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Circadian clock: Time for novel anticancer strategies?
Accepted Manuscript Title: Circadian clock: time for novel anticancer strategies? Author: Luisa Ercolani Alessio Ferrari Claudia De Mei Chiara Parodi Mark Wade Benedetto Grimaldi PII: DOI: Reference:
S1043-6618(15)00176-0 http://dx.doi.org/doi:10.1016/j.phrs.2015.08.008 YPHRS 2905
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
12-8-2015 12-8-2015 12-8-2015
Please cite this article as: Ercolani Luisa, Ferrari Alessio, Mei Claudia De, Parodi Chiara, Wade Mark, Grimaldi Benedetto.Circadian clock: time for novel anticancer strategies?.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Circadian clock: time for novel anticancer strategies? Luisa Ercolani*, Alessio Ferrari*, Claudia De Mei*, Chiara Parodi*, Mark Wade#@, Benedetto Grimaldi*@
* Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy #
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via
Adamello 16, 20139, Milano, Italy @
Corresponding authors: email: [email protected] email: [email protected]
Graphical abstract fx1
Abstract
Disruption of the circadian clock is associated with a variety of human pathologies, including cancer. Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression observed in many tumors may serve for cancer cell survival. This scenario suggests the provocative hypothesis that
pharmacological modulation of clock-related proteins may be suitable as an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REVERBα and REV-ERBβ, in cancer cell survival and describe the potential development of small molecule compounds that modulate REV-ERB activity as novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies. Keywords: anticancer agents, ARN5187, autophagy, cancer, circadian, combined anticancer strategy, dual inhibitor, metabolism, lysosomotropic agents, nuclear receptors, REV-ERB, REV-ERB antagonist, REV-ERB agonist, SR9011.
1. Introduction 1.1 The circadian clock Many physiological processes obey a biological clock that has an intrinsic period of approximately 24 hours (circadian)(1, 2). The molecular mechanism underlying these circadian rhythms is based on interconnected transcriptional–translational feedback loops in which specific factors repress the transcription of their own genes(3). The anatomical center of the mammalian circadian clock, composed of approximately 15,000 neurons, resides in a region of the anterior hypothalamus of the brain called the suprachiasmatic nucleus (SCN) (1). Nevertheless, a finding that profoundly modified our view of circadian clocks is that most peripheral tissues contain intrinsically independent pacemakers (1). Although the clock of the SCN plays a fundamental role in synchronizing whole-body circadian rhythm, tissue-specific cell-autonomous molecular clocks choreograph the rhythmic processes in peripheral tissues(1, 4).
At the molecular level, the endogenous clock can be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the transcription of clock controlled genes (CCGs) (Figure 1). The proteins CLOCK and BMAL1 interact to form a clock activator complex promoting transcription of CCGs. These genes also code for proteins belonging to the clock repressor factors (PERs, CRYs and REV-ERBs proteins). The function of these repressor proteins is to decrease CLOCK/BMAL1-dependent transcription. Finally, rhythmic cell-specific or tissue-specific gene expression is achieved through the cooperative action of the core clock genes with local transcription factors and chromatin remodeling enzymes (5). The overall balance between clock-activator and clock-repressor complexes results in the circadian expression of around 10-15% of the mammalian genome (6, 7).
Figure 1. The circadian clock machinerycan be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the
transcription of clock controlled genes (CCGs). In mammals, the proteins BMAL1/BMAL2 (shown as BMAL) and CLOCK/NAPS2 (shown as CLOCK) are transcriptional activators acting to enhance the expression of the CCGs. These factors also induce the expression of the transcriptional repressor proteins, PER1/PER2/PER3 (shown as PER), CRY1/CRY2 (shown as CRY) and REV-ERBα/REV-ERBβ (shown as REV-ERB). The overall balance of the fight between transcriptional activators and repressors results in rhythmic expression of a number of genes involved in a variety of physiological responses.
Clock-regulated transcripts also include a number of pharmacological targets, as well as key enzymes involved in drug metabolism (8). Circadian expression of drug-related genes and differential circadian profiles between normal and pathological cellular states are well documented.These phenomena may have profound consequences for the efficacy and safety of new and existing therapeutic agents, as they are likely to determine the timing, dosage, and duration of optimal drug exposures(8). As an example, the anabolic enzymes that generate the cytotoxic forms of 5-Fluorouracil (5FU) display a circadian expression pattern in both rodents and human (9). Moreover, the major on-target side effects of the DNA synthesis inhibitor 5-FU in highly proliferative tissues (such as bone marrow, skin, and the oral and rectal mucosa) can be reduced by treating patients at night rather than during the daytime (9). This is since proliferation is under circadian control in these tissues, and is more rapid during daylight hours. The improved therapeutic index due to administration of 5-FU during the night, is a clear example of the benefits of chronotherapy. Disruption of the circadian clock is associated with a variety of human pathologies, including cancer, and the expression of several clock genes is perturbed in many tumors (10)(11).
Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression in tumors likely plays a causal role in the development of cancer and the survival of tumor cells. For instance, several epidemiological studies indicated that the incidence of breast cancer is higher among women who spent more years and hours per week working nightshifts(12). These observations suggest the provocative hypothesis that pharmacological modulation of clock-related proteins may be an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REVERBα and REV-ERBβ, in cancer cell survival and describe thepotential development of small molecule compounds that modulate REV-ERB activityas novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies.
1.2 The circadian nuclear receptors, REV-ERBs The circadian regulator REV-ERBα (also known as NR1D1) and its variant isoform REVERBβ (also known as NR1D2) belong to the nuclear receptor superfamily, which is composed of a large family of ligand-sensitive transcription factors(13). Unlike most other nuclear receptors, REV-ERB proteins lack a transcriptional activation domain and instead repress target genes bearing Rev-erb Responsive Elements (RevREs) within the promoter. REV-ERB transcriptional repression is modulated by the interaction of the receptor Ligand Binding Domain (LBD) with a heme moiety (14). REV-ERBα and REV-ERBβ regulateinter-connected processes, such as circadian rhythm and metabolism (Figure 2) (15).
Studies in rodents revealed that the two REV-ERB variants compensate for one another in the repression of many common target genes. These studies also indicatedthat, in several conditions, abrogation of both REV-ERBα and REV-ERBβ function is required to profoundly affect REV-ERB-mediated physiological responses (15, 16). A pharmacological screen identified the SR6452 (GSK4112) compound as the first REVERBα synthetic agonist (17). Accordingly, GSK4112 increased REV-ERBα-mediated repression in cell-based assays and modulated REV-ERBα regulated adipogenesis of 3T3-L1 cells (18).
Figure 2. REV-ERBα and REV-ERBβ nuclear receptors (shown as REV-ERB) are hemebinding proteins that repress transcription of target genes containing a REV-ERB responsive element (RevRE) within their promoters. Upon heme binding, the nuclear co-repressor NcoR and the histone de-acetyltransferase HDAC3results are recruited on the promoter of REVERB-regulated genes to shut-down transcription. REV-ERB targets include the master clock
regulator, BMAL1, and the gluconeogenetic phosphoenolpyruvate carboxykinase enzyme, PEPCK. Because of the intimate link between the circadian clock and cellular metabolism, REV-ERB-mediated regulation of the molecular clock machinery further places REV-ERB proteins at a strategic position in metabolism.
