characterizing-dna-repair-processes-at-transient-long-lasting-double

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

using-next-generation-sequencing-to-identify-mutations-associated

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Kansas State University

Persistent high-risk genus human  (HPV) infections cause nearly every cervical carcinoma and a subset of tumors in the oropharyngeal tract. During the decades required for HPV-associated tumorigenesis, the cellular genome becomes significantly destabilized. Our analysis of cervical tumors from four separate data sets found a significant upregulation of the homologous-recombination (HR) pathway genes. The increased abundance of HR proteins can be replicated in primary cells by expression of the two HPV oncogenes (E6 and E7) required for HPV-associated transformation. HPV E6 and E7 also enhanced the ability of HR proteins to form repair foci, and yet both E6 and E7 reduce the ability of the HR pathway to complete double-strand break (DSB) repair by about 50%. The HPV oncogenes hinder HR by allowing the process to begin at points in the cell cycle when the lack of a sister chromatid to serve as a homologous template prevents completion of the repair. Further, HPV E6 attenuates repair by causing RAD51 to be mislocalized away from both transient and persistent DSBs, whereas HPV E7 is only capable of impairing RAD51 localization to transient lesions. Finally, we show that the inability to robustly repair DSBs causes some of these lesions to be more persistent, a phenotype that correlates with increased integration of episomal DNA. Together, these data support our hypothesis that HPV oncogenes contribute to the genomic instability observed in HPV-associated malignancies by attenuating the repair of damaged DNA. This study expands the understanding of HPV biology, establishing a direct role for both HPV E6 and E7 in the destabilization of the host genome by blocking the homologous repair of DSBs. To our knowledge, this is the first time that both viral oncogenes were shown to disrupt this DSB repair pathway. We show that HPV E6 and E7 allow HR to initiate at an inappropriate part of the cell cycle. The mislocalization of RAD51 away from DSBs in cells expressing HPV E6 and E7 hinders HR through a distinct mechanism. These observations have broad implications. The impairment of HR by HPV oncogenes may be targeted for treatment of HPV malignancies. Further, this attenuation of repair suggests HPV oncogenes may contribute to tumorigenesis by promoting the integration of the HPV genome, a common feature of HPV-transformed cells. Our data support this idea since HPV E6 stimulates the integration of episomes.

High-Risk Alphapapillomavirus Oncogenes Impair the Homologous Recombination Pathway.

While the role of genus alpha human papillomaviruses in the tumorigenesis and tumor maintenance of anogenital and oropharyngeal cancers is well-established, the role of genus beta human papilloviruses (β-HPVs) in non-melanoma skin cancers (NMSCs) is less certain. Persistent β-HPV infections cause NMSCs in sun-exposed skin of people with a rare genetic disorder, epidermodysplasia verruciformis. However, β-HPV infections in people without epidermodysplasia verruciformis are typically transient. Further, β-HPV gene expression is not necessary for tumor maintenance in the general population as on average there is fewer than one copy of the β-HPV genome per cell in NMSC tumor biopsies. Cell culture, epidemiological, and mouse model experiments support a role for β-HPV infections in the initiation of NMSCs through a "hit and run" mechanism. The virus is hypothesized to act as a cofactor, augmenting the genome destabilizing effects of UV. Supporting this idea, two β-HPV proteins (β-HPV E6 and E7) disrupt the cellular response to UV exposure and other genome destabilizing events by abrogating DNA repair and deregulating cell cycle progression. The aberrant damage response increases the likelihood of oncogenic mutations capable of driving tumorigenesis independent of a sustained β-HPV infection or continued viral protein expression. This review summarizes what is currently known about the deleterious effects of β-HPV on genome maintenance in the context of the virus's putative role in NMSC initiation.

Loss of Genome Fidelity: Beta HPVs and the DNA Damage Response.

The curious case of APOBEC3 activation by cancer-associated human papillomaviruses.

High risk human papillomavirus (HPV) infections are the causative agent in virtually every cervical cancer as well as a host of other anogenital and oropharyngeal malignancies. These viruses must activate DNA repair pathways to facilitate their replication, while avoiding the cell cycle arrest and apoptosis that can accompany DNA damage. HPV oncoproteins facilitate each of these goals, but also reduce genome stability. Our data dissect the cytotoxic and cytoprotective characteristics of HPV oncogenes in cervical cancer cells. These data show that while the transformation of keratinocytes by HPV oncogene leaves these cells more sensitive to UV, the oncogenes also protect against UV-induced apoptosis. Cisplatin and UV resistant cervical cancer cell lines were generated and probed for their sensitivity to genotoxic agents. Cervical cancer cells can acquire resistance to one DNA crosslinking agent (UV or cisplatin) without gaining broad tolerance of crosslinked DNA. Further, cisplatin resistance may or may not result in sensitivity to PARP1 inhibition.

