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Keywords:

  • UV ;
  • DNA damage;
  • DNA repair;
  • cell cycle checkpoint

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

The ultraviolet radiation (UVR) component of sunlight is the major environmental risk factor for melanoma, producing DNA lesions that can be mutagenic if not repaired. The high level of mutations in melanomas that have the signature of UVR-induced damage indicates that the normal mechanisms that detect and repair this damage must be defective in this system. With the exception of melanoma-prone heritable syndromes which have mutations of repair genes, there is little evidence for somatic mutation of known repair genes. Cell cycle checkpoint controls are tightly associated with repair mechanisms, arresting cells to allow for repair before continuing through the cell cycle. Checkpoint signaling components also regulate the repair mechanisms. Defects in checkpoint mechanisms have been identified in melanomas and are likely to be responsible for increased mutation load in melanoma. Loss of the checkpoint responses may also provide an opportunity to target melanomas using a synthetic lethal approach to identify and inhibit mechanisms that compensate for the defective checkpoints.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

There are a number of major challenges associated with the treatment of melanoma, and while five-year relative survival rates among patients diagnosed with localized disease is over 90%, melanoma in its advanced stage is one of the most deadly solid cancers (Jemal et al., 2010). Regional recurrence occurs in up to 30% of patients, with median survival in patients with advanced disease ranging between 6 and 10 months (Shepherd et al., 2010). Over the past 40 yr, there has been little improvement in survival for these patients and metastatic melanoma remains a significant clinical problem (AIHW and AACR, 2012; Balch et al., 2001). Recent targeted therapies have increased response rates and durations (Belden and Flaherty, 2012; Chapman et al., 2011; Falchook et al., 2012), however, rapidly developed resistance through molecularly diverse mechanisms is a major roadblock to significantly improved survival benefits (Corcoran et al., 2012; Nazarian et al., 2010; Paraiso et al., 2011; Poulikakos and Rosen, 2011; Smalley et al., 2008b; Villanueva et al., 2010; Yadav et al., 2012). The identification of new selective therapies for melanoma, increasing the efficacy of current therapies, and understanding the molecular basis of the resistance are critical steps toward improving outcomes for patients.

The importance of DNA damage response has long been recognized in the field of oncology. Damage to the DNA can be produced by a range of stressors, including environmental (UVR and ionizing radiation) and internal (oxygen radicals and replication and recombination errors). Many conventional chemotherapeutic drugs produce DNA damage. Each genotoxic stress produces a specific range of DNA damage which is repaired by damage type specific mechanisms; for example, ultraviolet radiation (UVR) produces 6–4 photoproducts (6–4PP) and cyclobutane pyrimidine dimers (CPD) which are repaired by nucleotide excision repair (NER) (Figure 1A). In addition to initiating repair, the normal cellular responses to damage are cell cycle arrest through checkpoint mechanisms to allow time for repair to be completed and where damage is too extensive either senescence or cell death to ensure that unrepaired DNA damage is not transmitted to future generations (Figure 1B). Two of the hallmarks of cancer are uncontrolled proliferation, a consequence of loss of normal cell cycle control, and increased genomic instability and mutation (Hanahan and Weinberg, 2011). The primary cell cycle response to DNA damage is a checkpoint signaling of a cell cycle arrest, the mechanism utilized being dependent on the type of damage and the phase of the cell cycle at which the damage is detected (Kaufmann, 2007; Medema and Macurek, 2012; Smith et al., 2010). The tight connection between DNA repair mechanisms and cell cycle checkpoints suggests that in many cases, loss of one may be reflected in defects in the other.

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Figure 1. DNA responses. (A) Different forms of DNA damage are repaired by specific repair mechanisms. (B) The extent of damage and success of repair determine the outcome, either cell cycle checkpoint arrest to allow for repair and return to proliferation or where damage is too extensive for successful repair, senescence, or death.

