Human papillomavirus type 16 E6 amino acid 83 variants enhance E6-mediated MAPK signaling and differentially regulate tumorigenesis by notch signaling and oncogenic Ras

Oncogenically high-risk human papillomaviruses (HPVs) are causally associated with the progression of major human neoplasia-like cancers of the cervix. Several studies have defined functions of the key E6 and E7 oncoproteins in epithelial cell immortalization. The roles of these oncogenes in the progression of immortalized epithelial cells to invasive tumors are still poorly understood. Here, we establish a novel link between the E6 oncoprotein and activation of mitogen-activated protein kinase (MAPK) signaling and show that this signaling involves Rap1. We find that activated MAPK signaling cooperates with deregulated Notch1 signaling to recreate features of HPV-driven invasive cervical carcinomas. We extend our analysis to evaluate an E6 (amino acid [aa] 83) variant that has been linked to invasive tumors. The variant enhances MAPK signaling and cooperative transformation with deregulated Notch1 signaling. Unlike E6, this variant surprisingly inhibits oncogenic Ras-mediated transformation. Our data reveal that the quantitative differences in activation of MAPK signaling by E6 and its variant correlate with differences in cooperative transformation with other signaling pathways, thus suggesting that thresholds of MAPK activation may define permissive conditions for other signaling pathways in tumorigenesis. Epidemiological studies have suggested the importance of E6 aa 83 variants in invasive carcinomas; our data support a key deterministic role for this variant in human cervical tumorigenesis. These observations, along with our recent data showing that deregulated Notch signaling activates phosphatidylinositol 3-kinase signaling, strengthen the possibility of the existence of Ras-independent mechanisms to recreate signaling through classical Ras effector pathways.     

E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na(+)/H(+) exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3'-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway

Activated Notch1 (AcN1) alleles cooperate with oncogenes from DNA tumor viruses in transformation of epithelial cells. AcN1 signaling has pleiotropic effects, and suggested oncogenic roles include driving proliferation through cyclin D1 or the generation of resistance to apoptosis on matrix withdrawal through a phosphatidylinositol 3-kinase (PI3K)-PKB/Akt-dependent pathway. Here, we extend the antiapoptotic role for AcN1 by showing inhibition of p53-induced apoptosis and transactivation. Chemical inhibitors of the PI3K pathway block AcN1-induced inhibition of p53-dependent apoptosis and nuclear localization of Hdm2. We show that expression of wild-type p53 does not inhibit synergistic transformation by AcN1 and human papillomavirus E6 and E7 oncogenes. We suggest that activation of Notch signaling may serve as an additional mechanism to inhibit wild-type p53 function in papillomavirus-associated neoplasia.     

Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.     

Degradation of p53, not telomerase activation, by E6 is required for bypass of crisis and immortalization by human papillomavirus type 16 E6/E7

Bypass of two arrest points is essential in the process of cellular immortalization, one of the components of the transformation process. Expression of human papillomavirus type 16 E6 and E7 together can escape both senescence and crisis, processes which normally limit the proliferative capacity of primary human keratinocytes. Crisis is thought to be mediated by telomere shortening. Because E6 stimulates telomerase activity and exogenous expression of the TERT gene with E7 can immortalize keratinocytes, this function is thought to be important for E6 to cooperate with E7 to bypass crisis. However, it has also been reported that E6 dissociates increased telomerase activity from maintenance of telomere length and that a dominant-negative p53 molecule can substitute for E6 in cooperative immortalization of keratinocytes with E7. Thus, to determine which functions of E6 are required to allow bypass of crisis and immortalization of keratinocytes with E7, immortalization assays were performed using specific mutants of E6, in tandem with E7. In these experiments, every clone expressing an E6 mutant capable of degrading p53 was able to bypass crisis and immortalize, regardless of telomerase induction. All clones containing E6 mutants incapable of degrading p53 died at crisis. These results suggest that the ability of E6 to induce degradation of p53 compensates for continued telomere shortening in E6/E7 cells and demonstrate that degradation of p53 is required for immortalization by E6/E7, while increased telomerase activity is dispensable.     

Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation.

The transforming function of human papillomavirus type 16 (HPV16) E7 has been shown to depend on activities additional to the ability to bind RB. In this paper we describe two further properties of E7 which may also contribute to transformation, an association with a histone H1 kinase at the G2/M phase of the cell cycle and an ability to bind the RB-related protein p107. The region of E7 identified previously as important for RB binding was found to be involved in the association with the kinase and complex formation with p107, although analysis of E7 point mutants within this region revealed a difference in the precise sequence requirement for RB and p107 binding. Association with the kinase activity correlated with the ability to bind RB, but the restriction of the kinase association to the G2/M phase of the cell cycle implies that this activity might not be directly mediated by RB binding. Since kinase-binding-deficient E7 mutants are also transformation defective, this may represent an independent function of E7 which plays a role in the G2/M phase of the cell cycle.     

The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties.

The E7 early viral protein of the oncogenic human papillomavirus type 16 (HPV-16) has been strongly implicated in the maintenance of the malignant phenotype in cervical cancers and cancer-derived cell lines. HPV-16 E7 is a nuclear phosphoprotein that can cooperate with ras to transform baby rat kidney cells, transactivates the adenovirus E2 promoter, and binds to the retinoblastoma (RB) protein. The E7 phosphoprotein of the nononcogenic HPV-6b, which is generally associated with benign genital warts, is similar to the HPV-16 E7 in amino acid sequence but differs dramatically in migration in sodium dodecyl sulfate-polyacrylamide gels, sedimentation in nondenaturing glycerol gradients, and the ability to bind the RB protein. Our results indicate that the RB protein preferentially binds the phosphorylated form of HPV-6b E7, which comprises a minor fraction of the total E7 expressed in transiently transfected COS-7 cells. These characteristics may help to explain the difference in the oncogenic potential of the oncogenic and nononcogenic types of genital papillomaviruses.