Cervical cancer cell lines are sensitive to sub-erythemal UV exposure.

Nicholas Wallace studies how human papillomaviruses cause cancer throughout the genital and oropharyngeal tracts as well as in the skin. These viruses inhibit host DNA repair to promote their life style and in doing increase the risk of oncogenic mutations. In this mSphere of Influence article, he reflects on how two papers influenced him. "Human Papillomaviruses Activate the ATM DNA Damage Pathway for Viral Genome Amplification upon Differentiation" by C. A. Moody and L. Laimins (PLoS Pathog 5:e10000605, 2009, https://doi.org/10.1371/journal.ppat.1000605) reminded him of the power of straightforward approaches, while "Forty-Five Years of Cell-Cycle Genetics" by B. Reid et al. (B. J. Reid, J. G. Culotti, R. S. Nash, and J. R. Pringle, Mol Biol Cell 26:4307-4312, 2015, https://doi.org/10.1091/mbc.E14-10-1484) gave him the inspiration for his lab management style.

mSphere of Influence: the Value of Simplicity in Experiments and Solidarity among Lab Members.

To replicate, the human papillomaviruses (HPVs) that cause anogenital and oropharyngeal malignancies must simultaneously activate DNA repair pathways and avoid the cell cycle arrest that normally accompanies DNA repair. For years it seemed that HPV oncogenes activated the homologous recombination pathway to facilitate the HPV lifecycle. However, recent developments show that, although homologous recombination gene expression and markers of pathway activation are increased, homologous recombination itself is attenuated. This review provides an overview of the diverse ways that HPV oncogenes manipulate homologous recombination and ideas on how the resulting dysregulation and inhibition offer opportunities for improved therapies and biomarkers.

Catching HPV in the Homologous Recombination Cookie Jar.

Given the high prevalence of cutaneous genus beta human papillomavirus (β-HPV) infections, it is important to understand how they manipulate their host cells. This is particularly true for cellular responses to UV damage, since our skin is continually exposed to UV. The E6 protein from β-genus HPV (β-HPV E6) decreases the abundance of two essential UV-repair kinases (ATM and ATR). Although β-HPV E6 reduces their availability, the impact on downstream signaling events is unclear. We demonstrate that β-HPV E6 decreases ATM and ATR activation. This inhibition extended to XPA, an ATR target necessary for UV repair, lowering both its phosphorylation and accumulation. β-HPV E6 also hindered POLη accumulation and foci formation, critical steps in translesion synthesis. ATM's phosphorylation of BRCA1 is also attenuated by β-HPV E6. While there was a striking decrease in phosphorylation of direct ATM/ATR targets, events further down the cascade were not reduced. In summary, despite being incomplete, β-HPV 8E6's hindrance of ATM/ATR has functional consequences.

β-HPV 8E6 Attenuates ATM and ATR Signaling in Response to UV Damage.

Beta genus human papillomaviruses (β-HPVs) cause cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. However, β-HPVs are not necessary for tumor maintenance in the general population. Instead, they may destabilize the genome in the early stages of cancer development. Supporting this idea, β-HPV's 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper offers mechanistic insight into how β-HPV E6 causes this change in cell signaling. An  screen and characterization of HCT 116 cells lacking p300 suggested that the histone acetyltransferase is a negative regulator of Hippo pathway (HP) gene expression. HP activation restricts growth in response to stimuli, including failed cytokinesis. Loss of p300 resulted in increased HP gene expression, including proproliferative genes associated with HP inactivation. β-HPV 8E6 expression recapitulates some of these phenotypes. We used a chemical inhibitor of cytokinesis (dihydrocytochalasin B [H2CB]) to induce failed cytokinesis. This system allowed us to show that β-HPV 8E6 reduced activation of large tumor suppressor kinase (LATS), an HP kinase. LATS is required for p53 accumulation following failed cytokinesis. These phenotypes were dependent on β-HPV 8E6 destabilizing p300 and did not completely attenuate the HP. It did not alter H2CB-induced nuclear exclusion of the transcription factor YAP. β-HPV 8E6 also did not decrease HP activation in cells grown to a high density. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge, this marks the first time that a β-HPV E6 protein has been shown to hinder HP signaling. β-HPVs contribute to cSCC development in immunocompromised populations. However, it is unclear if these common cutaneous viruses are tumorigenic in the general population. Thus, a more thorough investigation of β-HPV biology is warranted. If β-HPV infections do promote cSCCs, they are hypothesized to destabilize the cellular genome.  data support this idea by demonstrating the ability of the β-HPV E6 protein to disrupt DNA repair signaling events following UV exposure. We show that β-HPV E6 more broadly impairs cellular signaling, indicating that the viral protein dysregulates the HP. The HP protects genome fidelity by regulating cell growth and apoptosis in response to a myriad of deleterious stimuli, including failed cytokinesis. After failed cytokinesis, β-HPV 8E6 attenuates phosphorylation of the HP kinase (LATS). This decreases some, but not all, HP signaling events. Notably, β-HPV 8E6 does not limit senescence associated with failed cytokinesis.

