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Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins.

ABSTRACT: The high-risk subtype human papillomaviruses (hrHPVs) infect and oncogenically transform basal epidermal stem cells associated with the development of squamous-cell epithelial cancers. The viral E6 oncoprotein destabilizes the p53 tumor suppressor, inhibits p53 K120-acetylation by the Tat-interacting protein of 60 kDa (TIP60, or Kat5), and prevents p53-dependent apoptosis. Intriguingly, the p53 gene is infrequently mutated in HPV + cervical cancer clinical isolates which suggests a possible paradoxical role for this gatekeeper in viral carcinogenesis. Here, we demonstrate that E6 activates the TP53-induced glycolysis and apoptosis regulator (TIGAR) and protects cells against oncogene-induced oxidative genotoxicity. The E6 oncoprotein induces a Warburg-like stress response and activates PI3K/PI5P4K/AKT-signaling that phosphorylates the TIGAR on serine residues and induces its hypoxia-independent mitochondrial targeting in hrHPV-transformed cells. Primary HPV + cervical cancer tissues contain high levels of TIGAR, p53, and c-Myc and our xenograft studies have further shown that lentiviral-siRNA-knockdown of TIGAR expression inhibits hrHPV-induced tumorigenesis in vivo. These findings suggest the modulation of p53 pro-survival signals and the antioxidant functions of TIGAR could have key ancillary roles during HPV carcinogenesis.

SUBMITTER: Yapindi L

PROVIDER: S-EPMC10527176 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins.

Yapindi Lacin L Bowley Tetiana T Kurtaneck Nick N Bergeson Rachel L RL James Kylie K Wilbourne Jillian J Harrod Carolyn K CK Hernandez Brenda Y BY Emerling Brooke M BM Yates Courtney C Harrod Robert R

Virology 20230522

The high-risk subtype human papillomaviruses (hrHPVs) infect and oncogenically transform basal epidermal stem cells associated with the development of squamous-cell epithelial cancers. The viral E6 oncoprotein destabilizes the p53 tumor suppressor, inhibits p53 K120-acetylation by the Tat-interacting protein of 60 kDa (TIP60, or Kat5), and prevents p53-dependent apoptosis. Intriguingly, the p53 gene is infrequently mutated in HPV + cervical cancer clinical isolates which suggests a possible para ...[more]

PMID: 37257253

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Project description:UnlabelledTranscripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy.ImportanceHPV16 is the most prevalent HPV genotype, being responsible for 60% of invasive cervical cancer cases worldwide. What makes HPV16 so potent in the development of cervical cancer remains a mystery. We discovered in this study that, besides producing two well-known oncoproteins, E6 and E7, seen in other high-risk HPVs, HPV16 produces E6^E7, a novel splice isoform of E6 and E7. E6^E7, in addition to self-interacting, binds cellular chaperone proteins, HSP90 and GRP78, and viral E6 and E7 to increase the steady-state levels and half-lives of viral oncoproteins, leading to cell proliferation. The splicing cis elements in the regulation of HPV16 E6^E7 production are highly conserved in 11 oncogenic or possibly oncogenic HPVs, and we confirmed the production of HPV18 E6^E7 in HPV18-infected cells. This study provides new insight into the mechanism of splicing, the interplay between different products of the polycistronic viral message, and the role of the host chaperones as they function.

Project description:BackgroundThe oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells.MethodsEnvisioning clinical application of the best characterized anti-HPV16 E6 and -HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed.ResultsWe showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway.ConclusionBased on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Project description:Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

Dataset Information

Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins.

Publications

Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins.

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