There are myriad mechanisms by which oncogenic viruses cooperate with and deregulate host cellular growth/proliferative-signaling pathways to cause human cancers. The human papillomaviruses (HPVs) are members of the Papovaviridae family and are small spherical, non-enveloped dsDNA viruses that infect basal epithelial stem cells associated with the development of benign localized skin lesions (common or plantar warts; papillomas). However, many of the alpha-genus, high-risk HPV subtypes (hrHPVs; e.g., HPV16, HPV18, HPV31, HPV33, and HPV45) are sexually transmitted and can infect urogenital and oropharyngeal mucosal squamous epithelia and have been etiologically linked to uterine cervical dysplasia and a number of HPV+epithelial malignancies, including cervical, vaginal, penile, and anogenital carcinomas, as well as certain subsets of head-and-neck cancers (Mix et al., 2021; Graham, 2017b; The Cancer Genome Atlas Network, 2015; Castellsagué et al., 2016). The viral genome is maintained and replicates as non-integrated circular episomes within the nuclei of HPV-infected cells. The switch from latency to vegetative genomic replication for the production of infectious particles is intricately linked to the keratinocyte differentiation program and is mediated by a viral regulatory nucleotide sequence, known as the long control region (LCR), and the early proteins: E1, E2, E4, E5, E6, and E7 (Münger et al., 2004; Graham, 2017a; Longworth and Laimins, 2004; Jones and Münger, 1997; Genther et al., 2003; Wang et al., 2009). Whereas the E1 and E2 proteins are responsible for episomal replication in HPV-infected basal epithelial cells, the E5, E6, and E7 proteins regulate vegetative genomic replication within infected suprabasal keratinocytes and differentiated spinous epithelial cells by reprogramming these cells to express S-phase-promoting components, such as Myc, Piwil2, and cyclin/cyclin-dependent kinases, which promote nucleotide biogenesis and DNA-replication (Genther et al., 2003; Wang et al., 2009; Feng et al., 2016; Vande Pol and Klingelhutz, 2013; Roman and Munger, 2013; Malanchi et al., 2002; Martin et al., 1998).
The viral E6 and E7 oncoproteins deregulate the cell-cycle by interfering with the functions of the tumor suppressor proteins, p53 and retinoblastoma (Rb; Vande Pol and Klingelhutz, 2013; Roman and Munger, 2013; Scheffner et al., 1993; Huibregtse et al., 1991; Münger et al., 1989; Boyer et al., 1996). The amino terminus of E7 binds to the pocket protein Rb and induces its ubiquitin-dependent degradation and dissociates Rb-E2F inhibitory complexes to promote the G1/S transition in infected differentiated keratinocytes (Münger et al., 1989; Boyer et al., 1996). The hrHPV E7 oncoprotein also degrades the protein tyrosine phosphatase PTPN14, independent of its ability to inactivate Rb, which inhibits keratinocyte differentiation and could contribute to cellular immortalization and viral carcinogenesis (Hatterschide et al., 2019). Although the p53 gene is infrequently mutated in primary HPV+cervical cancer clinical isolates (Schultheis et al., 2021; Lee et al., 1994), the E6 oncoprotein ubiquitinates and reduces the expression of p53 through interactions with the E6-associated protein (E6AP) and inhibits acetylation of the p53 protein on lysine residue K120 and prevents p53-dependent cellular apoptosis by destabilizing the Tat-interacting protein of 60 kDa (TIP60, or Kat5) through interactions with the E3 ubiquitin ligase, EDD1/UBR5 (Scheffner et al., 1993; Huibregtse et al., 1991; Martinez-Zapien et al., 2016; Jha et al., 2010; Subbaiah et al., 2016). The hrHPV E6/E7 oncoproteins also cooperate with cellular protooncogenes and augment the expression and transactivation functions of the basic domain/leucine zipper (bZIP) transcription factor, Myc. Strickland and Vande Pol (2016) have demonstrated that the E7 protein enhances the cap-independent translation of c-Myc by increasing the utilization of an internal ribosome entry site (IRES) within c-myc mRNA transcripts. Further, the E6 oncoprotein directly interacts with Myc and recruits E6/Myc/Max complexes to E-box enhancer elements within the human telomerase reverse transcriptase (htert) gene promoter associated with Myc-dependent transactivation and keratinocyte-immortalization (Zhang et al., 2017; McMurray and McCance, 2003; Liu et al., 2008). As mounting evidence suggests the remaining p53 protein present in HPV-transformed epithelial cells is transcriptionally active, and p53 is a downstream target of the Myc protooncogene (Butz et al., 1995; Banuelos et al., 2003; Kimple et al., 2013; Reisman et al., 1993; Roy et al., 1994; Zindy et al., 1998), it remains to be fully determined how E6 cooperates with Myc to induce the G1/S transition in HPV-infected keratinocytes, while overcoming the oxidative stress and p53-dependent genotoxicity associated with the unscheduled activation of Myc pro-replicative signals (Zindy et al., 1998; Vafa et al., 2002; Hermeking and Eick, 1994; Chen et al., 2013; Gorrini et al., 2007).
The TP53-induced glycolysis and apoptosis regulator (TIGAR) is a 2,6-bis-fructose-phosphatase that localizes to the outer membranes of mitochondria under conditions of hypoxia, glucose-deprivation, or ischemic injury and contributes to mitochondrial quality control and prevents ischemia-induced mitophagy and the accumulation of damaging reactive oxygen species (ROS) by increasing the intracellular levels of NADPH and reduced glutathione (Bensaad et al., 2006, 2009; Cheung et al., 2012, 2013; Li et al., 2014; Zhou et al., 2016). The mitochondrial targeting of TIGAR occurs through a mechanistic process that requires activation of hypoxia-inducible factor-1 alpha (HIF-1α) and molecular interactions with hexokinase-2 (HK2; Cheung et al., 2012). The TIGAR protein also increases the production of ribose-5-phosphate through glucose catabolism by the pentose-phosphate pathway which could promote nucleotide biosynthesis in proliferating cells (Bensaad et al., 2006). Several studies have implicated the overexpression of TIGAR as a key determinant of therapy-responsiveness and indicator of aggressive disease progression and poor clinical outcomes for many types of cancers, including gliomas, colorectal, esophageal, nasopharyngeal, and renal cell carcinomas, gastric cancers, multiple myeloma, chronic lymphocytic leukemia, acute myeloid leukemia, adult T-cell leukemia, lymphoblastic leukemia, and non-Hodgkin's lymphoma (Cheung et al., 2013; Tang and He, 2019; Maurer et al., 2019; Chu et al., 2020; Wong et al., 2015; Wang et al., 2020; Liu et al., 2019; Yin et al., 2014; Hong et al., 2016; Qian et al., 2016, Qian et al., 2016; Romeo et al., 2018; Hutchison et al., 2018). The TIGAR has also been shown to contribute to the epithelial-to-mesenchymal transition (EMT), tumor cell migration/invasiveness, and metastasis related to Met-signaling in non-small-cell lung cancers (Shen et al., 2018). Here, we demonstrate that hrHPV E6 oncoproteins activate HIF-1α/HIF-2α and cellular kinases which phosphorylate the TIGAR protein on serine residues to induce its hypoxia-independent mitochondrial targeting and prevent c-Myc-induced oxidative DNA-damage, associated with enhanced oncogenic cellular transformation in vitro and HPV tumorigenesis in vivo. Hypoxia has been reported to transcriptionally inhibit the expression of the viral E6/E7 oncoproteins in HPV-infected cells through the phosphatidylinositol-3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) pathway (Bossler et al., 2019) and, therefore, our studies allude to a functionally distinct role for E6-induced HIF-1α/HIF-2α pseudohypoxic signaling for the activation of mitochondrial TIGAR. These findings shed new light on the molecular mechanisms by which hrHPVs cooperate with cellular growth/proliferative signals to reprogram differentiated cells and suggest that the E6 oncoprotein augments p53 pro-survival functions by inducing a pseudohypoxic stress response to counter the metabolic cytotoxicity and replicative stress caused by aberrant protooncogene activation which could contribute to viral carcinogenesis. These studies further provide the first evidence that phospho-signaling regulates the mitochondrial localization and antioxidant functions of TIGAR which could lead to new therapeutic strategies to target this key pro-oncogenic factor in human cancers.
