Expression of HPV16 E5 down-modulates the TGFbeta signaling pathway
- Deborah French†1, 2,
- Francesca Belleudi†1,
- Maria Vittoria Mauro1,
- Francesca Mazzetta1,
- Salvatore Raffa1, 2,
- Vincenza Fabiano1,
- Antonio Frega2 and
- Maria Rosaria Torrisi1, 2Email author
© French et al.; licensee BioMed Central Ltd. 2013
Received: 9 January 2013
Accepted: 26 April 2013
Published: 7 May 2013
Infection with high-risk human papillomavirus (HR-HPV) genotypes, mainly HPV16 and HPV18, is a major risk factor for cervical cancer and responsible for its progression. While the transforming role of the HPV E6 and E7 proteins is more characterized, the molecular mechanisms of the oncogenic activity of the E5 product are still only partially understood, but appear to involve deregulation of growth factor receptor expression. Since the signaling of the transforming growth factor beta (TGFbeta) is known to play crucial roles in the epithelial carcinogenesis, aim of this study was to investigate if HPV16 E5 would modulate the TGF-BRII expression and TGFbeta/Smad signaling.
The HPV16 E5 mRNA expression pattern was variable in low-grade squamous intraepithelial lesions (LSIL), while homogeneously reduced in high-grade lesions (HSIL). Parallel analysis of TGFBRII mRNA showed that the receptor transcript levels were also variable in LSILs and inversely related to those of the viral protein. In vitro quantitation of the TGFBRII mRNA and protein in human keratinocytes expressing 16E5 in a dose-dependent and time-dependent manner showed a progressive down-modulation of the receptor. Phosphorylation of Smad2 and nuclear translocation of Smad4 were also decreased in E5-expressing cells stimulated with TGFbeta1.
Taken together our results indicate that HPV16 E5 expression is able to attenuate the TGFbeta1/Smad signaling and propose that this loss of signal transduction, leading to destabilization of the epithelial homeostasis at very early stages of viral infection, may represent a crucial mechanism of promotion of the HPV-mediated cervical carcinogenesis.
KeywordsHuman papillomavirus HPV16 E5 TGFβ signaling TGFβRII SMAD
The infection with high-risk human papillomavirus (HR-HPV) genotypes, particularly the HPV16 and HPV18 viruses, is a major risk factor for cervical cancer and appears to be responsible for its progression [1–3]. While the transforming role of the HPV E6 and E7 proteins is well characterized, the molecular mechanisms of the oncogenic activity of the E5 product of the virus are still only partially understood for a recent review, see . HPV16 E5 expression is lost during tumor progression as a result of viral genome integration: we have recently reported that the E5 expression pattern is extremely variable in low-grade squamous intraepithelial lesions (LSIL), while it is reduced and more homogeneous in high-grade lesions (HSIL) . When expressed, the E5 protein is known to play critical roles at the early stages of infection by co-operation with E6 and E7 affecting proliferation, differentiation and apoptosis  and these multiple functions appear to be dependent on its ability to enhance the signaling pathways of mitogenic growth factor receptors such as the epidermal growth factor receptor (EGFR) . However, 16E5 protein is also capable to down-regulate the expression of receptors responsible for epithelial differentiation, such as the keratinocyte growth factor receptor (KGFR/FGFR2b), in order to perturb the physiological tissue homeostasis and cell stratification .
Signaling of the transforming growth factor β (TGFβ) is known to play crucial roles in the control of a number of key physiological cell processes, such as proliferation, differentiation, motility and death, and is involved in the pathogenesis of many different human diseases including cancer for a recent review, see . Members of the TGFβ family bind to cell surface heterodimers composed of type I and type II serine/threonine receptors (TGFβRI and TGFβRII) and this binding triggers an intracellular signal transduction mediated by SMAD proteins and regulated by their phosphorylation and activation for a recent review, see . The cellular response to TGFβ signaling is highly variable, ranging from tumor suppressive to tumor promoting functions, and depends on the cellular context and tissue microenvironment . Dysregulated expression and activity of TGFβ, TGFβRI/II and SMADs have been frequently described in human cancer in association with tumor progression . It has been proposed that altered expression of TGFβ might be involved in cervical carcinogenesis [10, 11] and that TGFβ1 promotes chromosomal instability in HPV-infected cervical cells . Interestingly, mice lacking TGFβRII develop spontaneous anal and genital squamous carcinomas , previewed in , demonstrating that the loss of TGFβ signaling promotes the carcinogenesis in those stratified epithelia, which represent the tissue targets of mucosal HPV infection. Consistent with the hypothesis that the molecular mechanisms of HPV transformation may involve the TGFβ/TGFβR axis, the E7 transforming protein of HPV16 is able to down-regulate the TGF-βRII expression and signaling in a E7 transgenic mouse model suggesting a key role of this pathway . Therefore, it is possible that, in early infection and in the LSIL/HSIL context, HPV E5 might exert its oncogenic activity through modulation of the TGF-βRII expression and TGFβ signaling.
Our present study was aimed to analyze in vivo the possible HPV E5-induced alteration of the TGFβ signaling pathway in human lesions as well as its in vitro modulation under the expression of the E5 oncogenic protein. Since the local tissue concentration of the cytokine TGFβ might be extremely variable, depending on the inflammatory and immunological microenvironment [11, 16], we focused both in vivo and in vitro on the expression of the receptor, since its levels are thought to strictly regulate the specificity of the TGFβ signaling and the biological activity of the cytokine . In addition, because the biological behaviour of human HaCaT keratinocytes is drastically affected by TGFβ/Smad signaling , we took advantage of this sensitive cellular model in which 16E5 expression alone can be induced by trasfection in dose and time controlled manner.
Findings and discussion
Modulation of HPV16 E5 and TGFβRII in LSILs and HSILs
16E5 expression induces TGFβRII down-modulation and attenuates TGFβ/Smad signaling
To further demonstrate the role of 16E5 expression in TGFβRII down-regulation, we transiently transfected HaCaT cells using increasing amounts (0.5 μg, 1 μg, 2 μg) of pCI-neo E5-HA expression vector  (HaCaT E5), in order to induce an increased expression of 16E5 in a dose-dependent manner (Figure 2B, left panel). HaCaT cells transfected with the empty vector pCI-neo (HaCaT pCI-neo) were used as negative control. Results demonstrated that the TGFβRII transcript levels progressively decreased with the increase of 16E5 mRNA amount (Figure 2A and B, right panels), demonstrating that 16E alone is able to down-modulate the receptor in a dose-dependent and time-dependent manner. Interestingly, this down-modulating effect appears more evident when the viral protein expression is obtained with 1 μg cDNA and is similar to the 16E5 expression in the W12 cell model, representing the most physiological condition.
We are very grateful to Dr. Anna Coppa for providing reagents and for helpful discussion. We thank Ms Silvia Caputo and Ms Cristina Scrofani for excellent technical assistance. This work was partially supported by grants from MIUR and from AIRC - Associazione Italiana per la Ricerca sul Cancro (IG 10272), Italy.
- Doorbar J: Papillomavirus life cycle organization and biomarker selection. Dis Markers. 2007, 23: 297-313.PubMed CentralView ArticlePubMedGoogle Scholar
- Woodman CB, Collins SI, Young LS: The natural history of cervical HPV infection: unresolved issues. Nat Rev Cancer. 2007, 7: 11-22. 10.1038/nrc2050View ArticlePubMedGoogle Scholar
- Stanley M: Pathology and epidemiology of HPV infection in females. Gynecol Oncol. 2010, 117: S5-S10. 10.1016/j.ygyno.2010.01.024View ArticlePubMedGoogle Scholar
- Moody CA, Laimins LA: Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer. 2010, 10: 550-560. 10.1038/nrc2886View ArticlePubMedGoogle Scholar
- Lorenzon L, Mazzetta F, Venuti A, Frega A, Torrisi MR, French D: In vivo HPV 16 E5 mRNA: expression pattern in patients with squamous intra-epithelial lesions of the cervix. J Clin Virol. 2011, 52: 79-83. 10.1016/j.jcv.2011.06.007View ArticlePubMedGoogle Scholar
- Venuti A, Paolini F, Nasir L, Corteggio A, Roperto S, Campo MS, Borzacchiello G: Papillomavirus E5: the smallest oncoprotein with many functions. Mol Cancer. 2011, 10: 140- 10.1186/1476-4598-10-140PubMed CentralView ArticlePubMedGoogle Scholar
- Belleudi F, Leone L, Purpura V, Cannella F, Scrofani C, Torrisi MR: HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic. Oncogene. 2011, 30: 4963-4976. 10.1038/onc.2011.203View ArticlePubMedGoogle Scholar
- Drabsch Y, ten Dijke P: TGF-beta signalling and its role in cancer progression and metastasis. Cancer Metastasis Rev. 2012, 31: 553-568. 10.1007/s10555-012-9375-7View ArticlePubMedGoogle Scholar
- Zi Z, Chapnick DA, Liu X: Dynamics of TGF-beta/Smad signaling. FEBS Lett. 2012, 586: 1921-1928. 10.1016/j.febslet.2012.03.063PubMed CentralView ArticlePubMedGoogle Scholar
