Impact of Replication Stress in Human Papillomavirus Pathogenesis

The inactivation of critical cell cycle checkpoints by the human papillomavirus (HPV) oncoprotein E7 results in replication stress (RS) that leads to genomic instability in premalignant lesions. Intriguingly, RS tolerance is achieved through several mechanisms, enabling HPV to exploit the cellular RS response for viral replication and to facilitate viral persistence in the presence of DNA damage.

ABSTRACT

The inactivation of critical cell cycle checkpoints by the human papillomavirus (HPV) oncoprotein E7 results in replication stress (RS) that leads to genomic instability in premalignant lesions. Intriguingly, RS tolerance is achieved through several mechanisms, enabling HPV to exploit the cellular RS response for viral replication and to facilitate viral persistence in the presence of DNA damage. As such, inhibitors of the RS response pathway may provide a novel approach to target HPV-associated lesions and cancers.

INTRODUCTION

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the cutaneous or mucosal epithelium. Persistent infection with high-risk mucosal HPV types (e.g., HPV16, -18, -31, and -45) causes 5% of human cancers, including cervical cancer, other genital malignancies, and an increasing number of head and neck cancers, with HPV16 being the predominant type (1–3). HPV-associated cancers are mainly driven by the E6 and E7 oncoproteins, which function primarily through protein-protein interactions that interfere with cell cycle regulation, differentiation, and apoptosis to provide a replication-competent environment (4, 5).

HPV infects the proliferating basal cells of the stratified epithelium, establishing a low copy number (50 to 100 episomes per cell) that is stably maintained by replicating along with cellular DNA (6, 7). Epithelial differentiation triggers productive replication, resulting in viral genome amplification to 100s to 1,000s of copies per cell (8, 9). Epithelial cells normally exit the cell cycle upon differentiation; however, HPV-infected cells reenter the cell cycle and progress to a G₂-like phase that supports productive replication, late gene expression, and virion production (5, 10). The E7 oncoprotein plays a critical role in this process by targeting the tumor suppressor Rb for degradation, resulting in the aberrant activation of E2F transcription factors that drive S phase entry (11). To avoid cell cycle arrest or apoptosis associated with unscheduled cell cycle entry, the E6 oncoprotein targets the p53 tumor suppressor for degradation (12).

To replicate in a G₂-arrested environment, high-risk HPVs require the activation of the ataxia telangiectasia mutated (ATM)- and ataxia telangiectasia and Rad3 related (ATR)-dependent DNA damage response (DDR) (13–15). ATM and ATR are serine/threonine kinases that, along with DNA-dependent protein kinase (DNA-PK), constitute the core of the DDR response (16). While ATM and DNA-PK respond to primarily double-stranded DNA breaks (DSBs) that are sensed by the MRN complex (Mre11, Rad50, and Nbs1) and the Ku70/80 complex, respectively, ATR responds to single-stranded DNA (ssDNA) that is generated by replication stress (RS). In this Gem, I highlight recent studies detailing how the E7 oncoprotein induces RS yet employs several mechanisms to facilitate RS tolerance, resulting in constitutive activation of an ATR-driven DDR that HPV exploits for viral replication. As a result, viral persistence in the presence of RS results in genomic instability (i.e., mutations and abnormal chromosome structures and numbers) that likely fuels cancer progression.

ONCOGENE-INDUCED REPLICATION STRESS

RS broadly defines impediments in DNA replication that lead to replication fork stalling and the formation of ssDNA that can generate DNA damage. A number of obstacles can slow or stall replication forks, including DNA secondary structures, transcription-replication collisions, limited nucleotides, and topological stress (17). The ATR-dependent response, also known as the RS response, is critical to prevent the accumulation of DNA damage and genomic instability (18). ssDNA formed at stalled replication forks bound by replication protein A (RPA) serves as the main trigger for ATR recruitment through its binding partner ATRIP and becomes fully activated through TOPBP1 or ETAA1 activity (19, 20). ATR then phosphorylates its primary substrate kinase Chk1 to facilitate cell cycle arrest, prevent excess origin firing, and stabilize/repair stalled replication forks (18, 21, 22). ATR DDR activation prevents excessive accumulation of ssDNA and subsequent exhaustion of RPA that can in turn result in DSB formation and collapsed replication forks that activate the ATM DDR (23).

An emerging source of RS is the expression of oncogenes, both viral and cellular, which override controls restricting entry into S phase, resulting in unscheduled proliferation and the perturbation of DNA replication (24, 25). Oncogene-induced RS can occur through several mechanisms, including the insufficient usage of replication origins, leading to underreplicated DNA, as well as the excessive usage of replication origins, resulting in an exhaustion of replicative factors that slows down replication fork progression. The higher number of active origins can also lead to an increased chance of collisions between replication and transcription machinery, which results in stalled replication forks and DNA damage (25). Oncogene-induced RS is thought to be the initial source of genomic instability, with ATR as the primary sensor of oncogene-induced DNA damage (26).

HIGH-RISK HPVs INDUCE REPLICATION STRESS

Numerous studies support a role for HPV oncogenes, particularly E7, in inducing RS. In line with this, high-risk HPV16 and HPV31 E7, and to a lesser extent, HPV31 E6, induce ATR pathway activation (15, 27). HPV16 E7-expressing cells display classic signs of RS, including the formation of ssDNA and the recruitment of RS response proteins (e.g., FANCD2 and BRCA2) to ssDNA foci in an ATR-dependent manner (28). Studies using DNA fiber analysis revealed that the coexpression of HPV16 E6/E7 results in stalled replication forks and DSBs through increased replication origin firing (29). In addition, E6/E7-induced RS results in a loss of heterozygosity (LOH; i.e., a loss of genetic information) preferentially at common fragile sites (CFSs), which are large chromosome regions that have difficulty completing replication and are prone to breakage under conditions of RS (29, 30). CFSs typically exhibit genomic instability at early states of cancer development, and increasing evidence suggests that a perturbation of DNA replication at these regions is a major cause of CFS instability (17, 29, 31). Recent studies indicate that CFS gene products are directly involved in the DDR and may participate in maintaining genomic integrity (32). Whether CFS LOH potentiates genomic instability in HPV-associated lesions is currently unclear. Interestingly, in HPV-associated cancers, the viral genome is often found integrated into CFSs in host DNA (33–35). Integration is a dead end for the virus life cycle but usually leads to deregulated E6/E7 expression, resulting in hyperproliferation and the accumulation of genomic instability (36). Genomic instability in CFSs as a consequence of E7-induced RS could inadvertently increase the frequency of viral genome integration, driving cancer development. Genomic instability is a hallmark of HPV-associated cancers but is also present in premalignant lesions, where the virus is maintained episomally (37–39). Keratinocytes maintaining HPV16 episomes exhibit RS and DSBs similar to that found in E6/E7-expressing cells, indicating that RS underlies the genomic instability observed in early lesions (29). Importantly, supplementation with exogenous nucleosides promotes recovery from E7-induced RS and the repair of damaged DNA, providing evidence that RS and genomic instability result from an insufficient supply of nucleotides to support extensive proliferation (29, 40).

