ABSTRACT
High-risk human papillomavirus (HPV) types 16 and 18 are associated with more than 70% of cervical cancer cases. The oncoprotein E6 is multifunctional and has numerous cellular partners. The best-known activity of E6 is the polyubiquination of the pro-apoptotic tumor suppressor p53, targeting it for degradation by the 26S proteasome. Loss of p53 triggers genomic instability and favors cancer development. Here, we generated recombinant adenovirus (Ad) vectors expressing artificial microRNAs directed against HPV16 E6 (Ad16_1) or HPV18 E6 (Ad18_2). E6-knockdown was observed in HeLa after treatment with Ad18_2 and in SiHa with Ad16_1. Western-blot experiments found an increase in p53 levels after treatment in both cell lines. Cell death was observed in both cell lines after knockdown of E6. Further analysis such as cleavage of caspases (3 and 7) as well as of PARP1 indicated that treated HeLa and SiHa cells underwent apoptosis. The growth of HeLa-derived tumors developed in nude mice was significantly reduced after intra-tumoral injection of Ad18_2. Therefore, vectorisation of artificial miRNA against E6 oncoprotein by means of recombinant adenoviruses might represent a valuable therapeutic approach for treating HPV-positive cancers.
Subject(s)
Apoptosis/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , MicroRNAs/genetics , Oncogene Proteins, Viral/antagonists & inhibitors , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/therapy , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Uterine Cervical Neoplasms/therapy , Adenoviridae/genetics , Animals , Cell Line , Female , Gene Knockdown Techniques , Genetic Therapy , Genetic Vectors , HeLa Cells , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Human papillomavirus 18/genetics , Human papillomavirus 18/pathogenicity , Humans , Mice , Mice, Nude , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virology , Xenograft Model Antitumor AssaysABSTRACT
The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, with both functions being crucial for HPV-induced oncogenesis. A chimeric protein was built by fusing a PDZ domain and an LxxLL motif, both known to bind E6. NMR spectroscopy, calorimetry and a mammalian protein complementation assay converged to show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all of the hrm-HPV E6â proteins tested but not to low-risk mucosal or cutaneous HPVâ E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspaseâ 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers.