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

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.

siRNA-Inhibition of TIGAR Hypersensitizes Human Papillomavirus-Transformed Cells to Apoptosis Induced by Chemotherapy Drugs that Cause Oxidative Stress.

The high-risk subtype Human Papillomaviruses (hrHPVs), including HPV16, HPV18, HPV31, HPV33, and HPV45, infect and oncogenically transform epithelial cells and cause squamous cell carcinomas and adenocarcinomas associated with the development of cervical cancer and subsets of vulvar, vaginal, penile, and anogenital cancers, as well as head-and-neck oropharyngeal carcinomas which often have poor clinical prognoses. Many cancers have been shown to contain elevated levels of the TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR)-a glycolytic enzyme and antioxidant effector which frequently correlates with an aggressive tumor phenotype and serves as a determinant of therapy-resistance. We therefore tested whether siRNA-inhibition of TIGAR protein expression could sensitize HPV18-transformed HeLa cells to genotoxic chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and 4-hydroxycyclophosphamide) that induce oxidative stress and DNA-damage. Here we demonstrate that the siRNA-knockdown of TIGAR hypersensitized HeLa cells to low, otherwise sub-inhibitory concentrations of these drugs and markedly induced cellular apoptosis, as compared to a scrambled RNA (scrRNA) oligonucleotide negative control or a non-transformed immortalized human fibroblast cell-line, HFL1. Importantly, these findings suggest that therapeutically inhibiting TIGAR could hypersensitize hrHPV+ cervical tumor cells to low-dosage concentrations of chemotherapy drugs that induce oxidative DNA-damage, which could potentially lead to more favorable clinical outcomes by reducing the adverse side-effects of these anticancer medications and making them more tolerable for patients. Our studies have further shown that siRNA-inhibition of TIGAR sensitizes HPV18+ HeLa cells to apoptosis induced by 4-hydroxycyclophosphamide-a DNA-alkylating agent these cells were reported to have resistance to, alluding to another possible benefit of targeting TIGAR in combinatorial treatment strategies against virus-induced cancers.

The Botanical Glycoside Oleandrin Inhibits Human T-cell Leukemia Virus Type-1 Infectivity and Env-Dependent Virological Synapse Formation.

At present, there are no antiretroviral drugs that inhibit incorporation of the envelope glycoprotein into newly-synthesized virus particles. The botanical glycoside, oleandrin, derived from extracts of Nerium oleander, has previously been shown to reduce the levels of the gp120 envelope glycoprotein on human immunodeficiency virus type-1 (HIV-1) particles and inhibit HIV-1 infectivity in vitro. We therefore tested whether oleandrin or an extract from N. oleander could also inhibit the infectivity of the human T-cell leukemia virus type-1 (HTLV-1): A related enveloped retrovirus and emerging tropical infectious agent. The treatment of HTLV-1+ lymphoma T-cells with either oleandrin or a N. oleander extract did not significantly inhibit viral replication or the release of p19Gag-containing particles into the culture supernatants. However, the collected virus particles from treated cells exhibited reduced infectivity on primary human peripheral blood mononuclear cells (huPBMCs). Unlike HIV-1, extracellular HTLV-1 particles are poorly infectious and viral transmission typically occurs via direct intercellular interactions across a virological synapse. We therefore investigated whether oleandrin or a N. oleander extract could inhibit virus transmission from a GFP-expressing HTLV-1+ lymphoma T-cell-line to huPBMCs in co-culture assays. These results demonstrated that both oleandrin and the crude phytoextract inhibited the formation of virological synapses and the transmission of HTLV-1 in vitro. Importantly, these findings suggest oleandrin may have broad antiviral activity against enveloped viruses by reducing the incorporation of the envelope glycoprotein into mature particles, a stage of the infection cycle not targeted by modern HAART.

The human T-cell leukemia virus type-1 tax oncoprotein dissociates NF-κB p65RelA-Stathmin complexes and causes catastrophic mitotic spindle damage and genomic instability.

Genomic instability is a hallmark of many cancers; however, the molecular etiology of chromosomal dysregulation is not well understood. The human T-cell leukemia virus type-1 (HTLV-1) oncoprotein Tax activates NF-κB-signaling and induces DNA-damage and aberrant chromosomal segregation through diverse mechanisms which contribute to viral carcinogenesis. Intriguingly, Stathmin/oncoprotein-18 (Op-18) depolymerizes tubulin and interacts with the p65RelA subunit and functions as a cofactor for NF-κB-dependent transactivation. We thus hypothesized that the dissociation of p65RelA-Stathmin/Op-18 complexes by Tax could lead to the catastrophic destabilization of microtubule (MT) spindle fibers during mitosis and provide a novel mechanistic link between NF-κB-signaling and genomic instability. Here we report that the inhibition of Stathmin expression by the retroviral latency protein, p30II, or knockdown with siRNA-stathmin, dampens Tax-mediated NF-κB transactivation and counters Tax-induced genomic instability and cytotoxicity. The Tax-G148V mutant, defective for NF-κB activation, exhibited reduced p65RelA-Stathmin binding and diminished genomic instability and cytotoxicity. Dominant-negative inhibitors of NF-κB also prevented Tax-induced multinucleation and apoptosis. Moreover, cell clones containing the infectious HTLV-1 ACH. p30II mutant provirus, impaired for p30II production, exhibited increased multinucleation and the accumulation of cytoplasmic tubulin aggregates following nocodozole-treatment. These findings allude to a mechanism whereby NF-κB-signaling regulates tubulin dynamics and mitotic instability through the modulation of p65RelA-Stathmin/Op-18 interactions, and support the notion that p30II enhances the survival of Tax-expressing HTLV-1-transformed cells.

The TP53-Induced Glycolysis and Apoptosis Regulator mediates cooperation between HTLV-1 p30II and the retroviral oncoproteins Tax and HBZ and is highly expressed in an in vivo xenograft model of HTLV-1-induced lymphoma.

The human T-cell leukemia virus type-1 (HTLV-1) is an oncoretrovirus that infects and transforms CD4+ T-cells and causes adult T-cell leukemia/lymphoma (ATLL) -an aggressive lymphoproliferative disease that is highly refractive to most anticancer therapies. The HTLV-1 proviral genome encodes several regulatory products within a conserved 3' nucleotide sequence, known as pX; however, it remains unclear how these factors might cooperate or dynamically interact in virus-infected cells. Here we demonstrate that the HTLV-1 latency-maintenance factor p30II induces the TP53-induced glycolysis and apoptosis regulator (TIGAR) and counters the oxidative stress, mitochondrial damage, and cytotoxicity caused by the viral oncoproteins Tax and HBZ. The p30II protein cooperates with Tax and HBZ and enhances their oncogenic potential in colony transformation/foci-formation assays. Further, we have shown that TIGAR is highly expressed in HTLV-1-induced tumors associated with oncogene dysregulation and increased angiogenesis in an in vivo xenograft model of HTLV-1-induced T-cell lymphoma. These findings provide the first evidence that p30II likely collaborates as an ancillary factor for the major oncoproteins Tax and HBZ during retroviral carcinogenesis.