HPV TOLERATES REPLICATION STRESS THROUGH EPIGENETIC MECHANISMS

Oncogene-induced RS triggers a sustained DDR that results in apoptosis or senescence, acting as an intrinsic barrier to tumorigenesis (41, 42). However, high-risk HPV-infected cells remain proliferative despite constitutive activation of the ATR as well as ATM DDR (13–15, 43). Oncogene-induced senescence (OIS) can be activated by the expression of the KDM6B H3K27 demethylase, which epigenetically derepresses the expression of p16(INK4A), a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor that induces G₁ arrest and senescence through the activation of Rb (44, 45). Although KDM6B and p16 are elevated in response to E7 expression (46), HPV avoids OIS through E7’s ability to target Rb for degradation (Fig. 1). Intriguingly, E7-expressing cells become addicted to p16(INK4A) expression through the inhibition of CDK4/6 (47). E7 also induces the expression of the KDM6A H3K27 demethylase, resulting in elevated levels of the CDK inhibitor p21, which blocks cell cycle progression and replication by binding to and inhibiting CDK2 and PCNA, respectively (40, 46). Surprisingly, Soto et al. found that the depletion of KDM6A or p21 in E7-expressing cells results in enhanced RS and DSBs that lead to cell death, indicating that HPV hijacks the KDM6A/p21 pathway to tolerate persistent E7-induced RS (40). Tolerance to E7-induced RS is achieved through p21’s ability to inhibit PCNA-dependent replication, which is seemingly counterintuitive for a virus that requires entry into the cell cycle to replicate (40). Furthermore, E7 has been reported to bind tightly to p21 and inhibit its growth suppressive effects (48–50). However, p21 is present at such high levels in response to E7 expression that its inhibition is thought to be stoichiometric (48, 49, 51). E7 can reduce p21’s inhibitory effect on PCNA as well as on CDK2 to enable sustained proliferation, while a small pool of p21 remains active to limit RS and DNA damage (Fig. 1) (40, 48, 49). Outside of p21 inhibition, E7 has additional ways to sustain CDK2 activity and also maintains high levels of PCNA through Rb/E2F deregulation (11, 52–54). Epigenetic reprogramming by KDM6A/KDM6B in response to E7 expression, coupled with CDK2 hyperactivity and Rb loss, enables HPV to persist in the presence of RS and DNA damage, facilitating the accumulation of genomic instability.

FIG 1.

HPV induces, yet tolerates, replication stress (RS) to promote viral replication through the ATR-dependent RS response. Expression of E7 induces RS that activates the ATR/Chk1/E2F1 pathway. Increased expression of KDM6A and KDM6B represents a cellular defense to E7 expression, though whether this occurs in response to E7-induced RS is unclear. Despite high levels of KDM6B and p16 in E7-expressing cells, HPV circumvents oncogene-induced senescence (OIS) through Rb degradation, promoting aberrant proliferation. Increased expression of KDM6A leads to elevated levels of p21 that facilitate tolerance to RS by inhibiting PCNA. E7 maintains CDK2 activity as well as PCNA expression in the face of p21 activity, resulting in hyperproliferation and RS that constitutively activates an ATR-dependent DDR that HPV commandeers for replication. Dashed gray lines represent links that have not been examined experimentally.

HPV HARNESSES THE REPLICATION STRESS RESPONSE FOR REPLICATION

Several studies indicate that HPV hijacks the constitutively active ATR DDR for viral replication. The inhibition of ATR or Chk1 activity results in fewer copies of HPV16 and HPV31 episomes in undifferentiated cells and a block in productive replication of HPV31 upon differentiation (13, 15, 43). RPA phosphorylated on Ser33, a target of ATR, localizes to HPV31 replication foci, suggesting that RS occurs on viral DNA (55). Indeed, several factors involved in HR repair as well as in replication fork protection and restart (e.g., FANCD2, BRCA1, and Rad51) are bound to HPV genomes and are necessary for viral replication (56–61). ATR effectors are also recruited to HPV18 replication foci during transient replication in U20S cells, which mimics establishment replication, suggesting that RS occurs on viral DNA during the initial amplification (62). While the source of RS on viral DNA is currently unclear, a recent study demonstrated that R-loops (stable RNA/DNA hybrids) are present on HPV31 genomes, which can form as a result of RNA/DNA polymerase collisions and lead to RS (58, 63). Interestingly, recent studies from the McBride lab revealed that HPV replication foci tend to form adjacent to CFSs, which, as mentioned, exhibit RS and DNA damage in response to E7 expression (64). Viral replication in close proximity to CFSs undergoing RS may provide HPV access to required DNA repair factors but may also increase the incidence of accidental viral integration into cellular DNA and, in turn, promote tumorigenesis (65).

