Virology

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.

Lassa-VSV chimeric virus targets and destroys human and mouse ovarian cancer by direct oncolytic action and by initiating an anti-tumor response

Ovarian cancer is the third most common female cancer, with poor survival in later stages of metastatic spread. We test a chimeric virus consisting of genes from Lassa and vesicular stomatitis viruses, LASV-VSV; the native VSV glycoprotein is replaced by the Lassa glycoprotein, greatly reducing neurotropism. Human ovarian cancer cells in immunocompromised nude mice were lethal in controls. Chemotherapeutic paclitaxel and cisplatin showed modest cancer inhibition and survival extension. In contrast, a single intraperitoneal injection of LASV-VSV selectively infected and killed ovarian cancer cells, generating long-term survival. Mice with human ovarian cancer cells in brain showed rapid deterioration; LASV-VSV microinjection into brain blocked cancer growth, and generated long-term survival. Treatment of immunocompetent mice with infected mouse ovarian cancer cells blocked growth of non-infected ovarian cancer cells peritoneally and in brain. These results suggest LASV-VSV is a viable candidate for further study and may be of use in the treatment of ovarian cancer.

Vaccination with human alphapapillomavirus-derived L2 multimer protects against human betapapillomavirus challenge, including in epidermodysplasia verruciformis model mice

Human alphapapillomaviruses (αHPV) infect genital mucosa, and a high-risk subset is a necessary cause of cervical cancer. Licensed L1 virus-like particle (VLP) vaccines offer immunity against the nine most common αHPV associated with cervical cancer and genital warts. However, vaccination with an αHPV L2-based multimer vaccine, α11-88x5, protected mice and rabbits from vaginal and skin challenge with diverse αHPV types. While generally clinically inapparent, human betapapillomaviruses (βHPV) are possibly associated with cutaneous squamous cell carcinoma (CSCC) in epidermodysplasia verruciformis (EV) and immunocompromised patients. Here we show that α11-88x5 vaccination protected wild type and EV model mice against HPV5 challenge. Passive transfer of antiserum conferred protection independently of Fc receptors (FcR) or Gr-1+ phagocytes. Antisera demonstrated robust antibody titers against ten βHPV by L1/L2 VLP ELISA and neutralized and protected against challenge by 3 additional βHPV (HPV49/76/96). Thus, unlike the licensed vaccines, α11-88x5 vaccination elicits broad immunity against αHPV and βHPV.

Cationic liposomes overcome neutralizing antibodies and enhance reovirus efficacy in ovarian cancer

Reovirus (Reo) has shown promising potential in specifically killing tumor cells, and offering new possibilities for ovarian cancer (OC) treatment. However, neutralizing antibodies in the ascites from OC patients greatly limit the further application of Reo. In this study, we employed cationic liposomes (Lipo) to deliver Reo, significantly enhancing its ability to enter OC cells and its effectiveness in killing these cells under ascitic conditions. Pre-treatment with the MβCD inhibitor notably decreased Reo-mediated tumor cell death, indicating that Lipo primarily enables Reo's cellular uptake through caveolin-mediated endocytosis. Our results demonstrate that Lipo effectively facilitates the entry of Reo into the cytoplasm and triggers cell apoptosis. The above findings provide a new strategy to overcome the obstacle of neutralizing antibodies in the clinical application of Reo.

Alteration of Na/H exchange regulatory factor-1 protein levels in anogenital lesions positive for mucosal high-risk human papillomavirus type 16

Mucosal high-risk (HR) human papillomaviruses (HPV) are associated with anogenital carcinogenesis. The products of two early genes, E6 and E7, act as major viral oncoproteins. Functional studies in experimental models showed that HPV16 E6 induces degradation of the PDZ protein, the Na+/H+ exchanger regulatory factor-1 (NHERF-1). Here, we determined NHERF-1 protein levels by immunohistochemistry (IHC) in (i) benign anogenital warts (n = 8) (ii) premalignant lesions (L-SIL and H-SIL) (n = 43) and (iii) invasive cervical squamous cell carcinomas (SCC) (n = 17). A decrease of NHERF-1 protein level was not observed in genital warts in comparison to healthy epithelium. Conversely, a clearly decrease in NHERF-1 protein levels was observed in HPV16-positive pre-malignant and malignant lesions, while the phenomenon was much attenuated in lesions induced by other HR HPV types. In conclusion, these findings show that mucosal HPV types differently impact on NHERF-1 protein level in benign and malignant anogenital lesions.

Distribution of human papillomavirus among Vietnamese women with cervical cancer and unusual genetic variability of HPV16

HPV16, with typical mutations that differ in geographical distribution and carcinogenic potency, has implications for cervical cancer screening, clinical diagnosis, and treatment. DNASTAR and MEGA were used to identify HPV16 variants and construct a phylogenetic tree. The most prevalent HPV genotypes were HPV16 (63.9%), HPV18 (26.7%), and other HPV (6.9%). HPV16 alterations were found in all E6, E7, and L1 genes, including 15 missense and 18 synonymous mutations. Missense mutations include R10G, Q14H, D25E, H78Y, L83V (E6); M29V, R35K, L78R, L95P (E7); H73Y, T176 N, N178T, T317P, T386S, L472F/I (L1). HPV16 sublineages include A1 (17.2%), A2 (0.9%), A3 (56.0%), A4 (19.0%), D1 (4.3%), and D3 (2.6%). Although several mutations in the oncoproteins E6, E7, and L1 have been detected, mutations known to be associated with cervical cancer risk, such as D25E and L83V, occur at a relatively low frequency. This suggests that HPV16 mutations are associated with cervical cancer through a complicated mechanism.

CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 or E7 induces senescence in cervical cancer cells

Human papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, anogenital and oropharyngeal sites. Continuous expression of the HPV oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 or E7 genes can potentially treat HPV-related cancers. Here we report that CRISPR/Cas9-based knockout of E6 or E7 can trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, E6 or E7-inactivated HeLa cells exhibited characteristic senescence markers like enlarged cell surface area, increased β-galactosidase expression and loss of lamin B1. Since E6 and E7 are bicistronic transcripts, inactivation of HPV18 E6 resulted in knockout of both E6 and E7 and increasing levels of p53/p21 and pRb/p21, respectively. Knockout of HPV18 E7 resulted in decreased E6 expression with activation of pRb/p21 pathway. Taken together, our study demonstrates cellular senescence as an alternative outcome of HPV oncogene inactivation by CRISPR/Cas9.