Further studies revealed that GSK4112 was not preferentially selective for either REV-ERB variant (19), which is a common feature of the synthetic REV-ERB modulators described so far. Although the pharmacokinetic properties of GSK4112 limit its pharmacological uses, the commercial availability of this compound makes it an accessible and useful probe for cellular REV-ERB activity. In attempt to optimize the drug-like properties of GSK4112, Solt and co-workers identified two analogs, SR9009 and SR9011, with sufficient pharmacokinetic properties to examine their activity in vivo(19). Administration of these REV-ERB agonists alters circadian behavior and the circadian pattern of core clock gene expression in mice. In addition, both SR9009 and SR9011 decreased obesity and improved dyslipidemia and hyperglycemia in a diet-induced obese mouse model (19). However, the specificity of SR9009 and SR9011 for REV-ERB receptors has recently been questioned. The tertiary amine chemotype common to both GSK4112 and a number of its analogs (Table 1) has known activity on LXRα, a nuclear receptor that regulates antiinflammatory and metabolic pathways that overlap with those modulated by REV-ERB receptors, such as metabolism and inflammation(20)(21). Remarkably, Trump and co-workers showed that while the parent compound, GSK4112, was 10 times more selective for REVERBα than LXRα, SR9009 and SR9011 displayed a completely opposite behavior, showing a
marked selectivity for LXRα (22) (Table 1). In attempt to optimize the selectivity of the tertiary amine series of REV-ERBα agonists, Trump et al.developed a novel GSK4112 analog, GSK2945, that was 10 times more potent against REV-ERB than the parental compound and displayed a 1000-fold selectivity for REV-ERB versus LXRα (Table 1).
In addition, GSK2945 showed a pharmacokinetic profile suitable for chronic in vivo dosing via both oral and intra-venous routes (22). Analysis of compounds within the GSK4112 tertiary amine scaffold also led to the identification of SR8278, a compound with REV-ERB antagonist activity(23). SR8278 relieved
REV-ERB-mediated transcriptional repression in luciferase-based cellular assays in a dosedependent manner and HEP-G2 cells treated with SR8278 increased the expression of endogenous REV-ERB target genes (23). Similar to the REV-ERB agonist, GSK4112, the pharmacokinetic properties of SR8278 limit its pharmacological uses, but the commercial availability of this compound makes it an accessible probe for investigating cellular REV-ERB activity. Recently, a novel class of REV-ERB antagonists has been identified (24). The members of this class of secondary diphenyl cycloalkylamines also inhibit discrete stages of lysosomemediated autophagy and have in vitro anticancer activity against several human tumor cell lines of diverse tissue origin (see below).
2. REV-ERB nuclear receptors and cancer 2.1Expression of REV-ERBs in cancer REV-ERBα derives its name fromitschromosomal position, as it is located on chromosome 17 on the strand opposite that of the alpha-thyroid hormone receptor (TR) gene (THRA1 also known as ERBA)(25). This region of chromosome 17 is a frequent target of rearrangements in breast cancer. In particular, large scale amplification at the 17q12-q21 chromosomal region leads to increased copy numbers of multiple genes; this includes ERBB2, a bona fide oncogene that is over-expressed in about 30% of breast tumors (26). Notably, REV-ERBα is co-amplified with ERBB2 in both ERBB2-positive primary breast tumors and breast cancer cell lines (27).Remarkably, however, REV-ERBα andother genes in this region showed reduced expression when gained (28).
In line with this observation, REV-ERBα transcript levels are significantly lower in a number of breast cancer cell lines compared with non-cancer human mammary epithelial HMEC cells (29). This apparentREV-ERBα down-regulationin breast cancer cells is independent of cellular ERBB2 status. Unexpectedly, De Mei and colleagues reported that REV-ERBβ (which is transcribed from a different locus on chromosome 3) was the predominantly expressed REV-ERB variant in a number of breast cancer cell lines, as well as in tumor cell lines derived from the skin, liver and prostate (29). Consistent with its expression pattern in cancer cell lines, while REV-ERBα was more abundant than REV-ERBβ in various normal human tissues, the latter was more highly expressed in several primary tumor samples of different origin (29). Knockdown experiments revealed that the switch to REV-ERBβ overexpression in cancer cell lines corresponded with a predominant functional role of this variant in regulation of REVERB-dependent gene expression. Thus, REV-ERBβ silencing significantly enhanced the expression of circadian and metabolic genes, including the master clock regulator BMAL1, whereas REV-ERBα silencing had no such effect (29). ‘Isoform switching’ is a mechanism employed by many human tumor types in order to rewire cellular metabolic pathways and adapt them to the demands of transformation. A case in point is the switching of pyruvate kinase from the PKM1 to the PKM2 in order to support the high glycolytic rate of tumor cells (30). Although the precise biological role of REV-ERBβ overexpression in cancer remains to be determined, it is tempting to speculate that the switch to this nuclear receptor isoform is required to regulate circadian or metabolic gene expression and thereby facilitate tumorigenesis.
2.2The controversial role of REV-ERBα in breast cancer A functional RNA interference (RNAi) screen targeting 141 of the 154 genes co-amplified with ERBB2 in breast cancer BT-474 cells identified 9 genes that significantly decreased proliferation when silenced (31). REV-ERBα was included in this group of genes, and its silencing appeared to inhibit the proliferation of additional ERBB2-positive breast cancer lines, while it had negligible effects on both ERBB2-negative cells and non-cancer human mammary epithelial cells (HMEC)(31). However, the activity of REV-ERBα in supporting breast cancer cell viability has recently been questioned, as two groups independently reported that REV-ERBα silencing in ERBB2positive BT-474 breast cancer cells did not affect apoptosis or proliferation (29, 32). In addition, treatment of this cell line with a REV-ERB antagonist had negligible effects on cell viability (29). Nevertheless, Sahlberg and co-workers reported that REV-ERBα silencing led to apoptosis in JIMT-1, a breast cancer cell line that is insensitive to HER-2-inhibitors including trastuzumab (Herceptin) and pertuzumab (2C4) (33). Notably, REV-ERBα silencing in these cells also inhibited activation of AKT. Remarkably,the trastuzumab-insensitivity of JIMT-1 cells is attributed to the failure of the drug to reduce AKT phosphorylation(33). In summary, these data suggest that the ability of REV-ERBα to modulate AKT activity is likely to be cell type- and context-dependent.
2.3REV-ERBβ and autophagy inhibition as a suitable anticancer strategy Autophagy is a self-degradation process by which cells consume parts of themselves to survive starvation and stress (34, 35). Autophagy likely fulfills the metabolic needs of cancer cells during oncogene activation or nutrient limitation by providing a mechanism to recycle
intra-cellular carbon and nitrogen (36). Autophagy inhibition is emerging as a promising anticancer strategy. Indeed, lysosomotropic autophagy inhibitors, such as chloroquine (CQ), displayed anticancer properties in both cultured cells and xenograft models(37).These lysosomotropic compoundsinterferewith the autophagy flux by inhibiting lysosomal function, thusavoiding the final maturation stage of autophagosomes (38). As mentioned above, REV-ERBβ is the main REV-ERB variant expressed in cancer cells, and is the principal transcriptional regulator of circadian REV-ERB targets in transformed cells. REV-ERBβ does not seem to contribute to cancer cell viability per se, since its genetic and pharmacological inhibition does not have a significant impact on cell death or proliferation(29). However, this nuclear receptor plays an unexpected role in supporting cancer cell viability when autophagy is compromised. Accordingly, genetic inhibition of REV-ERBβ sensitizes to cytotoxicity induced by CQ(24, 29). The fact that a crucial regulator of circadian and metabolism affects the sensitivity to autophagy inhibition in cancer cells has several therapeutic implications. The efficacy and the collateral toxicity of many drugs (including antitumor agents) are greatly affected by circadian timing and recent studies have revealed that adopting an anticancer chronotherapeutic strategy leads to better therapeutic outcomes (39). Because REV-ERBβ expression follows a circadian oscillatory pattern, chronopharmacokinetic studies of autophagy inhibitors may improve their antitumor efficacy. In addition, a combinatory inhibition of both REV-ERBβ and autophagy may offer a novel pharmacological approach to induce cytotoxicity in cancer cells. Following this idea, De mei and co-workers identified a novel REV-ERBβ antagonist, ARN5187, which also possessed lysosomotropic properties (29). More precisely, ARN5187 is a novel autophagy inhibitor that disrupts lysosomal function and blocks the latter stages of autophagy. In addition, ARN5187
inhibits REV-ERBβ activity and relieves REV-ERB-mediated transcriptional repression. Although ARN5187 and CQ have similar inhibitory activity toward autophagy, ARN5187 showed a significant higher in vitro anticancer activity than CQ against breast cancer BT-474 cells, while it had negligible effects on HMEC viability (29). Further structure-activity studies, based around ARN5187, disclosed the first class of dual inhibitors of REV-ERB and autophagy and identified analogs with improved in vitro anticancer activity(24). Remarkably, the improved cytotoxicity of these analogs was related to their enhanced REV-ERBβ-inhibitory activity. By contrast, their lysosomotropic activities were not enhanced, and remained similar to that of CQ. Nevertheless, the most potent compound,30, induced death in a panel of tumor cell lines at doses 5–50 times lower than an equitoxic amount of CQ, but did not affect the viability of HMECs (Figure 3). Many cancer cells require high micromolar concentrations of CQ to block autophagy in vitro, yet such levels are rarely achieved inpatients (37). Although improved lysosomotropic agents have been synthetized (40), the homeostatic roles of lysosome and autophagosome in normal tissue impose carefully consideration of potential side effects in healthy organs (41).
Figure 3. Dual inhibitors of the circadian nuclear receptor REV-ERBβ and autophagy with improved in vitro anticancer activity compared to the medically relevant single autophagy inhibitor, chloroquine. In particular, the most potent inhibitor of the class, compound 30, decreases the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than chloroquine.
Consequently, the fact that dual REV-ERBβ/autophagy inhibitors have greater cytotoxicity than CQ, but similar autophagy inhibitory activity,supports their future development as novel anticancer agents with a potential improved therapeutic index. Although the molecular basis of REV-ERBβ cytoprotective function requires further investigation, the central position of this nuclear receptor in the regulation of metabolism suggests that inhibition of both REV-ERBβ and autophagy will cooperate to induce a massive metabolic dysfunction that is incompatible with cell viability (Figure 4).
Figure 4. Inhibition of both REV-ERB and autophagy cooperate to induce a metabolic dysfunction that is incompatible with cancer cell viability. REV-ERB antagonism will affect the metabolism at different levels, regulating the expression of key circadian and metabolic genes. The resulting compromise in metabolic pathways renders cancer cells susceptible to a lethal “second shoot” deriving from the blockade of autophagy.
In support of a pro-metabolic role of REV-ERB, studies in rodents have shown that multiple genes involved in glycolysis/gluconeogenesis, the tricarboxylic acid (TCA) cycle, and lipid metabolism are REV-ERB targets (42)(15). Thus, further studies on REV-ERB role in cancer metabolism may reveal novel interconnections between the circadian clock, autophagy, and cellular homeostasis that are required for cancer cell survival. Furthermore, we suggest that REV-ERBβ inhibition may be particularly suitable for combination therapies together with drugs that target cancer cell metabolism; the latter class of drugs remains the focus of intense research efforts(43).
2.4REV-ERB agonists and breast cancer Recently, cyclin A2 was identified as a novel REV-ERB regulated gene. Accordingly, overexpression of REV-ERBα variant in breast cancer SK-BR-3 cells repressedCCNA2 gene expression, while REV-ERBβ silencing generated an opposite effect (44). Cyclin A2 binds to and activates its catalytic partners, Cdk2 and Cdk1. Cdk/cyclin A2 complexes phosphorylate pocket proteins (Rb, p107, p130) and other proteins involved in DNA synthesis, thereby driving S-phase progression. Aberrant expression of cyclin A2 has been detected in a variety of cancers, and deregulation of cyclin A2 appears to be closely related to chromosomal instability and tumor proliferation (45). The treatment of several breast cancer cell lines with the REV-ERB agonist SR9011 resulted in a significant decrease in Cyclin A2 levels and caused a dose-dependent reduction in cell viability, independently from the ER, PR or HER2 status (44). As mentioned above, the specificity of SR9011 for REV-ERB proteins has been recently questioned, and this compound appears to act preferentially as an LXRa agonist (22).
Considering the well-characterized anti-proliferative activity of LXRa agonists in breast cancer cell lines(46), further studies adopting the more selective REV-ERB agonist, GSK2945, will help to unravel the potential use of REV-ERB activators as novel anticancer agents.
3. Stop the clock: circadian rhythm-based synthetic lethal approaches in cancer therapy The concept of synthetic lethality in multicellular organisms originates from seminal genetic studies in Drosophila (47).Around 50 years later, Drosophila genetics were used to isolate the first clock mutant (48).Several genetic screens focused on identifying regulators of the circadian machinery have since been performed in the fruit fly (49)(50) and in mammalian cells (51, 52). However, in virtually all cases the screens were designed to identify novel regulators of the canonical circadian signaling pathways, rather than to pinpoint synthetic lethal relationships. Given the emerging experimental data discussed above, future screens of circadian regulators with either lethality, or cancer-associated signaling cascades as the readout, may suggest novel mechanisms that govern the crosstalk between biological clock proteins and cancer. Phosphorylation-dependent degradation of PER, one of the major negative regulators of CLOCK/BMAL-driven circadian gene expression, is accelerated by DOUBLETIME (DBT) in Drosophila(53). Intriguingly, the mammalian DBT homologs are casein kinases epsilon and delta (CSNK1E and CSNK1D, respectively). CSNK1E is already validated as a target in oncology, since its functional knockout is synthetic lethal with overexpression of the CMYC and RAS oncogenes(54)(55).Since loss of CSNK1E is expected to block CLOCK/BMAL activity, these observations support the hypothesis that inhibition of circadian signaling could
be of potential therapeutic utility. Indeed, a recent study suggests that small molecule inhibition of CSNK1D/E (56) induces phase shifts in circadian transcriptional output. This may be leveraged in combination with existing therapeutics in order to optimize the timing of treatment and thus increase the therapeutic index (see discussion of 5-FU dosing above). Genetic screens designed to identify additional regulators of PER stability may provide yet more targets that are synthetic lethal in the context of oncogene overexpression. In this regard, we note that another casein kinase family member, CSNK2A1, was reported to regulate PER2 levels (52). There is some evidence that inhibition of CSNK2A1 increases survival in a murine model of glioblastoma (57). In contrast to CSNK1E, however, CSNK2A1 appears to stabilize PER2 and promote its nuclear accumulation. The finding that both CSNK1E and CSNK2A1 inhibition have antitumor effects, yet have opposite effects on PER stability, highlights the complexity of this biological system. Further analysis of the transcriptional programs that are disrupted in cancer cells that are differentially sensitive to CSNK inhibitors under these conditions may provide additional pathway-based insight. Factors that govern stability of REV-ERB proteins have also been identified, and may represent targets that complement the current small molecule modulators of these receptors. Intriguingly, the context-dependent tumor suppressor DBC1, which stabilizes and activates p53 (58), is also reported to stabilize REV-ERBα(59).Additionally, GSK3B-dependent phosphorylation of REV-ERBα stabilizes the protein (60), although theunderlying molecular mechanism remains unclear. However, it may be due to inhibition of either ArfBP1 (also known as MULE/HUWE1) orPAM (also known as MYCBP2), two E3 ubiquitin ligases that are reported to mediate REV-ERBα degradation(61). The links between these two ligases and REV-ERBα further emphasizes the importance of crosstalk between circadian regulators and
other signaling pathways.Moreover, although most of the work has been focused on REVERBα, identical signaling may be eventually adapted to REV-ERBβ. Analogous to the effect of CSNK1E inhibition (see above), the loss of ArfBP1 would be expected to stabilize REV-ERBα and thereby perturb transcription of CLOCK/BMAL targets. This is indeed the case. If such perturbations engender a transcriptional state that ‘primes’ cells for synthetic lethality, one might expect that loss of ArfBP1 in oncogene-overexpressing cells might hasten their demise. In support of this, ArfBP1 loss was synthetic lethal with RAS overexpression in colon carcinoma cells (62). What evidence is there to suggest that current drugs targeting metabolic pathways might display synthetic lethal activity with circadian regulators? Several drugs that are currently in clinical trials target proteins that are subject to circadian regulation. For example, AZD3965 inhibits MCT1, a lactate acid transporter that is required for tumor cell survival (63). Since MCT1 expression follows a circadian pattern (64), it is possible that co-treatment with circadian agonists/antagonists could increase the therapeutic index of AZD3965. The pyruvate kinase isoform PKM2 is another cancer-relevant target: it is upregulated in tumor cells where both dimeric and tetrameric formscontribute to tumor cell survival and tumor maintenance. This is due to PKM2-dependent rewiring of metabolites into anabolic processes in cancer. Thus, PKM2 inhibitors have been evaluated by several companies (65). However, it has been proposed that resistance to PKM2 inhibitors can develop due to activation of glutaminolysis, another rich source of anabolic cancer cell metabolites(66). Therefore, concomitant treatment with agents that disrupt glutaminolysis may abrogate such resistance. Targeting glutaminolysis has been investigated extensively, and the reader is referred to several reviews on the subject(67)(68).
Recent data indicate that the process is under circadian control. For example,glutaminase expression and activity follows a circadian pattern in hepatocarcinomas in vivo(69). This opens up the possibility that targeted inhibition of circadian regulators could thwart cancer cell growth by attenuating their glutamine utilization.
4. Conclusion In summary, recent studies are revealing that a pharmacological targeting of circadian-related proteins may be a suitable anticancer strategy. Although evidences that the single action on the molecular clock would be an effective approach are still controversial, the “engage” of the clock machinery for increasing the activity of existing anticancer drugs may be an effective approach. In addition to the discussed example related to REV-ERB and autophagy, it is worth mentioning that genetic inhibition of the circadian cryptochromes sensitizes p53-null cells to the anticancer drug oxaliplatin (70), and cryptochrome inhibitors have been recently identified (71). Furthermore, considering the intimate relationship between circadian signaling and metabolism (5), we suggest that circadian modulators may be particularly suitable for combination therapies together with drugs that target cancer cell metabolism; the latter class of drugs remains the focus of intense research efforts (43). In this scenario, synthetic lethality screens represent a valuable tool for the identification of novel avenues of pharmacological and therapeutic intervention.
Further studies will surely aimed to evaluate and optimize the drug-like properties of clock regulating compounds, such as their pharmacodynamics, specify and safety. Thus, a long path needs to be paved before “chrono-durgs” enter in clinical trials. Nonetheless, this seems a hopeful journey to find novel ways for fighting the timeless war against a disease that is the second leading cause of mortality in industrialized countries.
Conflict of interest None.
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Figure 1. The circadian clock machinerycan be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the transcription of clock controlled genes (CCGs). In mammals, the proteins BMAL1/BMAL2 (shown as BMAL) and CLOCK/NAPS2 (shown as CLOCK) are transcriptional activators acting to enhance the expression of the CCGs. These factors also induce the expression of
the transcriptional repressor proteins, PER1/PER2/PER3 (shown as PER), CRY1/CRY2 (shown as CRY) and REV-ERBα/REV-ERBβ (shown as REV-ERB). The overall balance of the fight between transcriptional activators and repressors results in rhythmic expression of a number of genes involved in a variety of physiological responses.
Figure 2. REV-ERBα and REV-ERBβ nuclear receptors (shown as REV-ERB) are hemebinding proteins that repress transcription of target genes containing a REV-ERB responsive element (RevRE) within their promoters. Upon heme binding, the nuclear co-repressor NcoR and the histone de-acetyltransferase HDAC3results are recruited on the promoter of REVERB-regulated genes to shut-down transcription. REV-ERB targets include the master clock regulator, BMAL1, and the gluconeogenetic phosphoenolpyruvate carboxykinase enzyme, PEPCK. Because of the intimate link between the circadian clock and cellular metabolism, REV-ERB-mediated regulation of the molecular clock machinery further places REV-ERB proteins at a strategic position in metabolism.
Figure 3. Dual inhibitors of the circadian nuclear receptor REV-ERBβ and autophagy with improved in vitro anticancer activity compared to the medically relevant single autophagy inhibitor, chloroquine. In particular, the most potent inhibitor of the class, compound 30, decreases the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than chloroquine.
Figure 4. Inhibition of both REV-ERB and autophagy cooperate to induce a metabolic dysfunction that is incompatible with cancer cell viability. REV-ERB antagonism will affect the metabolism at different levels, regulating the expression of key circadian and metabolic genes. The resulting compromise in metabolic pathways renders cancer cells susceptible to a lethal “second shoot” deriving from the blockade of autophagy.
Table 1. Structure, specificity and in vivo suitability of tertiary amine chemotype-based REVERB agonists.
S1043-6618(15)00176-0 http://dx.doi.org/doi:10.1016/j.phrs.2015.08.008 YPHRS 2905
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
12-8-2015 12-8-2015 12-8-2015
Please cite this article as: Ercolani Luisa, Ferrari Alessio, Mei Claudia De, Parodi Chiara, Wade Mark, Grimaldi Benedetto.Circadian clock: time for novel anticancer strategies?.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Circadian clock: time for novel anticancer strategies? Luisa Ercolani*, Alessio Ferrari*, Claudia De Mei*, Chiara Parodi*, Mark Wade#@, Benedetto Grimaldi*@
* Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy #
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via
Adamello 16, 20139, Milano, Italy @
Corresponding authors: email: [email protected] email: [email protected]
Graphical abstract fx1
Abstract
Disruption of the circadian clock is associated with a variety of human pathologies, including cancer. Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression observed in many tumors may serve for cancer cell survival. This scenario suggests the provocative hypothesis that
pharmacological modulation of clock-related proteins may be suitable as an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REVERBα and REV-ERBβ, in cancer cell survival and describe the potential development of small molecule compounds that modulate REV-ERB activity as novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies. Keywords: anticancer agents, ARN5187, autophagy, cancer, circadian, combined anticancer strategy, dual inhibitor, metabolism, lysosomotropic agents, nuclear receptors, REV-ERB, REV-ERB antagonist, REV-ERB agonist, SR9011.
1. Introduction 1.1 The circadian clock Many physiological processes obey a biological clock that has an intrinsic period of approximately 24 hours (circadian)(1, 2). The molecular mechanism underlying these circadian rhythms is based on interconnected transcriptional–translational feedback loops in which specific factors repress the transcription of their own genes(3). The anatomical center of the mammalian circadian clock, composed of approximately 15,000 neurons, resides in a region of the anterior hypothalamus of the brain called the suprachiasmatic nucleus (SCN) (1). Nevertheless, a finding that profoundly modified our view of circadian clocks is that most peripheral tissues contain intrinsically independent pacemakers (1). Although the clock of the SCN plays a fundamental role in synchronizing whole-body circadian rhythm, tissue-specific cell-autonomous molecular clocks choreograph the rhythmic processes in peripheral tissues(1, 4).
At the molecular level, the endogenous clock can be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the transcription of clock controlled genes (CCGs) (Figure 1). The proteins CLOCK and BMAL1 interact to form a clock activator complex promoting transcription of CCGs. These genes also code for proteins belonging to the clock repressor factors (PERs, CRYs and REV-ERBs proteins). The function of these repressor proteins is to decrease CLOCK/BMAL1-dependent transcription. Finally, rhythmic cell-specific or tissue-specific gene expression is achieved through the cooperative action of the core clock genes with local transcription factors and chromatin remodeling enzymes (5). The overall balance between clock-activator and clock-repressor complexes results in the circadian expression of around 10-15% of the mammalian genome (6, 7).
Figure 1. The circadian clock machinerycan be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the
transcription of clock controlled genes (CCGs). In mammals, the proteins BMAL1/BMAL2 (shown as BMAL) and CLOCK/NAPS2 (shown as CLOCK) are transcriptional activators acting to enhance the expression of the CCGs. These factors also induce the expression of the transcriptional repressor proteins, PER1/PER2/PER3 (shown as PER), CRY1/CRY2 (shown as CRY) and REV-ERBα/REV-ERBβ (shown as REV-ERB). The overall balance of the fight between transcriptional activators and repressors results in rhythmic expression of a number of genes involved in a variety of physiological responses.
Clock-regulated transcripts also include a number of pharmacological targets, as well as key enzymes involved in drug metabolism (8). Circadian expression of drug-related genes and differential circadian profiles between normal and pathological cellular states are well documented.These phenomena may have profound consequences for the efficacy and safety of new and existing therapeutic agents, as they are likely to determine the timing, dosage, and duration of optimal drug exposures(8). As an example, the anabolic enzymes that generate the cytotoxic forms of 5-Fluorouracil (5FU) display a circadian expression pattern in both rodents and human (9). Moreover, the major on-target side effects of the DNA synthesis inhibitor 5-FU in highly proliferative tissues (such as bone marrow, skin, and the oral and rectal mucosa) can be reduced by treating patients at night rather than during the daytime (9). This is since proliferation is under circadian control in these tissues, and is more rapid during daylight hours. The improved therapeutic index due to administration of 5-FU during the night, is a clear example of the benefits of chronotherapy. Disruption of the circadian clock is associated with a variety of human pathologies, including cancer, and the expression of several clock genes is perturbed in many tumors (10)(11).
Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression in tumors likely plays a causal role in the development of cancer and the survival of tumor cells. For instance, several epidemiological studies indicated that the incidence of breast cancer is higher among women who spent more years and hours per week working nightshifts(12). These observations suggest the provocative hypothesis that pharmacological modulation of clock-related proteins may be an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REVERBα and REV-ERBβ, in cancer cell survival and describe thepotential development of small molecule compounds that modulate REV-ERB activityas novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies.
1.2 The circadian nuclear receptors, REV-ERBs The circadian regulator REV-ERBα (also known as NR1D1) and its variant isoform REVERBβ (also known as NR1D2) belong to the nuclear receptor superfamily, which is composed of a large family of ligand-sensitive transcription factors(13). Unlike most other nuclear receptors, REV-ERB proteins lack a transcriptional activation domain and instead repress target genes bearing Rev-erb Responsive Elements (RevREs) within the promoter. REV-ERB transcriptional repression is modulated by the interaction of the receptor Ligand Binding Domain (LBD) with a heme moiety (14). REV-ERBα and REV-ERBβ regulateinter-connected processes, such as circadian rhythm and metabolism (Figure 2) (15).
Studies in rodents revealed that the two REV-ERB variants compensate for one another in the repression of many common target genes. These studies also indicatedthat, in several conditions, abrogation of both REV-ERBα and REV-ERBβ function is required to profoundly affect REV-ERB-mediated physiological responses (15, 16). A pharmacological screen identified the SR6452 (GSK4112) compound as the first REVERBα synthetic agonist (17). Accordingly, GSK4112 increased REV-ERBα-mediated repression in cell-based assays and modulated REV-ERBα regulated adipogenesis of 3T3-L1 cells (18).
Figure 2. REV-ERBα and REV-ERBβ nuclear receptors (shown as REV-ERB) are hemebinding proteins that repress transcription of target genes containing a REV-ERB responsive element (RevRE) within their promoters. Upon heme binding, the nuclear co-repressor NcoR and the histone de-acetyltransferase HDAC3results are recruited on the promoter of REVERB-regulated genes to shut-down transcription. REV-ERB targets include the master clock
regulator, BMAL1, and the gluconeogenetic phosphoenolpyruvate carboxykinase enzyme, PEPCK. Because of the intimate link between the circadian clock and cellular metabolism, REV-ERB-mediated regulation of the molecular clock machinery further places REV-ERB proteins at a strategic position in metabolism.
Further studies revealed that GSK4112 was not preferentially selective for either REV-ERB variant (19), which is a common feature of the synthetic REV-ERB modulators described so far. Although the pharmacokinetic properties of GSK4112 limit its pharmacological uses, the commercial availability of this compound makes it an accessible and useful probe for cellular REV-ERB activity. In attempt to optimize the drug-like properties of GSK4112, Solt and co-workers identified two analogs, SR9009 and SR9011, with sufficient pharmacokinetic properties to examine their activity in vivo(19). Administration of these REV-ERB agonists alters circadian behavior and the circadian pattern of core clock gene expression in mice. In addition, both SR9009 and SR9011 decreased obesity and improved dyslipidemia and hyperglycemia in a diet-induced obese mouse model (19). However, the specificity of SR9009 and SR9011 for REV-ERB receptors has recently been questioned. The tertiary amine chemotype common to both GSK4112 and a number of its analogs (Table 1) has known activity on LXRα, a nuclear receptor that regulates antiinflammatory and metabolic pathways that overlap with those modulated by REV-ERB receptors, such as metabolism and inflammation(20)(21). Remarkably, Trump and co-workers showed that while the parent compound, GSK4112, was 10 times more selective for REVERBα than LXRα, SR9009 and SR9011 displayed a completely opposite behavior, showing a
marked selectivity for LXRα (22) (Table 1). In attempt to optimize the selectivity of the tertiary amine series of REV-ERBα agonists, Trump et al.developed a novel GSK4112 analog, GSK2945, that was 10 times more potent against REV-ERB than the parental compound and displayed a 1000-fold selectivity for REV-ERB versus LXRα (Table 1).
In addition, GSK2945 showed a pharmacokinetic profile suitable for chronic in vivo dosing via both oral and intra-venous routes (22). Analysis of compounds within the GSK4112 tertiary amine scaffold also led to the identification of SR8278, a compound with REV-ERB antagonist activity(23). SR8278 relieved
REV-ERB-mediated transcriptional repression in luciferase-based cellular assays in a dosedependent manner and HEP-G2 cells treated with SR8278 increased the expression of endogenous REV-ERB target genes (23). Similar to the REV-ERB agonist, GSK4112, the pharmacokinetic properties of SR8278 limit its pharmacological uses, but the commercial availability of this compound makes it an accessible probe for investigating cellular REV-ERB activity. Recently, a novel class of REV-ERB antagonists has been identified (24). The members of this class of secondary diphenyl cycloalkylamines also inhibit discrete stages of lysosomemediated autophagy and have in vitro anticancer activity against several human tumor cell lines of diverse tissue origin (see below).
2. REV-ERB nuclear receptors and cancer 2.1Expression of REV-ERBs in cancer REV-ERBα derives its name fromitschromosomal position, as it is located on chromosome 17 on the strand opposite that of the alpha-thyroid hormone receptor (TR) gene (THRA1 also known as ERBA)(25). This region of chromosome 17 is a frequent target of rearrangements in breast cancer. In particular, large scale amplification at the 17q12-q21 chromosomal region leads to increased copy numbers of multiple genes; this includes ERBB2, a bona fide oncogene that is over-expressed in about 30% of breast tumors (26). Notably, REV-ERBα is co-amplified with ERBB2 in both ERBB2-positive primary breast tumors and breast cancer cell lines (27).Remarkably, however, REV-ERBα andother genes in this region showed reduced expression when gained (28).
In line with this observation, REV-ERBα transcript levels are significantly lower in a number of breast cancer cell lines compared with non-cancer human mammary epithelial HMEC cells (29). This apparentREV-ERBα down-regulationin breast cancer cells is independent of cellular ERBB2 status. Unexpectedly, De Mei and colleagues reported that REV-ERBβ (which is transcribed from a different locus on chromosome 3) was the predominantly expressed REV-ERB variant in a number of breast cancer cell lines, as well as in tumor cell lines derived from the skin, liver and prostate (29). Consistent with its expression pattern in cancer cell lines, while REV-ERBα was more abundant than REV-ERBβ in various normal human tissues, the latter was more highly expressed in several primary tumor samples of different origin (29). Knockdown experiments revealed that the switch to REV-ERBβ overexpression in cancer cell lines corresponded with a predominant functional role of this variant in regulation of REVERB-dependent gene expression. Thus, REV-ERBβ silencing significantly enhanced the expression of circadian and metabolic genes, including the master clock regulator BMAL1, whereas REV-ERBα silencing had no such effect (29). ‘Isoform switching’ is a mechanism employed by many human tumor types in order to rewire cellular metabolic pathways and adapt them to the demands of transformation. A case in point is the switching of pyruvate kinase from the PKM1 to the PKM2 in order to support the high glycolytic rate of tumor cells (30). Although the precise biological role of REV-ERBβ overexpression in cancer remains to be determined, it is tempting to speculate that the switch to this nuclear receptor isoform is required to regulate circadian or metabolic gene expression and thereby facilitate tumorigenesis.
2.2The controversial role of REV-ERBα in breast cancer A functional RNA interference (RNAi) screen targeting 141 of the 154 genes co-amplified with ERBB2 in breast cancer BT-474 cells identified 9 genes that significantly decreased proliferation when silenced (31). REV-ERBα was included in this group of genes, and its silencing appeared to inhibit the proliferation of additional ERBB2-positive breast cancer lines, while it had negligible effects on both ERBB2-negative cells and non-cancer human mammary epithelial cells (HMEC)(31). However, the activity of REV-ERBα in supporting breast cancer cell viability has recently been questioned, as two groups independently reported that REV-ERBα silencing in ERBB2positive BT-474 breast cancer cells did not affect apoptosis or proliferation (29, 32). In addition, treatment of this cell line with a REV-ERB antagonist had negligible effects on cell viability (29). Nevertheless, Sahlberg and co-workers reported that REV-ERBα silencing led to apoptosis in JIMT-1, a breast cancer cell line that is insensitive to HER-2-inhibitors including trastuzumab (Herceptin) and pertuzumab (2C4) (33). Notably, REV-ERBα silencing in these cells also inhibited activation of AKT. Remarkably,the trastuzumab-insensitivity of JIMT-1 cells is attributed to the failure of the drug to reduce AKT phosphorylation(33). In summary, these data suggest that the ability of REV-ERBα to modulate AKT activity is likely to be cell type- and context-dependent.
2.3REV-ERBβ and autophagy inhibition as a suitable anticancer strategy Autophagy is a self-degradation process by which cells consume parts of themselves to survive starvation and stress (34, 35). Autophagy likely fulfills the metabolic needs of cancer cells during oncogene activation or nutrient limitation by providing a mechanism to recycle
intra-cellular carbon and nitrogen (36). Autophagy inhibition is emerging as a promising anticancer strategy. Indeed, lysosomotropic autophagy inhibitors, such as chloroquine (CQ), displayed anticancer properties in both cultured cells and xenograft models(37).These lysosomotropic compoundsinterferewith the autophagy flux by inhibiting lysosomal function, thusavoiding the final maturation stage of autophagosomes (38). As mentioned above, REV-ERBβ is the main REV-ERB variant expressed in cancer cells, and is the principal transcriptional regulator of circadian REV-ERB targets in transformed cells. REV-ERBβ does not seem to contribute to cancer cell viability per se, since its genetic and pharmacological inhibition does not have a significant impact on cell death or proliferation(29). However, this nuclear receptor plays an unexpected role in supporting cancer cell viability when autophagy is compromised. Accordingly, genetic inhibition of REV-ERBβ sensitizes to cytotoxicity induced by CQ(24, 29). The fact that a crucial regulator of circadian and metabolism affects the sensitivity to autophagy inhibition in cancer cells has several therapeutic implications. The efficacy and the collateral toxicity of many drugs (including antitumor agents) are greatly affected by circadian timing and recent studies have revealed that adopting an anticancer chronotherapeutic strategy leads to better therapeutic outcomes (39). Because REV-ERBβ expression follows a circadian oscillatory pattern, chronopharmacokinetic studies of autophagy inhibitors may improve their antitumor efficacy. In addition, a combinatory inhibition of both REV-ERBβ and autophagy may offer a novel pharmacological approach to induce cytotoxicity in cancer cells. Following this idea, De mei and co-workers identified a novel REV-ERBβ antagonist, ARN5187, which also possessed lysosomotropic properties (29). More precisely, ARN5187 is a novel autophagy inhibitor that disrupts lysosomal function and blocks the latter stages of autophagy. In addition, ARN5187
inhibits REV-ERBβ activity and relieves REV-ERB-mediated transcriptional repression. Although ARN5187 and CQ have similar inhibitory activity toward autophagy, ARN5187 showed a significant higher in vitro anticancer activity than CQ against breast cancer BT-474 cells, while it had negligible effects on HMEC viability (29). Further structure-activity studies, based around ARN5187, disclosed the first class of dual inhibitors of REV-ERB and autophagy and identified analogs with improved in vitro anticancer activity(24). Remarkably, the improved cytotoxicity of these analogs was related to their enhanced REV-ERBβ-inhibitory activity. By contrast, their lysosomotropic activities were not enhanced, and remained similar to that of CQ. Nevertheless, the most potent compound,30, induced death in a panel of tumor cell lines at doses 5–50 times lower than an equitoxic amount of CQ, but did not affect the viability of HMECs (Figure 3). Many cancer cells require high micromolar concentrations of CQ to block autophagy in vitro, yet such levels are rarely achieved inpatients (37). Although improved lysosomotropic agents have been synthetized (40), the homeostatic roles of lysosome and autophagosome in normal tissue impose carefully consideration of potential side effects in healthy organs (41).
Figure 3. Dual inhibitors of the circadian nuclear receptor REV-ERBβ and autophagy with improved in vitro anticancer activity compared to the medically relevant single autophagy inhibitor, chloroquine. In particular, the most potent inhibitor of the class, compound 30, decreases the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than chloroquine.
Consequently, the fact that dual REV-ERBβ/autophagy inhibitors have greater cytotoxicity than CQ, but similar autophagy inhibitory activity,supports their future development as novel anticancer agents with a potential improved therapeutic index. Although the molecular basis of REV-ERBβ cytoprotective function requires further investigation, the central position of this nuclear receptor in the regulation of metabolism suggests that inhibition of both REV-ERBβ and autophagy will cooperate to induce a massive metabolic dysfunction that is incompatible with cell viability (Figure 4).
Figure 4. Inhibition of both REV-ERB and autophagy cooperate to induce a metabolic dysfunction that is incompatible with cancer cell viability. REV-ERB antagonism will affect the metabolism at different levels, regulating the expression of key circadian and metabolic genes. The resulting compromise in metabolic pathways renders cancer cells susceptible to a lethal “second shoot” deriving from the blockade of autophagy.
In support of a pro-metabolic role of REV-ERB, studies in rodents have shown that multiple genes involved in glycolysis/gluconeogenesis, the tricarboxylic acid (TCA) cycle, and lipid metabolism are REV-ERB targets (42)(15). Thus, further studies on REV-ERB role in cancer metabolism may reveal novel interconnections between the circadian clock, autophagy, and cellular homeostasis that are required for cancer cell survival. Furthermore, we suggest that REV-ERBβ inhibition may be particularly suitable for combination therapies together with drugs that target cancer cell metabolism; the latter class of drugs remains the focus of intense research efforts(43).
2.4REV-ERB agonists and breast cancer Recently, cyclin A2 was identified as a novel REV-ERB regulated gene. Accordingly, overexpression of REV-ERBα variant in breast cancer SK-BR-3 cells repressedCCNA2 gene expression, while REV-ERBβ silencing generated an opposite effect (44). Cyclin A2 binds to and activates its catalytic partners, Cdk2 and Cdk1. Cdk/cyclin A2 complexes phosphorylate pocket proteins (Rb, p107, p130) and other proteins involved in DNA synthesis, thereby driving S-phase progression. Aberrant expression of cyclin A2 has been detected in a variety of cancers, and deregulation of cyclin A2 appears to be closely related to chromosomal instability and tumor proliferation (45). The treatment of several breast cancer cell lines with the REV-ERB agonist SR9011 resulted in a significant decrease in Cyclin A2 levels and caused a dose-dependent reduction in cell viability, independently from the ER, PR or HER2 status (44). As mentioned above, the specificity of SR9011 for REV-ERB proteins has been recently questioned, and this compound appears to act preferentially as an LXRa agonist (22).
Considering the well-characterized anti-proliferative activity of LXRa agonists in breast cancer cell lines(46), further studies adopting the more selective REV-ERB agonist, GSK2945, will help to unravel the potential use of REV-ERB activators as novel anticancer agents.
3. Stop the clock: circadian rhythm-based synthetic lethal approaches in cancer therapy The concept of synthetic lethality in multicellular organisms originates from seminal genetic studies in Drosophila (47).Around 50 years later, Drosophila genetics were used to isolate the first clock mutant (48).Several genetic screens focused on identifying regulators of the circadian machinery have since been performed in the fruit fly (49)(50) and in mammalian cells (51, 52). However, in virtually all cases the screens were designed to identify novel regulators of the canonical circadian signaling pathways, rather than to pinpoint synthetic lethal relationships. Given the emerging experimental data discussed above, future screens of circadian regulators with either lethality, or cancer-associated signaling cascades as the readout, may suggest novel mechanisms that govern the crosstalk between biological clock proteins and cancer. Phosphorylation-dependent degradation of PER, one of the major negative regulators of CLOCK/BMAL-driven circadian gene expression, is accelerated by DOUBLETIME (DBT) in Drosophila(53). Intriguingly, the mammalian DBT homologs are casein kinases epsilon and delta (CSNK1E and CSNK1D, respectively). CSNK1E is already validated as a target in oncology, since its functional knockout is synthetic lethal with overexpression of the CMYC and RAS oncogenes(54)(55).Since loss of CSNK1E is expected to block CLOCK/BMAL activity, these observations support the hypothesis that inhibition of circadian signaling could
be of potential therapeutic utility. Indeed, a recent study suggests that small molecule inhibition of CSNK1D/E (56) induces phase shifts in circadian transcriptional output. This may be leveraged in combination with existing therapeutics in order to optimize the timing of treatment and thus increase the therapeutic index (see discussion of 5-FU dosing above). Genetic screens designed to identify additional regulators of PER stability may provide yet more targets that are synthetic lethal in the context of oncogene overexpression. In this regard, we note that another casein kinase family member, CSNK2A1, was reported to regulate PER2 levels (52). There is some evidence that inhibition of CSNK2A1 increases survival in a murine model of glioblastoma (57). In contrast to CSNK1E, however, CSNK2A1 appears to stabilize PER2 and promote its nuclear accumulation. The finding that both CSNK1E and CSNK2A1 inhibition have antitumor effects, yet have opposite effects on PER stability, highlights the complexity of this biological system. Further analysis of the transcriptional programs that are disrupted in cancer cells that are differentially sensitive to CSNK inhibitors under these conditions may provide additional pathway-based insight. Factors that govern stability of REV-ERB proteins have also been identified, and may represent targets that complement the current small molecule modulators of these receptors. Intriguingly, the context-dependent tumor suppressor DBC1, which stabilizes and activates p53 (58), is also reported to stabilize REV-ERBα(59).Additionally, GSK3B-dependent phosphorylation of REV-ERBα stabilizes the protein (60), although theunderlying molecular mechanism remains unclear. However, it may be due to inhibition of either ArfBP1 (also known as MULE/HUWE1) orPAM (also known as MYCBP2), two E3 ubiquitin ligases that are reported to mediate REV-ERBα degradation(61). The links between these two ligases and REV-ERBα further emphasizes the importance of crosstalk between circadian regulators and
other signaling pathways.Moreover, although most of the work has been focused on REVERBα, identical signaling may be eventually adapted to REV-ERBβ. Analogous to the effect of CSNK1E inhibition (see above), the loss of ArfBP1 would be expected to stabilize REV-ERBα and thereby perturb transcription of CLOCK/BMAL targets. This is indeed the case. If such perturbations engender a transcriptional state that ‘primes’ cells for synthetic lethality, one might expect that loss of ArfBP1 in oncogene-overexpressing cells might hasten their demise. In support of this, ArfBP1 loss was synthetic lethal with RAS overexpression in colon carcinoma cells (62). What evidence is there to suggest that current drugs targeting metabolic pathways might display synthetic lethal activity with circadian regulators? Several drugs that are currently in clinical trials target proteins that are subject to circadian regulation. For example, AZD3965 inhibits MCT1, a lactate acid transporter that is required for tumor cell survival (63). Since MCT1 expression follows a circadian pattern (64), it is possible that co-treatment with circadian agonists/antagonists could increase the therapeutic index of AZD3965. The pyruvate kinase isoform PKM2 is another cancer-relevant target: it is upregulated in tumor cells where both dimeric and tetrameric formscontribute to tumor cell survival and tumor maintenance. This is due to PKM2-dependent rewiring of metabolites into anabolic processes in cancer. Thus, PKM2 inhibitors have been evaluated by several companies (65). However, it has been proposed that resistance to PKM2 inhibitors can develop due to activation of glutaminolysis, another rich source of anabolic cancer cell metabolites(66). Therefore, concomitant treatment with agents that disrupt glutaminolysis may abrogate such resistance. Targeting glutaminolysis has been investigated extensively, and the reader is referred to several reviews on the subject(67)(68).
Recent data indicate that the process is under circadian control. For example,glutaminase expression and activity follows a circadian pattern in hepatocarcinomas in vivo(69). This opens up the possibility that targeted inhibition of circadian regulators could thwart cancer cell growth by attenuating their glutamine utilization.
4. Conclusion In summary, recent studies are revealing that a pharmacological targeting of circadian-related proteins may be a suitable anticancer strategy. Although evidences that the single action on the molecular clock would be an effective approach are still controversial, the “engage” of the clock machinery for increasing the activity of existing anticancer drugs may be an effective approach. In addition to the discussed example related to REV-ERB and autophagy, it is worth mentioning that genetic inhibition of the circadian cryptochromes sensitizes p53-null cells to the anticancer drug oxaliplatin (70), and cryptochrome inhibitors have been recently identified (71). Furthermore, considering the intimate relationship between circadian signaling and metabolism (5), we suggest that circadian modulators may be particularly suitable for combination therapies together with drugs that target cancer cell metabolism; the latter class of drugs remains the focus of intense research efforts (43). In this scenario, synthetic lethality screens represent a valuable tool for the identification of novel avenues of pharmacological and therapeutic intervention.
Further studies will surely aimed to evaluate and optimize the drug-like properties of clock regulating compounds, such as their pharmacodynamics, specify and safety. Thus, a long path needs to be paved before “chrono-durgs” enter in clinical trials. Nonetheless, this seems a hopeful journey to find novel ways for fighting the timeless war against a disease that is the second leading cause of mortality in industrialized countries.
Conflict of interest None.
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Figure 1. The circadian clock machinerycan be conceptualized as two transcriptional complexes regulating each other’s activity, and operating in an antagonistic manner on the transcription of clock controlled genes (CCGs). In mammals, the proteins BMAL1/BMAL2 (shown as BMAL) and CLOCK/NAPS2 (shown as CLOCK) are transcriptional activators acting to enhance the expression of the CCGs. These factors also induce the expression of
the transcriptional repressor proteins, PER1/PER2/PER3 (shown as PER), CRY1/CRY2 (shown as CRY) and REV-ERBα/REV-ERBβ (shown as REV-ERB). The overall balance of the fight between transcriptional activators and repressors results in rhythmic expression of a number of genes involved in a variety of physiological responses.
Figure 2. REV-ERBα and REV-ERBβ nuclear receptors (shown as REV-ERB) are hemebinding proteins that repress transcription of target genes containing a REV-ERB responsive element (RevRE) within their promoters. Upon heme binding, the nuclear co-repressor NcoR and the histone de-acetyltransferase HDAC3results are recruited on the promoter of REVERB-regulated genes to shut-down transcription. REV-ERB targets include the master clock regulator, BMAL1, and the gluconeogenetic phosphoenolpyruvate carboxykinase enzyme, PEPCK. Because of the intimate link between the circadian clock and cellular metabolism, REV-ERB-mediated regulation of the molecular clock machinery further places REV-ERB proteins at a strategic position in metabolism.
Figure 3. Dual inhibitors of the circadian nuclear receptor REV-ERBβ and autophagy with improved in vitro anticancer activity compared to the medically relevant single autophagy inhibitor, chloroquine. In particular, the most potent inhibitor of the class, compound 30, decreases the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than chloroquine.
Figure 4. Inhibition of both REV-ERB and autophagy cooperate to induce a metabolic dysfunction that is incompatible with cancer cell viability. REV-ERB antagonism will affect the metabolism at different levels, regulating the expression of key circadian and metabolic genes. The resulting compromise in metabolic pathways renders cancer cells susceptible to a lethal “second shoot” deriving from the blockade of autophagy.
Table 1. Structure, specificity and in vivo suitability of tertiary amine chemotype-based REVERB agonists.