- Baritaki S, Sifakis S, Huerta-Yepez S, Neonakis IK, Soufla G, Bonavida B, Spandidos DA: Overexpression of VEGF and TGF-beta1 mRNA in Pap smears correlates with progression of cervical intraepithelial neoplasia to cancer: implication of YY1 in cervical tumorigenesis and HPV infection. Int J Oncol. 2007, 31: 69-79.PubMedGoogle Scholar
- Peghini BC, Abdalla DR, Barcelos AC, Teodoro L, Murta EF, Michelin MA: Local cytokine profiles of patients with cervical intraepithelial and invasive neoplasia. Hum Immunol. 2012, 73: 920-926. 10.1016/j.humimm.2012.06.003View ArticlePubMedGoogle Scholar
- Deng W, Tsao SW, Kwok YK, Wong E, Huang XR, Liu S, Tsang CM, Ngan HY, Cheung AN, Lan HY: Transforming growth factor beta1 promotes chromosomal instability in human papillomavirus 16 E6E7-infected cervical epithelial cells. Cancer Res. 2008, 68: 7200-7209. 10.1158/0008-5472.CAN-07-6569View ArticlePubMedGoogle Scholar
- Guasch G, Schober M, Pasolli HA, Conn EB, Polak L, Fuchs E: Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia. Cancer Cell. 2007, 12: 313-327. 10.1016/j.ccr.2007.08.020PubMed CentralView ArticlePubMedGoogle Scholar
- Wakefield LM, Stuelten C: Keeping order in the neighborhood: new roles for TGFbeta in maintaining epithelial homeostasis. Cancer Cell. 2007, 12: 293-295. 10.1016/j.ccr.2007.10.002View ArticlePubMedGoogle Scholar
- Diaz-Chavez J, Hernandez-Pando R, Lambert PF, Gariglio P: Down-regulation of transforming growth factor-beta type II receptor (TGF-betaRII) protein and mRNA expression in cervical cancer. Mol Cancer. 2008, 7: 3- 10.1186/1476-4598-7-3PubMed CentralView ArticlePubMedGoogle Scholar
- Boccardo E, Lepique AP, Villa LL: The role of inflammation in HPV carcinogenesis. Carcinogenesis. 2010, 31 (11): 1905-1912. 10.1093/carcin/bgq176View ArticlePubMedGoogle Scholar
- Rojas A, Padidam M, Cress D, Grady WM: TGF-beta receptorlevels regulate the specificity of signaling pathway activation and biological effects of TGF-beta. Biochim Biophys Acta. 2009, 1793 (7): 1165-1173. 10.1016/j.bbamcr.2009.02.001PubMed CentralView ArticlePubMedGoogle Scholar
- Buschke S, Stark HJ, Cerezo A, Pratzel-Wunder S, Boehnke K, Kollar J, Langbein L, Heldin CH, Boukamp P: A decisive function of transforming growth factor–β/SMAD signaling in tissue morphogenesis and differentiation of human HaCaT keratinocytes. Mol Biol Cell. 2011, 22 (6): 782-794. 10.1091/mbc.E10-11-0879PubMed CentralView ArticlePubMedGoogle Scholar
- Stanley MA, Browne HM, Appleby M, Minson AC: Properties of a non-tumorigenic human cervical keratinocyte cell line. Int J Cancer. 1989, 43: 672-676. 10.1002/ijc.2910430422View ArticlePubMedGoogle Scholar
- Pett MR, Alazawi WO, Roberts I, Dowen S, Smith DI, Stanley MA, Coleman N: Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes. Cancer Res. 2004, 64: 1359-1368. 10.1158/0008-5472.CAN-03-3214View ArticlePubMedGoogle Scholar
- Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE: Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988, 106: 761-771. 10.1083/jcb.106.3.761View ArticlePubMedGoogle Scholar
- Oelze I, Kartenbeck J, Crusius K, Alonso A: Human papillomavirus type 16 E5 protein affects cell-cell communication in an epithelial cell line. J Virol. 1995, 69: 4489-4494.PubMed CentralPubMedGoogle Scholar
- Ashrafi GH, Haghshenas MR, Marchetti B, O'Brien PM, Campo MS: E5 protein of human papillomavirus type 16 selectively downregulates surface HLA class I. Int J Cancer. 2005, 113: 276-283. 10.1002/ijc.20558View ArticlePubMedGoogle Scholar
- Wetherill LF, Holmes KK, Verow M, Muller M, Howell G, Harris M, Fishwick C, Stonehouse N, Foster R, Blair GE, Griffin S, Macdonald A: High-risk human papillomavirus E5 oncoprotein displays channel-forming activity sensitive to small-molecule inhibitors. J Virol. 2012, 86: 5341-5351. 10.1128/JVI.06243-11PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.