pyrimidinones and Papillomavirus-Infections

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.

Topics: CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; DNA Damage; Forkhead Box Protein M1; Head and Neck Neoplasms; Humans; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Repressor Proteins; Squamous Cell Carcinoma of Head and Neck; Up-Regulation

Although the majority of patients with HPV(+) oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV(+) head and neck squamous cell carcinoma (HNSCC).. Clonogenic survival assays and an orthotopic mouse model of HPV(+) oral cancer were used to examine the in vitro and in vivo sensitivity of HPV(+) HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms.. We found that AZD-1775 displays single-agent activity and enhances the response of HPV(+) HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV(+) HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV(+) HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV(-) HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment.. AZD-1775 selectively sensitizes HPV(+) HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV(+) HNSCC tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, p53; Head and Neck Neoplasms; Humans; Inhibitory Concentration 50; Male; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nuclear Proteins; Papillomavirus Infections; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Squamous Cell Carcinoma of Head and Neck; Tumor Burden; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

Non-surgical, antiviral treatment options are desirable for HPV-related lesions within the genitourinary and upper digestive tract. We compared the toxicity of three zinc finger-ejecting (ZFE) compounds (4,4-dithiodimorpholine, azodicarbonamide, and diamide) to the HIV protease inhibitor lopinavir using HPV-positive SiHa, CaSki, HeLa, ME180, and HPV-negative C33A cervical carcinoma cell lines as well as primary human foreskin keratinocytes (PHFKs). Colorimetric growth assays revealed selective toxicity when treated with lopinavir. All carcinoma cell lines, except CaSki, were sensitive to 20 μM lopinavir whereas primary PHFKs were highly resistant. In contrast, 4,4-dithiodimorpholine was uniformly toxic to all cells tested while azodicarbonamide and diamide showed no effect at all. It is concluded that lopinavir may be an attractive candidate to treat pre-cancerous and cancerous HPV-positive lesions.

Topics: Antiviral Agents; Azo Compounds; Cell Line, Tumor; Diamide; Female; HIV Protease Inhibitors; Humans; Keratinocytes; Lethal Dose 50; Lopinavir; Microbial Sensitivity Tests; Morpholines; Papillomaviridae; Papillomavirus Infections; Pyrimidinones

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism.. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used.. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression.. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

Topics: Antiviral Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Endoribonucleases; Female; HIV Protease Inhibitors; Human papillomavirus 16; Humans; Lopinavir; Papillomavirus Infections; Pyrimidinones; Up-Regulation; Uterine Cervical Neoplasms

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Topics: Cell Fractionation; Cell Line, Tumor; Chromatography, Liquid; Female; HIV Protease Inhibitors; Human papillomavirus 16; Humans; Indinavir; Lopinavir; Oncogene Proteins, Viral; Papillomavirus Infections; Principal Component Analysis; Pyrimidinones; Repressor Proteins; Spectrometry, Mass, Electrospray Ionization; Spectrum Analysis, Raman; Transfection; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

HPV infections have become a major problem in immunocompromised patients, particularly in HIV-positive subjects. HPV lesions are observed more frequently in the ano-genital area and rarely in different body areas, such as the skin and oral cavity. However, in HIV-positive subjects there is an increased risk of oral condylomas. We describe the case of an HIV-positive Nigerian young woman, who came to our notice due to the appearance of small labial and mouth mucous membrane lesions, related to HPV infection, as shown by a biopsy. These lesions were not evident in the genital area. After two years in which the patient no longer received therapy, there was a progressive reduction in CD4 count, associated with the development of the oral condylomas. Hence the patient began a new HAART combination, but after seven months, although a slight improvement emerged in the CD4 count with the disappearance of HIV-RNA, there has been no regression of oral condylomas.

Topics: Adenine; Adult; Alphapapillomavirus; Anti-HIV Agents; Antiretroviral Therapy, Highly Active; Deoxycytidine; Dideoxynucleosides; Emtricitabine; Female; HIV Infections; Humans; Italy; Lamivudine; Lopinavir; Mouth Mucosa; Nigeria; Organophosphonates; Papillomavirus Infections; Pyrimidinones; Ritonavir; Sex Work; Stomatitis; Tenofovir; Treatment Refusal; Zidovudine