Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA ...crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of the downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was associated with the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.
The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β ...human papillomavirus genus (β-HPVs). Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by promoting p300 degradation, resulting in a reduction in ATR levels, which leads to increased thymine dimer persistence and increased UVB induced DSBs.
Abstract In general, the interplay among viruses and DNA damage repair (DDR) pathways can be divided based on whether the interaction promotes or inhibits the viral lifecycle. The propagation of ...human papillomaviruses is both promoted and inhibited by DDR proteins. As a result, HPV proteins both activate repair pathways, such as the ATM and ATR pathways, and inhibit other pathways, most notably the p53 signaling pathway. Indeed, the role of HPV proteins, with regard to the DDR pathways, can be divided into two broad categories. The first set of viral proteins, HPV E1 and E2 activate a DNA damage response and recruit repair proteins to viral replication centers, where these proteins are likely usurped to replicate the viral genome. Because the activation of the DDR response typically elicits a cell cycle arrest that would impeded the viral lifecycle, the second set of HPV proteins, HPV E6 and E7, prevents the DDR response from pausing cell cycle progression or inducing apoptosis. This review provides a detailed account of the interactions among HPV proteins and DDR proteins that facilitate HPV propagation.
Merkel cell polyomavirus (MCPyV) accounts for 80% of all Merkel cell carcinoma (MCC) cases through expression of two viral oncoproteins: the truncated large T antigen (LT-t) and small T antigen (ST). ...MCPyV ST is thought to be the main driver of cellular transformation and has also been shown to increase LT protein levels through the activity of its Large-T Stabilization Domain (LSD). The ST LSD was reported to bind and sequester several ubiquitin ligases, including Fbw7 and β-TrCP, and thereby stabilize LT-t and several other Fbw7 targets including c-Myc and cyclin E. Therefore, the ST LSD is thought to contribute to transformation by promoting the accumulation of these oncoproteins. Targets of Fbw7 and β-TrCP contain well-defined, conserved, phospho-degrons. However, as neither MCPyV LT, LT-t nor ST contain the canonical Fbw7 phospho-degron, we sought to further investigate the proposed model of ST stabilization of LT-t and transformation. In this study, we provide several lines of evidence that fail to support a specific interaction between MCPyV T antigens and Fbw7 or β-TrCP by co-immunoprecipitation or functional consequence. Although MCPyV ST does indeed increase LT protein levels through its Large-T Stabilization domain (LSD), this is accomplished independently of Fbw7. Therefore, our study indicates a need for further investigation into the role and mechanism(s) of MCPyV T antigens in viral replication, latency, transformation, and tumorigenesis.
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.
Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate ...the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.
The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by ...c-Myc to induce oncogenesis remain elusive. Here we show that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions.
Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. This study sought to identify biomarkers that improve ...prognostic accuracy and provide insight into MCC biology.
Gene expression profiles of 35 MCC tumors were clustered based on prognosis. The cluster of genes overexpressed in good-prognosis tumors was tested for biologic process enrichment. Relevant mRNA expression differences were confirmed by quantitative polymerase chain reaction and immunohistochemistry. An independent set of 146 nonoverlapping MCC tumors (median follow-up, 25 months among 116 living patients) was employed for biomarker validation. Univariate and multivariate Cox regression analyses were performed.
Immune response gene signatures were prominent in patients with good prognoses. In particular, genes associated with cytotoxic CD8+ lymphocytes were overexpressed in tumors from patients with favorable prognoses. In the independent validation set, cases with robust intratumoral CD8+ lymphocyte infiltration had improved outcomes (100% MCC-specific survival, n = 26) compared with instances characterized by sparse infiltration (60% survival, n = 120). Only stage and intratumoral CD8 infiltration (but not age, sex, or CD8+ lymphocytes localized to the tumor-stroma interface) were significant in both univariate and multivariate Cox regression analyses. Notably, traditional histologic identification of tumor-infiltrating lymphocytes was not a significant independent predictor of survival.
Intratumoral CD8+ lymphocyte infiltration can be readily assessed on paraffin-embedded tissue, is independently associated with improved MCC-specific survival, and therefore, may provide prognostic information that enhances established MCC staging protocols.
Merkel cell polyomavirus (MCPyV) expressing viral T antigens is a common feature of most Merkel cell carcinomas, a primary neuroendocrine skin tumor. MCPyV has been difficult to propagate in vitro. ...In this issue of Cell Host & Microbe, Liu et al. (2016) report that human dermal fibroblasts support productive MCPyV infection.
Merkel cell polyomavirus (MCPyV) expressing viral T antigens is a common feature of most Merkel cell carcinomas, a primary neuroendocrine skin tumor. MCPyV has been difficult to propagate in vitro. In this issue of Cell Host & Microbe, Liu et al. (2016) report that human dermal fibroblasts support productive MCPyV infection.
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