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Excessive exposure to the UVR component of sunlight is a major environmental factor contributing to skin cancers, including melanoma. Melanoma accounts for 10% of all cancers reported each year and 3% of cancer-related mortalities in Australia (AIHW and AACR, 2012). Ultraviolet radiation can directly damage DNA, and melanomas have been shown to contain significantly elevated numbers of UVR signature mutations compared with internal cancers (Pleasance et al., 2010), including mutations in cancer susceptibility genes such as the CDKN2a locus (coding for p16 INK4A and p19 ARF) (Pollock et al., 1996) and the recently identified mutations in the hTERT promoter (Horn et al., 2013; Huang et al., 2013). A series of recent studies have confirmed that that sun-exposed melanomas had markedly more UVR-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas and identified more driver mutations with UVR signatures (Berger et al., 2012; Hodis et al., 2012; Krauthammer et al., 2012). Increased exposure will produce more UVR-induced DNA damage than at shielded sites. As melanocytes in sun-exposed sites are constantly exposed to this mutagen, incomplete damage repair will result in increased mutation loads in these melanocytes. The consequence of this would likely be high levels of transformation, but this is not the case, even in melanoma-prone individuals. This suggests the existence of high fidelity damage detection and repair processes exist to ensure the maintenance of a mutation-free genome in melanocytes. The high level of UV signature mutations in melanomas indicates that the high fidelity detection or repair of the UVR-induced DNA damage must be defective in these melanomas. Despite this vast genome sequencing effort, no recurrent somatic mutations in known DNA repair genes that could explain this level of UVR signature mutations have been identified. Thus, a major unresolved question in the development of melanoma is how the UVR-induced DNA damage that is the source of the UVR signature mutations escapes repair. This review will focus on our current understanding of the cell cycle and DNA repair mechanisms involved in response to primarily UVR-induced DNA damage and will also encompass other DNA damage and cell cycle defects in melanoma. These repair and checkpoint defects are likely to be major contributors to melanomagenesis, but may also offer therapeutic opportunities, and recent advances in this area will also be discussed.

Cell cycle checkpoints

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

In response to internal and external stress such as genomic DNA damage and suboptimal growth conditions, proliferating cells trigger mechanisms called cell cycle checkpoints that assess and respond to the stress. Where the stress can compromise cell viability or the integrity of its genome, the checkpoint will block cell cycle progression and initiate processes to repair the damage detected. The checkpoint mechanism triggered, and cell cycle stage of arrest is dependent on the type of stress detected. For example, mitogenic signals are transduced through the Rb-CDK4/cyclin D pathway. DNA damage detected in G1 phase triggers either an ataxia telangiectasia mutated (ATM)-dependent or ataxia telangiectasia and Rad3-related protein (ATR)-dependent mechanism, dependent on whether it generates double-stranded breaks (DSB) or single-stranded DNA (ssDNA) intermediates, respectively (O'Connell and Cimprich, 2005). Replication defects generally result in ssDNA intermediates as a consequence of uncoupling of helicase unwinding from the DNA polymerase (Byun et al., 2005) and consequent ATR-dependent checkpoint arrest. In G2 phase, DNA damage triggers ATM- or ATR-dependent arrest to block entry into mitosis, while p53 signaling can stabilize the ATM-dependent arrest (Taylor and Stark, 2001). Mitotic defects are detected by the kinetochore-based spindle assembly checkpoint mechanism including the MAD and BUB proteins (Chan et al., 2005; Musacchio and Salmon, 2007) (Figure 2).

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Figure 2. Cell cycle checkpoints. Cell cycle checkpoints are triggered in response to specific stresses and utilize different signaling mechanisms to arrest the cells at specific points in the cell cycle until the stress has been resolved.

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Cell cycle checkpoint responses to UVR

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

Ultraviolet radiation can cause a range of DNA damage dependent on the wavelength and intensity of the exposure (Cadet et al., 2012). The major lesions are the CPD and 6–4PP produced by direct action of shorter wavelength UVC and UVB wavebands on DNA (Budden and Bowden, 2013; Cadet et al., 2012; Jhappan et al., 2003). The 6–4PP are less abundant and efficiently repaired, whereas the more abundant CPDs repair is slower (Budden and Bowden, 2013; Cadet et al., 2012; Jhappan et al., 2003; Mouret et al., 2008). Misrepair of these lesions produce the UVR signature C>T and CC>TT mutations (Pfeifer and Besaratinia, 2012). The UVC and much of the UVB waveband are absorbed by the earth's atmosphere, but the residual UVB radiation is primarily responsible for the UVR signature mutations that are responsible for the carcinogenic effects of sunlight exposure.

G1 phase checkpoints

The first cell cycle checkpoint protein associated with DNA damage response (DDR) to UVR insult was the p53 tumor suppressor. Its accumulation is associated with a G1 phase arrest and increased expression of the CDK inhibitor p21WAF1 (Petrocelli et al., 1996). P53 regulates the expression of UVR lesion recognition (DDB2) and repair (XPC) proteins (Soria and Gottifredi, 2010; Stoyanova et al., 2009). In skin, increased p53 expression is only detected at erythemal doses of UVR (Pavey et al., 1999; Ponten et al., 1995), and its transcriptional target p21WAF1 is actively downregulated at both transcript and protein levels in response to UVR to permit efficient NER (Soria and Gottifredi, 2010; Stoyanova et al., 2009). The role of p53 appears to be primarily in regulating DNA repair and survival signaling in response to erythemal (>1 minimal erythemal dose) UVR rather than cell cycle arrest (Cui et al., 2007; Enk et al., 2006). Although p53 mutation is not a common feature of melanoma, p53 is effectively defective in a majority of melanomas through a variety of mechanisms including overexpression of the p53 antagonist MDM4 (Gembarska et al., 2012; Smalley et al., 2007), thereby contributing to increased survival of damaged cells following UVR. At suberythemal UVR doses, cells will trigger ATR-dependent checkpoint signaling (Mailand et al., 2000), although little G1 phase arrest is detected at these low doses of UVR (Wigan et al., 2012). ATR signaling is also observed in non-cycling cells indicating a role for ATR in DNA repair as well as checkpoint arrest (Vrouwe et al., 2011).

S phase checkpoints

Cells irradiated in S phase or entering S phase with unrepaired UVR-induced lesions elicit a ATR-dependent intra-S phase arrest. This is triggered by either replication fork arrest on encountering a UVR lesion resulting in disengagement of helicase unwinding from the polymerase, which produces extended regions of ssDNA that are recognized by the ssDNA binding replication protein A (RPA) (Byun et al., 2005). The replication fork arrest can also trigger lesion bypass, a DNA damage tolerance mechanisms that allows replication to proceed relatively unimpeded and avoid replication fork collapse, which would likely lead to apoptosis (Chang and Cimprich, 2009). Lesion bypass mechanism involves repriming of the replication fork downstream of the damage, producing a gap in the replicated strand and leaving the lesion on the template strand unrepaired. Cells irradiated during S phase trigger an S phase delay through the ATR-dependent intra-S phase checkpoint, although the extent of the delay is dependent on the dose of UVR, low doses have little detected S phase delay (Auclair et al., 2010). Cells irradiated in G1 phase also fail to completely repair UVR lesions as a result of weaknesses in NER, such as the less efficient repair of CPDs than 6–4PPs (Mouret et al., 2008). In both cases, lesion bypass ensures the completion of replication. At higher doses of UVR, the translesion synthesis (TLS) polymerase Polη is a major contributor to S phase repair (Auclair et al., 2010; Bomgarden et al., 2006). At low doses, there is also a role for Werner's helicase (WRN) which is responsible for efficient replication fork complex bypass of unrepaired lesions (Wigan et al., 2012), resulting in accumulation of ssDNA gaps during S phase (Jansen et al., 2009b; Lopes et al., 2006; Novarina et al., 2011). The subsequent presence of the RPA-coated ssDNA foci recruits a range of checkpoint signaling proteins including mediator of DNA damage checkpoint protein 1 (MDC1), breast cancer 1 early onset (BRCA1), ATR-interacting protein (ATRIP) and ATR, thereby activating ATR and recruiting Claspin which acts as a cofactor in activation of checkpoint kinase 1 (CHK1) (Chang and Cimprich, 2009; Jansen et al., 2009a; Wigan et al., 2012) (Figure 3). The size of the ssDNA gaps may be dependent on whether the initial DNA damage occurred on the lagging or leading strand (Gangavarapu et al., 2007). Repriming on the lagging strand is an efficient process and likely to produce small ssDNA gaps. Repriming on the leading strand is an uncommon event and likely to be inefficient, generating larger (approximately 3 kb) ssDNA gaps (Jansen et al., 2009b; Lopes et al., 2006). Another determinant of ssDNA gaps size is activity of the exonuclease Exo1 which can increase the size of the gaps when either NER associated DNA polymerase gap filling activity is compromised or possibly when there are two closely paired UVR lesions on opposing strands (Sertic et al., 2011). The role of Exo1 is to ensure that the cell cycle checkpoint is engaged to permit repair of the original lesion and of the excision gap produced by NER, which at 30 nt is too short to efficiently induce a checkpoint arrest.

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Figure 3. ATR Checkpoint signaling. ATR is recruited to single-stranded DNA by the RPA ssDNA binding complex, with auxiliary proteins ATRIP and Claspin, BRCA1 and MDC1 to activate CHK1 which signals the cell cycle arrest.

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G2 phase checkpoints

Ultraviolet radiation can trigger G2 phase checkpoint signaling as a result of either irradiation of G2 phase cells or as a replicative repair response to unrepaired UVR lesions encountered by the replication fork in S phase. Both are dependent on ATR checkpoint signaling although the former is replication independent (Stiff et al., 2008), and the latter is replication dependent (Diamant et al., 2012; Wigan et al., 2012). In the replication-dependent checkpoint response, the replication fork is used to interrogate every base of the DNA during replication. When it encounters an unrepaired lesion replication bypass is triggered. This can be either TLS polymerase-dependent replication past the lesion or WRN-dependent repriming of the replication fork downstream of the lesion. The latter produces a ssDNA gap that attracts the ssDNA binding RPA which in turn recruits ATR-dependent checkpoint signaling (Figure 4) (Wigan et al., 2012). The ssDNA gaps appear to act as markers of the unrepaired lesions and attract not only checkpoint signaling components but also repair proteins for the ssDNA gaps and the original UVR lesion. There is evidence in yeast and humans that both TLS and recombination repair mechanisms have roles in post-replicative repair in response to UVR (Callegari et al., 2010; Daigaku et al., 2010; Diamant et al., 2012; Gohler et al., 2011; Karras and Jentsch, 2010; Lin et al., 2011). The TLS Polη also contributes to the repair of the ssDNA gaps as depletion or loss of Polη increases the ATR-dependent G2 phase delay after UVR (Bomgarden et al., 2006; Diamant et al., 2012), and ATR can directly regulate Polη activity (Gohler et al., 2011). BRCA1 has also been implicated in S and G2 phase post-replication repair where it is required for recruitment of GG-NER and homologous recombination repair pathway components, but has a role in inhibiting Polη recruitment (Pathania et al., 2011). This apparently conflicting evidence for the contribution of TLS in post-replicative repair and checkpoint signaling may be reconciled by consideration of the DNA strand on which the UVR lesion resides. Lesions on the leading strand are likely to produce gaps of several kb, whereas those on the lagging strand, due to the canonical use of Okazaki fragments to prime the synthesis of this strand, would yield much smaller gaps of 100–200 bp, the size of an Okazaki fragment (Lopes et al., 2006). It is possible that the larger and smaller gaps would be filled by different repair mechanism, TLS for the smaller gaps, and homologous recombination for the larger gaps to ensure the fidelity of repair. Against this is the lack of strand bias in the mutation rate observed with depletion of TLS components (Yoon et al., 2009).

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Figure 4. G2 phase checkpoint and repair response to UV lesions detected in S phase. Cells irradiated in G1 phase repair the majority of lesions (TT) using nucleotide excision repair (NER). Ultraviolet radiation also stimulates progression into S phase where the few unrepaired lesions are detected by the replication fork. This can lead to WRN helicase-dependent lesion bypass, with the replication fork complex reinitiating downstream of the lesion leaving a ssDNA gap opposite the lesion. The ssDNA attracts RPA which in turn recruits the ATR checkpoint signaling complex to arrest the cell cycle in G2 phase. The ssDNA gap is repaired by a post-replicative repair process while the UV lesion is likely to be repaired by GG-NER.

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Checkpoint and repair defects in melanoma

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

DDR Defects

The xeroderma pigmentosum (XP) syndrome genes are mutant in XP patients but are rarely targets for somatic mutation in melanoma. This includes the TLS polymerase Polη, which is encoded by PolH also known as the XPV gene (Masutani et al. 1999), and has a role in UVR lesion repair at the replication fork and in G2 phase repair (Auclair et al., 2010; Gohler et al., 2011). In addition to these, several NER components have been reported to have common SNPs significantly associated with melanoma. PolH has several SNPs with an increased risk of >2 (Di Lucca et al., 2009). Polymorphisms in XPD (Li et al., 2006; Millikan et al., 2006), XPF, and ERCC1 (Povey et al., 2007) are also associated with a modest (<2 fold) increased risk of melanoma. There is some evidence that there is reduced efficiency of NER associated with melanocortin-1 receptor (MC1R) variants responsible for the red hair phenotype (Kadekaro et al., 2010), but in most cases, NER capability of melanomas appears relatively unaffected (Gaddameedhi et al., 2010). In addition to these, MEN1 is expressed in low levels in a high proportion of melanomas and loss of MEN1 reduces the expression of homologous recombination genes as well as the homologous recombination repair (Fang et al., 2013).

Checkpoint defects

Defective G1 phase checkpoint signaling was found to be a common feature of melanoma cells. Approximately 70% of a large panel of melanoma cell lines screened for checkpoint functionality following exposure to ionizing radiation demonstrated a defective G1 checkpoint arrest (Carson et al., 2012; Kaufmann et al., 2008). A gene expression signature, including CDKN1A, DDB2, CDC7 and GEMININ, was associated with the defective G1 phase checkpoint, demonstrating that this was due to defective p53 function despite the relatively low level (20%) of p53 mutation in melanoma (Box and Terzian, 2008; Hodis et al., 2012). Moreover, the identified gene signature was prognostic of metastatic spread suggesting the involvement of p53 as protective against metastasis.

Another G1 phase mechanism that responds to extensive DNA damage is p16-mediated senescence (Robles and Adami, 1998; Shapiro et al., 1998). The melanoma susceptibility gene CDKN2A which encodes the cell cycle inhibitor p16 INK4A (p16) is commonly defective in melanoma (Bartkova et al., 1996; Castellano et al., 1997; Hayward, 2003). P16 inhibits CDK4/6-cyclin D activity and thereby blocks Rb inactivation promoting a G1 phase arrest. P16 is a major driver of senescence in melanocytes and is largely responsible for the oncogene-induced senescence in nevi driven by either BRAF or NRAS mutation (Gray-Schopfer et al., 2006; Haferkamp et al., 2009; Michaloglou et al., 2005). Melanoma associated mutations of p16 disrupt its ability to promote senescence arrest (Haferkamp et al., 2008). In addition to this role, increased p16 expression has been correlated with the G2 phase checkpoint arrest in response to suberythemal UVR (Abd Elmageed et al., 2009; Pavey et al., 1999, 2001; Wang et al., 1996). Loss or mutation of p16 or CDK4 is associated with loss of the checkpoint arrest (Milligan et al., 1998; Wigan et al., 2012), although the mechanistic relationship between p16 and the G2 phase arrest is not clear. ATR can phosphorylate and regulate the stability of p16 and its binding to CDK4/6 (Al-Khalaf et al., 2011; Gabrielli et al., 1999), and inhibition of CDK4 can promote a G2 phase delay (Burgess et al., 2006; Gabrielli et al., 1999), possibly through the regulation of the G2/M phase transcriptional regulator FOXM1 (Anders et al., 2011).

Other defects that may contribute to disruption of the p16-dependent responses are cyclin D overexpression and amplification in melanoma (Lazar et al., 2009; Utikal et al., 2005; Vizkeleti et al., 2012). CDK4R24 mutations that render CDK4 insensitive to p16 are uncommon (Soufir et al., 1998; Wolfel et al., 1995), and CDK4 is rarely overexpressed in melanomas (Muthusamy et al., 2006; Smalley et al., 2008a). Reduced Rb protein levels are also infrequently found in melanoma (Bartkova et al., 1996; Castellano et al., 1997; Roesch et al., 2005). In addition to p16-dependent senescence, there appear to be p16-independent mechanisms that are regulated through PTEN-PI3K-AKT signaling. This is demonstrated by the appearance of nevi on Tyr-BRAFV600E mice that are bred onto a p16 null background (Dhomen et al., 2009), and depletion of PTEN or inhibition of PI3K signaling was sufficient to overcome BRAFV600E induced senescence without affecting p16 levels (Vredeveld et al., 2012).

The G2 phase checkpoint response to ionizing radiation-induced DNA double-strand breaks has been reported to be defective in 1/3 of melanoma cell lines and was significantly associated with BRAF mutation (Kaufmann et al., 2008; Omolo et al., 2013). Likewise, the G2 phase decatenation checkpoint triggered in response to failure of the TopoII-dependent resolution of the DNA strand catenations that normally occurs during replication (Deming et al., 2001) and is defective in at least 1/3 of melanoma cell lines (Brooks et al., 2013a). The G2 phase checkpoint response to the post-replication repair of the ssDNA gaps produced by lesion bypass of unrepaired UVR lesions is defective in a higher proportion of melanoma cell lines (Wigan et al., 2012). Both the former responses are dependent on ATM for the checkpoint arrest (Bower et al., 2010), while the later is ATR dependent. GWAS studies have identified SNPs in the DNA damage response genes ATM, PARP1 and APEX1 with significant association with melanoma (Law et al., 2012). The SNP in ATM was found to be protective, suggesting that it is unlikely to contribute to the ATM-dependent checkpoint defects (Barrett et al., 2011).

Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

Chemotherapy and radiotherapy

Many of the conventional approaches to the treatment of advanced cancers are genotoxic and necessarily trigger a DNA damage response and commonly a cell cycle checkpoint response (Al-Ejeh et al., 2010; Curtin, 2012). Intact checkpoint responses can reduce sensitivity to these agents. Dacarbazine (5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide, DTIC) and the nitrosourea drugs (carmustine and lomustine) only yield response rates of 10 to 20% with no demonstrated impact on overall survival (Chapman et al., 1999; Wagner et al., 2000), with similar response rates observed with temozolomide, an orally available imidazotetrazine derivative of the alkylating agent dacarbazine (Middleton et al., 2000). These alkylating agents promote G1, S, and G2 phase arrests in melanoma depending on its p53 status. The efficacy of alkylating agents is reduced in p53 wild-type cells by p53-dependent upregulation of DNA damage repair components thereby increasing repair (Barckhausen et al., 2013). The role of adjuvant or definitive radiation therapy in the treatment of melanoma remains controversial, as melanoma has traditionally been viewed as a prototypical radioresistant cancer. Under certain clinical circumstances, there may be a significant role for radiation therapy in melanoma treatment (Burmeister et al., 2012; Macklis, 2012). Genotoxic therapies trigger ATM-/ATR-dependent checkpoint arrest, and the defects in checkpoint responses dependent on these signaling pathways suggest that stratifying patients on the basis of checkpoint signaling or repair defects in their melanomas could increase the response rates and survival benefit of these therapies. In breast cancer, cisplatin treatment was more effective in patients with BRCA mutant breast cancer which are defective for the homologous recombination repair of the cisplatin induced DNA damage (Curtin, 2012; Silver et al., 2010). This suggests that identification of the checkpoint and DDR status of melanomas may improve outcomes with conventional therapies.