All cells used for these studies were grown at 37 °C under 5% CO2 in a humidified incubator. The HPV18+ Hela cervical adenocarcinoma cell-line was cultured in Dulbecco's Modified Eagle's Medium (DMEM; No. 30-2002; ATCC, Manassas, VA), supplemented with 10% fetal bovine serum (FBS; Biowest, Riverside, MO), 100 U/ml penicillin and 100μg/ml streptomycin sulfate (Invitrogen, Waltham, MA). The HPV18+ C-4 I, HPV18/45+ MS751, and HPV16+ SiHa cervical carcinoma cell-lines were cultured in Eagle's
High-risk HPV E6 proteins induce hypoxia-independent mitochondrial targeting of the TIGAR
The expression of TIGAR is positively regulated by p53 and the myc protooncogene and protects proliferating cells from toxic metabolic byproducts, such as damaging ROS, by increasing the levels of NADPH and reduced glutathione (Bensaad et al., 2006, 2009; Romeo et al., 2018; Cheung et al., 2016). The TIGAR protein is predominantly cytoplasmic, although a significant fraction has been shown to relocate to the outer membranes of mitochondria under conditions of hypoxic stress or ischemic injury
The MYST-family acetyltransferase TIP60 (Kat5) is a transcriptional cofactor for both c-Myc and p53 (Patel et al., 2004; Frank et al., 2003; Awasthi et al., 2005; Tang et al., 2006; Sykes et al., 2006; Kurash et al., 2008); and the p53 gene is a downstream target of c-Myc (through the activation of E-box elements within the p53 promoter and protein stabilization by p14ARF) which has been shown to mediate oncogene-induced DNA-damage, genomic instability and cellular apoptosis (Reisman et al.,
These aggregate data demonstrate that hrHPVs modulate the expression of certain p53-regulated pro-survival genes by inhibiting TIP60-mediated p53-K120-acetylation (Jha et al., 2010; Subbaiah et al., 2016), and induce a pseudohypoxic stress response to promote the serine-phosphorylation and mitochondrial targeting of TIGAR which could help protect HPV-infected cells against oncogene-induced oxidative stress and genotoxicity associated with the development and progression of hrHPV+epithelial
This work was supported by National Cancer Institute/National Institutes of Health grants 1R15CA202265-01A1 and 1R15CA267892-01A1 and an SMU Dean's Research Council grant to RH. LY was supported by an SMU Moody School of Graduate and Advanced Studies Dissertation Fellowship, and TB was supported by an SMU Dean's Dissertation Fellowship and an American Society for Microbiology-Graduate Student Fellowship.
All data are contained within the article.
CRediT authorship contribution statement
Lacin Yapindi: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Visualization, Writing – review & editing. Tetiana Bowley: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – review & editing. Nick Kurtaneck: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation. Rachel L. Bergeson: Formal analysis, Investigation. Kylie James: Formal analysis, Investigation. Jillian
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
We kindly thank K. Vousden for providing the pcDNA3.1-TIGAR (FLAG) expression construct, Y. Nakatani for the pOZ-TIP60 expression vector, and B. Vogelstein for the pCEP-p53 (wt) and pCEP-p53-R175H plasmids. Other members of the Harrod lab: M. Saberi, N. Adams, and J. Savage, are thanked for their helpful technical assistance. We also thank L. Banks for generously providing the HPV16/18 E6 (HA) expression constructs and the Pathology Shared Resource of the University of Hawaii Cancer Center for
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