Recent studies demonstrated that HPV exploits the ATR RS response to maintain the activation of the E2F transcriptional network (13). In response to RS, sustained E2F-dependent transcription is important for cell survival, because many E2F targets function in replication, DNA repair, and nucleotide synthesis (66, 67). ATR/Chk1 maintains E2F activity through the phosphorylation/stabilization of E2F1 or the phosphorylation/inactivation of the atypical E2F factors (E2F6, E2F7, and E2F8), which repress E2F-dependent G₁/S transcription (68–70). A critical target of the ATR/Chk1/E2F1 response is RRM2, the small subunit of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of deoxynucleoside triphosphates (dNTPs) (69, 71, 72). ATR-driven accumulation of RRM2 provides dNTPs for DNA repair and is necessary for the survival of cells exhibiting high levels of RS (71). High-risk HPV31 utilizes the ATR/Chk1 pathway to increase the levels of E2F1, resulting in the accumulation of RRM2 and dNTPs that are necessary for viral replication (Fig. 1) (13). The activation of the ATR/Chk1/E2F1/RRM2 axis is especially important upon differentiation, where productive replication occurs in a G₂-arrested environment, providing limited substrates for replication (5, 10). The high levels of E2F1, RRM2, and dNTPs in high-risk HPV-infected cells suggest that increasing ATR and Chk1 activity may be an additional strategy utilized by HPV to replicate in the presence of persistent RS. The identification of additional targets of the ATR/Chk1/E2F1 pathway will provide further insight into how HPV commandeers the RS response for viral replication.

IS E7-INDUCED REPLICATION STRESS DEPENDENT ON RB DEGRADATION?

In addition to limiting DNA damage in response to RS, the aberrant activation of E2F transcription factors is thought to play a key role in facilitating replication perturbation by oncogenes (73). Indeed, the deregulation of E2F activity through Rb degradation contributes to E7-induced RS (28). However, E7 also maintains activation of E2F-responsive promoters in an Rb-independent manner through the activation of E2F1 and inhibition of E2F6 (74, 75). In addition, sequences in the C terminus of HPV16 E7 important for overcoming p21-mediated growth arrest have been reported to play a role in inducing RS (28). Furthermore, the upregulation of KDM6A and KDM6B in response to E7 expression occurs through an unknown but Rb-independent mechanism (46). While these studies suggest that Rb-dependent and -independent mechanisms underlie E7-induced RS, HPV requires E7’s Rb binding domain to effectively respond to RS and DNA damage. Numerous DNA repair factors are elevated in high-risk HPV-infected cells in an E7-dependent manner, including ATM, ATR, and Chk1, as well as the HR factors BRCA1 and Rad51, with mutation of the Rb binding domain abrogating this phenotype (76). E7 also induces expression of the ATR activator TOPBP1 through STAT5 activation, which is postulated to occur in an Rb-dependent manner (15). The E7-mediated increase in DDR proteins likely provides an additional level of protection against RS and DNA damage triggered by HPV oncogene expression.

FUTURE PERSPECTIVES

While much has been learned regarding the mechanisms by which the HPV oncogenes promote cancer development, many questions remain regarding the full impact of RS and activation of the RS response in HPV pathogenesis. If E7-induced RS serves as the initial oncogenic hit, it will be interesting to determine if the activation of the ATR pathway plays a central role in HPV’s ability to bypass OIS, potentially by providing dNTPs through E2F1/RRM2 activation (77). Cancer cells commonly feature intrinsically high RS, and the upregulation of ATR and Chk1 is essential to their survival (78, 79). It will be important to determine if continual activation of the ATR/Chk1 pathway, similar to KDM6A expression, enables HPV-infected cells to effectively respond to E7-induced RS, leading to a higher survival advantage and, in turn, the accumulation of genomic instability. Furthermore, the finding that ATR activity is required for viral replication raises the intriguing possibility that E7-induced RS facilitates viral persistence, a major risk factor in the development of cancer.

Substantial attention has been focused on the pathways that detect and signal DNA damage as potential drug targets for cancer therapy. Indeed, several studies have demonstrated a synthetic lethality between OIS and an impaired RS response (80). As such, high-risk HPV-associated lesions may be uniquely sensitive to inhibitors of the ATR pathway. Studies have established that E7-expressing cells are addicted to KDM6A by promoting tolerance to RS, offering further support that RS may serve as an Achilles heel for HPV-associated cancers that can be targeted therapeutically (40). Targeting the RS response in HPV infections is particularly intriguing in light of two recent clinical trials demonstrating the effectiveness of radiochemotherapy combined with the RRM2 inhibitor triapine in advanced cervical cancers (81). Further understanding of how HPV promotes cell survival in the face of persistent RS may identify novel ways to target HPV infections and associated malignancies.