthermore, in melanoma, major histocompatibility complex (MHC, HLA in humans) class I antigen expression is suppressed by overexpression of the c-Myc protooncogene and HLA-B locus, which is mainly affected by c-Myc, is downmodulated at the level of initiation of transcription [55]. Downregulation of MHC class I expression is a mechanism by which tumor cells escape from T cell-mediated lysis and these alterations have severe consequences for the recognition of these tumor cells by the immune system of the organism. HLA-B core promoter is repressed by wild-type p53, making p53 a candidate for mediating c-Myc-induced HLA-B downregulation [56]. However, transfection of c-Myc into p53-null cell lines still resulted in suppression of the basal HLA-B promoter, demonstrating that c-Myc and p53 repress the minimal HLA-B promoter through independent mechanisms [56].

The crucial molecular step in ionizing radiation-induced apoptosis by radiotherapy is the release of mitochondrial cytochrome c into the cytosol [57]. The precise ways the tumor suppressor protein p53, as well as the oncoprotein c-Myc, as well as Ras and Raf, and Bcl-2 gene can influence this process at different stages are still poorly understood [57]. Indeed, the result of activation of an oncoprotein on tumor radiosensitivity depends on its mechanism of action and on the presence of other prooncogenic factors, because complex interactions among many molecular factors determine the delicate balance between cell proliferation and cell death. The ongoing identification and characterization of these factors influencing this pathophysiologic scenario will eventually make it possible to predict tumor radiosensitivity and thereby improve cancer treatment [57].

c-Myc and Angiogenesis

The ability of neoplastic cells to recruit blood vessels is crucial to their survival and growth in the host organism. However, the evidence linking dominant oncogenes to the angiogenic switch remains incomplete. Interestingly, the oncoprotein c-Myc stimulates neovascularization in several malignancies [7,8,55,56,58]. For example, angiogenesis was identified as an early consequence of c-Myc overexpression in two models of retroviral lymphomagenesis; Avian leukosis virus induces bursal lymphoma in chickens after proviral c-Myc gene integration, whereas the HB-1 retrovirus carries a v-Myc oncogene and also induces metastatic lymphoma. Overexpression of c-Myc confers on the conditioned media the ability to promote migration of adjacent endothelial cells in vitro and corneal neovascularization in vivo, and furthermore, mobilization of estrogen-dependent c-Myc in vivo with the appropriate steroid provokes neovascularization of cell implants [59]. These data indicate that c-Myc is capable of triggering the angiogenic switch in vivo, and that secondary events may not be required for neovascularization of c-Myc-induced tumors.

Indeed, ectopic c-Myc overexpression in the low Myc B cell lines increased endothelial growth activity, indicating that c-Myc induces secretion of angiogenic factors from B cells [60]. These findings demonstrated that c-Myc overexpression in lymphocytes generates also an angiogenic phenotype in vitro as well as in vivo.

Overexpression of c-Myc causes downregulation of the thrombospondin-1 (tsp-1) gene, an important negative modulator of tumor angiogenesis, and c-Myc in combination with Max can bind, albeit with low affinity, to an E-box-like element in the tsp-1 promoter [61]. However, the 2.7-kb DNA segment containing both this noncanonical E-box and other promoter-like sequence does not constitute a c-Myc-responsive element in a transient expression system. Furthermore, c-Myc does not significantly affect the rate of initiation or elongation of the tsp-1 mRNA. Thus, in this case c-Myc does not seem to act as a canonical transcription factor. Instead, as demonstrated by blocking de novo RNA synthesis, downregulation of the tsp-1 gene by c-Myc occurs through increased mRNA turnover. Thus, gene regulation by c-Myc may involve mRNA destabilization.

Modulation of c-Myc may also partly account for the mechanism of action of some antiangiogenic drugs. Endostatin is a potent endogenous angiogenesis inhibitor that induces regression of tumors in mice. Neither an extracellular receptor for endostatin nor intracellular signals that result in the regression of tumor vascular beds have been identified. Endostatin, but not angiostatin, at concentrations comparable to those used in in vivo animal studies, rapidly downregulates many genes in exponentially growing endothelial cells including c-Myc [62]. Suppression of both apoptosis inhibitors and cell proliferation genes may have a limited contribution to the antiangiogenesis process because endostatin induces neither apoptosis nor growth inhibition unless studied under reduced serum conditions. In contrast, the antimigratory effect of endostatin was rapid and potent even under serum-supplemented conditions. Endostatin caused gene suppression and migration arrest exclusively in endothelial cells, most profoundly in microvascular endothelial cells. The c-Myc null fibroblasts obtained by targeted homologous recombination showed an attenuated migration rate compared with isogenic parental cells, whereas the introduction of the c-Myc gene into endothelial cells abrogated the antimigratory effect of endostatin [62]. Inhibition of E-box-driven transcription by overexpressing Max or Mad suppressed endothelial migration. Thus, rapid downregulation of genes by endostatin neither restores proliferating endothelial cells to their resting states nor induces apoptosis; rather, it potently inhibits endothelial cell migration partly through suppression of c-Myc expression. As described, in addition to angiogenesis, c-Myc may play other roles in neoplasia such as positive or negative signaling for apoptosis [7,8,47–49,50,51,55,56,63] and loss of growth inhibition [7,8,64] that may affect vascular cell signaling.

Conclusions

A growing bulk of evidence suggests that c-Myc may play a dual pathogenic role in neoplasia and cardiovascular diseases (Figure 1). Most of these studies carried in the cardiovascular system did not provide a causal role of c-Myc in the disease but these data are consistent with the hypothesis that alterations of the c-Myc-dependent signaling network are involved in the early pathogenesis of common cardiovascular diseases. The c-Myc gene produces an oncogenic transcription factor that affects diverse cellular processes involved in cell growth, cell proliferation, apoptosis, and cellular metabolism. Complete removal of c-Myc results in slowed cell growth and proliferation, suggesting that although c-Myc is not required for cell proliferation, it acts as an integrator and accelerator of cellular metabolism and proliferation [65]. Down-regulation of c-Myc induced by intervention with antioxidants [34,35,37] may induce relevant effects in the arterial wall [66–68] and in neoplastic tissues [34]. Similarly, the use of c-Myc antisense oligomers may reduce vascular diseases. Microarray can identify c-Myc-dependent target genes involved in cardiovascular disorders [67,68]. Further intervention studies are necessary to investigate the effects of tissue-specific block of c-Myc during certain neoplastic and cardiovascular diseases. To date, the PREVENT trial [26] provides a novel approach that reduced graft failure and c-Myc expression within vessel.

Figure 1.

Flow chart depicting the proposed sequela of events leading to the dual role of c-Myc activation in neoplasia and cardiovascular diseases. C-Myc is involved in several neoplasias as indicated. Restenosis through the activation of the cell cycle progression proteins such as cyclin E and ornithine decarboxylase, but it also increases transcription of proliferation genes such as c-Fos and c-Jun. During atherogenesis there is activation of several c-Myc-dependent transcription factors such as AP-1 (activation protein 1), E2F (elongation factor-2) and AP-2 (activation factor-2). Cardiac hyperthropy stimulates the activity of phosphoinositol-3-phosphate kinase (PI3K) and phospholipase C-gamma (PLC-γ). Finally, myocardial ischemia-reperfusion induces c-Myc-dependent activation of the transcription factor NFµB (nuclear factor kappa B) whereas during angiogenesis there is a concomitant activation of the thrombospondin-1 factor (tsp-1).