Beta Human Papillomavirus 8E6 Attenuates LATS Phosphorylation after Failed Cytokinesis.

Human papillomavirus (HPV) is a family of viruses divided into five genera: alpha, beta, gamma, mu, and nu. There is an ongoing discussion about whether beta genus HPVs (β-HPVs) contribute to cutaneous squamous cell carcinoma (cSCC). The data presented here add to this conversation by determining how a β-HPV E6 protein (β-HPV 8E6) alters the cellular response to cytokinesis failure. Specifically, cells were observed after cytokinesis failure was induced by dihydrocytochalasin B (H2CB). β-HPV 8E6 attenuated the immediate toxicity associated with H2CB but did not promote long-term proliferation after H2CB. Immortalization by telomerase reverse transcriptase (TERT) activation also rarely allowed cells to sustain proliferation after H2CB exposure. In contrast, TERT expression combined with β-HPV 8E6 expression allowed cells to proliferate for months following cytokinesis failure. However, this continued proliferation comes with genome destabilizing consequences. Cells that survived H2CB-induced cytokinesis failure suffered from changes in ploidy.

β-HPV 8E6 combined with TERT expression promotes long-term proliferation and genome instability after cytokinesis failure.

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity.

The incidence of head and neck squamous cell carcinomas (HNSCCs) is rising in developed countries. This is driven by an increase in HNSCCs caused by high-risk human papillomavirus (HPV) infections or HPV + HNSCCs. Compared to HNSCCs not caused by HPV (HPV- HNSCCs), HPV + HNSCCs are more responsive to therapy and associated with better oncologic outcomes. As a result, the HPV status of an HNSCC is an important determinant in medical management. One method to determine the HPV status of an HNSCC is increased expression of p16 caused by the HPV E7 oncogene. We identified novel expression changes in HPV + HNSCCs. A comparison of gene expression among HPV+ and HPV- HNSCCs in The Cancer Genome Atlas demonstrated increased DNA repair gene expression in HPV + HNSCCs. Further, DNA repair gene expression correlated with HNSCC survival. Immunohistochemical analysis of a novel HNSCC microarray confirmed that DNA repair protein abundance is elevated in HPV + HNSCCs.

DNA repair gene expression is increased in HPV positive head and neck squamous cell carcinomas.

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction.

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other β-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants.

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation.

High-risk human papillomavirus (HR HPV) causes nearly all cervical cancers, half of which are due to HPV type 16 (HPV16). HPV16 oncoprotein E6 (16E6) binds to NFX1-123, and dysregulates gene expression, but their clinical implications are unknown. Additionally, HPV16 E7's role has not been studied in concert with NFX1-123 and 16E6. HR HPVs express both oncogenes, and transformation requires their expression, so we sought to investigate the effect of E7 on gene expression. This study's goal was to define gene expression profiles across cervical precancer and cancer stages, identify genes correlating with disease progression, assess patient survival, and validate findings in cell models. We analyzed NCBI GEO datasets containing transcriptomic data linked with cervical cancer stage and utilized LASSO analysis to identify cancer-driving genes. Keratinocytes expressing 16E6 and 16E7 (16E6E7) and exogenous NFX1-123 were tested for LASSO-identified gene expression. Ten out of nineteen genes correlated with disease progression, including CEBPD, NOTCH1, and KRT16, and affected survival. 16E6E7 in keratinocytes increased CEBPD, KRT16, and SLPI, and decreased NOTCH1. Exogenous NFX1-123 in 16E6E7 keratinocytes resulted in significantly increased CEBPD and NOTCH1, and reduced SLPI. This work demonstrates the clinical relevance of CEBPD, NOTCH1, KRT16, and SLPI, and shows the regulatory effects of 16E6E7 and NFX1-123.

Nicholas A. Wallace

Division of Biology

Kansas State University

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter