Merkel cell polyomavirus small T antigen is oncogenic in transgenic mice - PubMed (original) (raw)

. 2015 May;135(5):1415-1424.

doi: 10.1038/jid.2014.446. Epub 2014 Oct 14.

Doris Mangelberger # 1, Paul W Harms 1 2, Tracy D Vozheiko 1, Jack W Weick 1, Dawn M Wilbert 1, Thomas L Saunders 3, Alexandre N Ermilov 1, Christopher K Bichakjian 1, Timothy M Johnson 1 4 5, Michael J Imperiale 6, Andrzej A Dlugosz 1 7

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Merkel cell polyomavirus small T antigen is oncogenic in transgenic mice

Monique E Verhaegen et al. J Invest Dermatol. 2015 May.

Abstract

Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor frequently associated with clonal integration of a polyomavirus, Merkel cell polyomavirus (MCPyV), and MCC tumor cells express putative polyomavirus oncoprotein small T antigen (sTAg) and truncated large T antigen. Here, we show robust transforming activity of sTAg in vivo in a panel of transgenic mouse models. Epithelia of preterm sTAg-expressing embryos exhibited hyperplasia, impaired differentiation, increased proliferation, and apoptosis, and activation of a DNA damage response. Epithelial transformation did not require sTAg interaction with the protein phosphatase 2A protein complex, a tumor suppressor in some other polyomavirus transformation models, but was strictly dependent on a recently described sTAg domain that binds Fbxw7, the substrate-binding component of the Skp1/Cullin1/F-box protein ubiquitin ligase complex. Postnatal induction of sTAg using a Cre-inducible transgene also led to epithelial transformation with development of lesions resembling squamous cell carcinoma in situ and elevated expression of Fbxw7 target proteins. Our data establish that expression of MCPyV sTAg alone is sufficient for rapid neoplastic transformation in vivo, implicating sTAg as an oncogenic driver in MCC and perhaps other human malignancies. Moreover, the loss of transforming activity following mutation of the sTAg Fbxw7 binding domain identifies this domain as crucial for in vivo transformation.

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Figures

Figure 1

Figure 1. Epithelial transformation in pre-term mouse embryo expressing MCPyV sTAg

A) Transgenic cassette including bovine K5 promoter, wild-type MCPyV sTAg and IRES-tdTomato (RFP), designated K5-sTAg. B) Histology of indicated epithelia from pre-term control and K5-sTAg embryos. Rectangles indicate regions shown at higher magnification. Note increased thickness of epithelia in_K5-sTAg_ mice and loss or reduction of differentiated granular (white asterisk) and cornified (black asterisk) cell layers seen in controls. White arrowheads indicate pyknotic nuclei with condensed chromatin suggestive of apoptosis in_K5-sTAg_ epithelia; black arrowheads identify suprabasal mitotic figures, which are normally restricted to basal or immediate suprabasal cell layers. Scale bars = 25 μm.

Figure 2

Figure 2. MCPyV sTAg alters epithelial differentiation, proliferation, and apoptosis

A) H&E and RFP immunostaining of acral skin and tongue showing disorganized, hyperplastic epithelia and widespread transgene (RFP) expression. B) Expansion of the basal layer marker K5 and impaired expression of the granular cell marker loricrin in sTAg-expressing epithelia. C) Striking upward expansion of Ki67-expressing proliferating cells and phospho-histone H3 (pHH3)-expressing mitotic cells in sTAg-expressing epidermis. D) Immunostaining for the apoptosis marker CC3 and DNA damage-response marker γH2AX in sTAg epidermis. Scale bars = 25 μm.

Figure 3

Figure 3. Epithelial transformation by a PP2A binding-deficient sTAg mutant

A) K5-sTAgL142A transgene construction identical to_K5-sTAg_ in Figure 1A except for L142A substitution which blocks sTAg binding to PP2A. B) H&E and RFP immunostaining in sections from acral skin, tongue, and palate, showing features of transformation similar to those in K5-sTAg mice and robust transgene expression. RFP immunostaining is shown in insets. Scale bars = 50 μm.

Figure 4

Figure 4. Lack of epithelial transformation by an LTAg stabilization domain (LSD) sTAg mutant

A) Transgene construction identical to K5-sTAg in Figure 1A except for 91-95A substitutions which block sTAg binding to Fbxw7. B) Similar histology of acral skin and tongue in sections from control and_K5-sT91-95A_ transgenic mice, despite robust RFP expression in basal layer cells of transgenic mice. C) Similar profile of epidermal markers and proliferation, and lack of CC3 and γH2AX immunostaining in control and_K5-sT91-95A_ transgenic acral skin. Scale bars = 50 μm.

Figure 5

Figure 5. Postnatal activation of sTAg induces epidermal transformation in adult mice

A) Design of KLEsT transgene expressing eGFP and dormant sTAg, in which Cre-mediated recombination and GFP excision allow K5-driven sTAg expression. B,C) Phenotype of K5-CreER;KLEst (iK5;KLEsT) bitransgenic mice 2-3 weeks post tamoxifen treatment at P21. Note massive epidermal hyperplasia, disorganized stratification, suprabasal mitoses (arrowheads), and hyperkeratotic regions (asterisk). D) Quantification of increased epidermal thickness at indicated sites (N=3 for each column, error bars indicate SEM, **p<0.005, ***p<0.0003). E) Similarity of_iK5;KLEsT_ epidermal phenotype and human SCC in situ. Both lesions show a severely disorganized epithelium, pale-staining atypical epidermal cells (arrowheads), apoptotic cells (arrows), and hyperkeratosis (asterisk). F) Expression of sTAg in lysates from iK5;KLEsT mice collected 11 days (snout) and 21 days (tail) after tamoxifen treatment. sTAg was detected by immunoprecipitation and immunoblotting using 2t2 monoclonal Ab. Scale bars = 50 μm.

Figure 6

Figure 6. Postnatal activation of sTAg induces markers of epidermal transformation and accumulation of Fbxw7 target proteins

A) Expansion of proliferative cell layers (Ki67), increased apoptosis (CC3), and DNA damage (γH2AX) in tail epidermis of sTAg-expressing iK5;KLEsT mice three weeks after treatment with tamoxifen. B) Expansion of cell layers expressing K5 and focal reductions in expression of the differentiation markers K10 and loricrin. C) Upregulation of hyperplasia-associated keratins K6 and K17 in epidermis of_iK5;KLEsT_ mice. Normal expression of these keratins is largely restricted to the hair follicle. D) Immunoblotting for Fbwx7 targets, PCNA, K17, and GFP in sets of snout lysates from control and iK5;KLEsT mice, collected 24 days (left panels) or 28 days (right panels) after tamoxifen treatment. Loss of GFP expression confirms efficient recombination of the KLEsT transgene. Scale bars = 50 μm.

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