MAPK-targeted drugs

A number of agents targeting the MAPK pathway have been developed and entered clinical trials (Belden and Flaherty, 2012). Mutation of BRAF has been estimated to occur in ~50% of melanomas (Davies et al., 2002; Jakob et al., 2012). BRAF inhibitors have proven activity in patients with metastatic melanoma harboring activating mutations in BRAF (V600E, V600K); however, acquired resistance remains the greatest limitation (Belden and Flaherty, 2012). A strategy to further improve response rates and delay resistance has been through the combination of BRAF and MEK1/2 inhibitors and is currently being assessed in clinical phase III trials (Flaherty et al., 2012). The cellular effect of these inhibitors is a G1 phase arrest through inhibition of MAPK pathway-dependent cyclin D expression (Haass et al., 2008; Smalley et al., 2006; Tsai et al., 2008; Vultur et al., 2011), although there is no evidence for any checkpoint or DDR involvement. Mutant BRAF and RAS have been reported to diminish the ATM-dependent G2 phase checkpoint arrest (Kaufmann et al., 2008; Knauf et al., 2006), but the relative lack of efficacy of conventional chemotherapeutics and radiation in melanoma suggests that these common mutations do significantly affect the checkpoint response to these genotoxic insults in patients.

CDK inhibitors

The alteration of the DNA damage and mitotic checkpoints frequently results in increased CDK activity that drives tumor cell cycles, with misregulated CDKs inducing unscheduled proliferation as well as genomic and chromosomal instability (Malumbres and Barbacid, 2009). This has suggested that CDKs are rational drug targets and there are a number of cell cycle inhibitors that are undergoing human clinical evaluation in a range of cancers. CDK inhibitors have produced modest results as single agents, whereas in combination with chemotherapy, they can improve cytotoxic efficacy and overcome drug resistance (Lapenna and Giordano, 2009). Wide-spectrum CDK inhibitors often display high toxicity in early clinical trials (Lapenna and Giordano, 2009; Shapiro, 2006). Alvocidib has been used in clinical trials as a monotherapy and combination with chemotherapy (Senderowicz, 2003), although the clinical trials have shown no significant activity and dose limiting toxicities were observed with use in combination with chemotherapy. P276-00 inhibits CDK4-cyclin D, CDK1-cyclin B, and CDK9-cyclin T1 and can induce either cell cycle arrest or apoptosis. It has significant antiproliferative effects in melanoma cell lines and has been used in a clinical trial in advanced melanoma positive for cyclin D1 expression (Joshi et al., 2007). SNS-032 selectively inhibits CDK1, 2, 7, and 9 and produces G2 and M phase cell cycle arrest and apoptosis. It has shown good preclinical activity and sensitizes radioresistant tumor cells to radiation; however, it showed a high toxicity when administered in vivo and is now discontinued (Lapenna and Giordano, 2009). Indisulam (E7070) targets the G1 phase by inhibiting the activation of CDK2/cyclin E, inducing p53 and p21, and inhibiting CDK2 phosphorylation (Terret et al., 2003). It has been used to treat melanoma; however, no objective responses were observed (Smyth et al., 2005).

Checkpoint inhibitors

Approaches targeting cell cycle checkpoints are currently being explored to increase the efficacy and selectivity of conventional treatments. This is being exploited in preclinical models with p53 mutant tumors. Loss of p53 results in cells being reliant on the G2 phase ATR-CHK1-dependent checkpoint in response to genotoxic drugs such as alkylating agents. This checkpoint is readily bypassed using CHK1 inhibitors (Dai and Grant, 2010; Ma et al., 2011). CHK1 inhibitors are being assessed as chemosensitizers, increasing the potency of the chemotherapeutic agents (Dai and Grant, 2010; Morgan et al., 2010; Myers et al., 2009). Although p53 is not commonly mutated in melanoma, it is commonly defective (Box and Terzian, 2008), suggesting that CHK1 inhibitors may have chemosensitizer activity in melanoma. CHK1 inhibitors also have activity as single agents in melanoma, with very potent activity in melanomas with high levels of replicative stress (Brooks et al., 2013a). ATR inhibitors similarly act as chemosensitizers but are also selectively cytotoxic in NER-defective cells (Fokas et al., 2013; Sultana et al., 2013).

Synthetic lethal combinations

More recently, the emphasis has moved toward a targeted molecular therapeutic approach, incorporating high throughput genomics and functional screening strategies for biomarker discovery. The use of unbiased, systematic screens may be a more efficient approach in identifying molecular targets/pathways and synthetically lethal combinatorial mechanisms (Chan and Giaccia, 2011; Kuiken and Beijersbergen, 2010; Reinhardt et al., 2009). The synthetic lethal approach is best exemplified in BRCA mutant breast cancers where base excision repair (BER) compensates for the loss of BRCA-dependent homologous recombination repair. Inhibiting BER with PARP inhibitors results in selective cytotoxicity of the BRCA mutant cancers (Ashworth, 2008). This approach has the advantage of utilizing endogenous DNA damage rather than an externally applied stressing agent, which increases the selectivity of this approach. Similarly, the use of CHK1 inhibitors is an example of synthetic lethal targeting, either in p53 defective cancers in combination with chemotherapeutics or as a single agents exploiting endogenous replication stress (Brooks et al., 2013a; Dai and Grant, 2010; Ma et al., 2011). The success of this approach to therapy has encouraged wider investigation of DNA damage repair defects as synthetic lethal targets in multiple cancer types (Curtin, 2012; Martin et al., 2008; Morandell and Yaffe, 2012). The close relationship between DNA repair and cell cycle checkpoints suggests that checkpoint defects may be also useful synthetic lethal targets (Gabrielli et al., 2012; Morandell and Yaffe, 2012). An example of synthetic lethal targeting defective checkpoints is the mechanism of histone deacetylase inhibitor (HDACi) selective killing of cancer cells. HDACi triggers a G2 phase checkpoint arrest in normal cells, whereas a high proportion of cancer cell lines, including melanomas, are defective for this G2 phase arrest (Qiu et al., 2000). Loss of this HDACi-sensitive checkpoint resulted in cells undergoing an aberrant mitosis triggering rapid apoptosis (Burgess et al., 2001; Qiu et al., 2000; Warrener et al., 2003), the consequence of HDACi-mediated mitotic slippage (Gabrielli and Brown, 2012; Stevens et al., 2008).

Future directions

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

The melanoma-prone syndromes such as XP have been used to identify many of the components of NER pathway and demonstrated the causal link between UVR-induced DNA lesions and melanoma. However, few somatic defects in DNA repair mechanisms have been identified, indicating that other mechanisms involved in DNA damage response are defective. Cell cycle checkpoint controls are intimately involved in repair, directly regulating repair components and providing time for the repair to be completed. Defective checkpoint control has been identified in melanomas and is associated with reduced UVR damage repair. The less efficient repair is responsible for the increased UVR signature mutation rate found in melanomas and also likely to contribute to the genomic instability that underlies the ability of melanomas to develop diverse drug resistance mechanisms. The defective checkpoint provides an advantage to the melanoma increasing its genetic adaptability. It is also a divergence from the normal and thus provides a target selective for the melanoma. Additionally, the defective checkpoint and repair must also be more reliant on other mechanisms to compensate for the defective normal response. Identification and targeting of this compensatory mechanism are likely to provide a selective lethal insult to checkpoint defective melanomas. The immediate task is to identify drugs that are synthetically lethal with the defective checkpoint and repair and identify clinically useful molecular markers of the defect that will identify patients' tumors that are most likely to be sensitive to this targeted therapy.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Cell cycle checkpoints
  5. Cell cycle checkpoint responses to UVR
  6. Checkpoint and repair defects in melanoma
  7. Can we target defective cell cycle checkpoint and repair mechanisms in melanoma?
  8. Future directions
  9. Acknowledgements
  10. References

SP is a recipient of a University of Queensland Research Fellowship for Women; BG is an National Health and Medical Research Council Senior Research Fellow; NKH is a recipient of the Cameron Fellowship from the Melanoma and Skin Cancer Research Institute/Melanoma Foundation/Dermatology Foundation, Australia. This work was supported by grants from National Health and Medical Research Council (APP1029260 to BG, APP1003637 to NKH), Cancer Council New South Wales (RG 13-06 to NKH), Cancer Australia/Cure Cancer Australia Foundation (1051996 to NKH), Cancer Council Queensland (1026513 to BG).

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  4. Cell cycle checkpoints
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  6. Checkpoint and repair defects in melanoma
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  8. Future directions
  9. Acknowledgements
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