leupeptins and Papillomavirus-Infections

Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells.. To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment.. RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells.. Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

Topics: Animals; Apoptosis; Aurora Kinase A; Aurora Kinase B; Azepines; Female; Head and Neck Neoplasms; Human papillomavirus 16; Humans; Leupeptins; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Papillomavirus E7 Proteins; Papillomavirus Infections; Proteolysis; Pyrimidines; RNA Interference; Xenograft Model Antitumor Assays

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Female; HeLa Cells; Humans; Leupeptins; Membrane Glycoproteins; Papillomaviridae; Papillomavirus Infections; Proteasome Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Recombinant Proteins; RNA Interference; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

The group of mucosal epithelia-infecting human papillomaviruses (HPV) can be subdivided in "low" and "high risk" HPV types. Both types induce benign neoplasia (condyloma), but only the infection with a "high risk" HPV type is causally associated with an increased risk of developing anogenital tumors. The oncogenic potential of high risk HPVs resides at least partially in the viral E6 protein. The E6 protein targets the cellular p53 protein for proteasome-dependent degradation, which is associated with the immortalizing and transforming functions of these viruses. Recently the E6-dependent proteasome-mediated destabilization of additional cellular proteins (E6TP1, c-myc, Bak, hMCM7, human scribble, E6AP, MAGI-1) has been described, but the cellular mechanisms controlling the viral E6 protein stability itself have been so far not analyzed. In this study, we transiently expressed the E6 genes of the high risk HPV type 16, the low risk HPV types 6a and 11, and the cutaneous epithelia-infecting HPV types 5 and 8 from a eucaryotic expression vector and compared the cellular steady-state levels of the expressed E6 proteins. We demonstrated that the high risk HPV 16 E6 protein possesses the lowest steady-state level in comparison to the low risk HPV type E6 proteins and the cutaneous epithelia-infecting HPV type E6 proteins. Inhibition of cellular proteasome-dependent protein degradation led to an increase in steady-state levels of high risk but not of low risk E6 proteins. Analysis of functionally deficient HPV 16 E6 proteins in p53 null- and p53 wild-type-expressing cell lines revealed that the cellular steady-state level of this protein is influenced neither by its p53- nor its E6AP-binding abilities.

Topics: Animals; Antineoplastic Agents; Chlorocebus aethiops; COS Cells; Cysteine Endopeptidases; Gene Deletion; Humans; Leupeptins; Ligases; Multienzyme Complexes; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Proteasome Endopeptidase Complex; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Virus Infections; Ubiquitin-Protein Ligases

The bovine papillomavirus E2 proteins regulate viral transcription, replication, and episomal genome maintenance. We have previously mapped the major phosphorylation sites of the E2 proteins to serine residues 298 and 301 and shown that mutation of serine residue 301 to alanine leads to a dramatic (10- to 20-fold) increase in viral DNA copy number. In this study we analyzed how phosphorylation regulates E2 protein function. S301 is located in a PEST sequence; these sequences are often found in proteins with a short half-life and can be regulated by phosphorylation. We show here that the E2 protein is ubiquitinated and degraded by the proteasome. Mutation of serine 301 to alanine increases the half-life of E2 from approximately 50 min to 160 min. Furthermore, the A301 E2 protein shows greatly reduced ubiquitination and degradation by the proteasome. These results suggest that the E2 protein level is regulated by phosphorylation, which in turn determines viral episomal copy number.

Topics: Alanine; Animals; Bovine papillomavirus 1; Cattle; Cell Line; Chlorocebus aethiops; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; DNA, Viral; Gene Dosage; Genome, Viral; Lactones; Leupeptins; Multienzyme Complexes; Mutagenesis, Site-Directed; Papilloma; Papillomavirus Infections; Phosphorylation; Proteasome Endopeptidase Complex; Serine; Viral Proteins; Warts

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

Topics: Acetylcysteine; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Humans; Leupeptins; Neuroblastoma; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Proto-Oncogene Proteins c-myc; Signal Transduction; Tumor Cells, Cultured; Tumor Virus Infections; Ubiquitins