Deregulation of eIF4E: 4E-BP1 in Differentiated Human Papillomavirus-Containing Cells Leads to High Levels of Expression of the E7 Oncoprotein

Center for Molecular Biology of Oral Diseases, College of Dentistry, M/C 860, University of Illinois at Chicago, 801 South Paulina Street, 60612, USA.
Journal of Virology (Impact Factor: 4.44). 08/2006; 80(14):7079-88. DOI: 10.1128/JVI.02380-05
Source: PubMed


Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.

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Available from: Kwang-Jin Oh, Jan 29, 2014
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    • "For analyzing the half-life of Rb in differentiating cells, the Caski cells were grown in semi-suspension condition in medium containing 1.6% methylcellulose for 14 h (Reusch et al., 1998). Incubation of Caski cells for 14 h in the semisolid medium induces growth arrest and differential expression of keratin linked with differentiation (Oh et al., 2006). The Rb proteolysis was enhanced in differentiating cells, and the half-life of Rb was reduced to less than 2 h (Fig. 3B). "
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    ABSTRACT: The HPV oncoprotein E7 promotes proteasomal degradation of the tumor suppressor protein Rb. In this study, we analyzed the regulation of E7-induced Rb proteolysis in HPV-containing Caski cervical cancer cells. We show that the Rb proteolysis is cell cycle dependent; in S phase Rb is stable while in post-mitotic early G1 phase cells and in differentiated cells, Rb is unstable. Similarly, the in vivo Rb/E7 interaction is not detected in S-phase cells, but is readily detected in differentiating Caski cells. The ubiquitinating enzymes involved in Rb proteolysis have not been identified. We find that the E3 ligase MDM2 is not involved in the Rb proteolysis in Caski cells. An in vivo analysis using multiple catalytic site mutant dominant negative E2 enzymes show that the C92A E2-25K most effectively blocks E7-induced Rb proteolysis. Taken together, these results show that E7 induces Rb proteolysis in growth-arrested cells and E2-25K is involved in the proteolysis.
    Virology 01/2010; 396(1-396):118-124. DOI:10.1016/j.virol.2009.10.018 · 3.32 Impact Factor
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    • "This sequence accords with our morphoproteomic findings of increased plasmalemmal expression of EGFR in HSIL and invasive SCC of the uterine cervix and with the correspondingly increased cell cycle activity, as described above. In addition, the constitutive activation and overexpression of the mTOR pathway in HSIL and invasive SCC in this study should result in the phosphorylative inactivation of 4E-BP1 and thereby, the release of eIF-4E to effect translational synthesis of proteins to include viral oncoprotein E7 from their corresponding messenger RNA [6]. As noted earlier, E7 oncoprotein has been directly linked to the growth and survival (immortalization) of the HPV-associated cancer cells [2] [3] [4] [5]. "
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    ABSTRACT: Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.
    International journal of clinical and experimental pathology 01/2009; 2(3):249-60. · 1.89 Impact Factor
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    • "Whilst there is strong evidence that human papillomavirus (HPV) is the principle aetiological agent in cervical neoplasia [13] [27] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64], some other sexually transmitted organisms may either contribute to or protect against cervical carcinogenesis [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] (Figure 1). "
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    ABSTRACT: Whilst there is strong evidence that human papillomavirus (HPV) is the principal aetiological agent in cervical neoplasia, some other sexually transmitted agents may either contribute or protect against cervical carcinogenesis, such as the herpes virus family (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) or Chlamydia trachomatis (CT). Epidemiological studies suggest that HSV may have a role in cervical neoplasia, but there is no clear supportive experimental evidence. Serological studies have also failed to reveal a difference in the prevalence of antibodies to CMV and EBV between patients with cervical cancer and controls. However, longitudinal seroepidemiological studies have provided evidence that CT is an independent risk factor for the development of cervical squamous carcinoma and this association is serotype specific. The increased risk of cervical neoplasia in patients infected with HIV has been recognised for over a decade and HIV may interact with HPV either by alternating HPV gene transcription or by immunosuppression. Finally extensive experimental and limited epidemiological evidence suggests that adeno-associated viruses (AAV) may have antioncogenic activity in man and may protect against the development of cervical cancer. At present the mechanism of this action is unclear but may relate to AAV-induced regulation of HPV gene expression and the HPV life cycle. In this review we summarize the current literature relating to the associations and mechanisms of cervical carcinogenesis by each of these infectious microorganisms.
    International journal of clinical and experimental pathology 02/2008; 2(1):48-64. · 1.89 Impact Factor
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