© 2014. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and WNT signaling in a
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rodent model
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Sadia Benamrouz 1, 2, Valerie Conseil 1, 2, Magali Chabé 2, 3, Marleen Praet 4, Christophe Audebert 6, 7, Renaud Blervaque 5, 6, Karine Guyot 2, Sophie Gazzola 2, Anthony Mouray 8, Thierry Chassat 8, Baptiste Delaire 9, Nathalie Goetinck 10, Nausicaa Gantois 2, Marwan Osman 2,11, Christian Slomianny 12 , Vanessa Dehennaut 13, Tony Lefebvre 13, Eric Viscogliosi 2, Claude Cuvelier 4, Eduardo Dei-Cas 2, 10, Colette Creusy 9, Gabriela Certad 2
Accepted manuscript
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Ecologie et biodiversité, Faculté Libre des Sciences et Technologies de Lille, Université Catholique de Lille, Université Lille Nord de France, Lille, France.
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Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d'Infection et d'Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8402, Université Lille Nord de France.
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Faculté de Pharmacie, Université Lille Nord de France, Lille, France.
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Academic Department of Pathology, Ghent University, Ghent, Belgium.
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Disease Models & Mechanisms DMM
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Transcriptomic and Applied Genomic (TAG), Centre d'Infection et d'Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8404, Université Lille Nord de France.
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PEGASE-Biosciences, Institut Pasteur de Lille, Lille, France.
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Gene Diffusion, Douai, France
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Plateforme d'Expérimentations et de Hautes Technologies Animales, Institut Pasteur de Lille, Lille, France.
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Service d’Anatomie et de Cytologie Pathologiques, Groupe Hospitalier de l’Université Catholique de Lille, France.
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Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, France.
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Centre AZM pour la Recherche en Biotechnologie et ses Applications, Laboratoire Microbiologie, Sante´ et Environnement, Universite´ Libanaise, Tripoli, Lebanon
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Inserm U1003, Laboratoire de Physiologie Cellulaire, Université Lille 1, 59655 Villeneuve d'Ascq cedex, France.
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Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, IFR 147, Université Lille1, Villeneuve d'Ascq, France.
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* Corresponding author : Dr Gabriela Certad - Biologie et Diversité des Pathogènes Eucaryotes Émergents, Institut Pasteur de Lille, 1, rue du Prof. Calmette, BP245, 59019 Lille, France. Tel : 33 320 87 71 57, Fax : 33 320 87 72 76 /
[email protected]
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DMM Advance Online Articles. Posted 20 March 2014 as doi: 10.1242/dmm.013292 Access the most recent version at http://dmm.biologists.org/lookup/doi/10.1242/dmm.013292
Accepted manuscript Disease Models & Mechanisms DMM
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SUMMARY
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Cryptosporidium species are worldwide spread apicomplexan protozoan. These parasites
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constitute a significant risk to humans and animals. They cause self-limited diarrhea in
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immunocompetent hosts and a life threatening disease in immunocompromised hosts.
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Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in
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humans. Consistently with a potential tumorigenic role of this parasite, in an original
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reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or
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untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and
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human origin) is able to induce digestive adenocarcinoma even in infections induced with
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very low inoculum. The aim of this study was to further characterize this animal model and to
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explore metabolic pathways potentially involved in the development of C. parvum-induced
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ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved
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in cell cycle, differentiation or cell migration, such as β-catenin, Apc, E-cadherin, Kras and
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p53. After infection of animals with C. parvum we demonstrated immunohistochemical
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abnormal localization of Wnt signaling pathway components and p53. Mutations in the
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selected loci of studied genes were not found after high-throughput sequencing. Furthermore,
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alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of
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C. parvum infected mice were recorded using transmission electron microscopy. In
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conclusion, we found for the first time that the Wnt signaling pathway, and particularly the
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cytoskeleton network seems to be pivotal for the development of C. parvum-induced
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neoplastic process and cell migration of transformed cells. Furthermore, this model is a
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valuable tool to contribute to the comprehension of the host-pathogen interactions associated
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to the intricate infection process due to this parasite, which is able to modulate host
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cytoskeleton activities and several host-cell biological processes and that remains a significant
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cause of infection worldwide.
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Accepted manuscript
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INTRODUCTION
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Cryptosporidium species are worldwide spread apicomplexan parasitic protists. The infection
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results from the ingestion of Cryptosporidium oocysts through the consumption of fecally
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contaminated food or water or through direct person-to-person or animal-to-person contact
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(Chalmers and Katzer, 2013). This ubiquitous, intracellular parasite constitutes a significant
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health risk to humans and animals. It causes self-limited diarrhea in immunocompetent
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persons and a life threatening disease in immunocompromised persons (Ramirez et al., 2004).
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Contaminated water is the major source of Cryptosporidium infections for humans. Large-
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scale outbreaks of human cryptosporidiosis were reported, often implicating contaminated
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drinking or recreational water (Ramirez et al., 2004,Rowan, 2011,Yoder and Beach, 2010).
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The ingestion of as few as ten oocysts can cause infection in immunocompetent persons
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(Okhuysen et al., 1999). This low infection threshold, together with the well-known resistance
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of Cryptosporidium oocysts to chlorine disinfection at concentrations typically applied in
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drinking water plants, facilitate the waterborne transmission of cryptosporidiosis (Rowan,
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2011,Yoder and Beach, 2010).
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Nevertheless, key aspects of cryptosporidiosis remain unclear. For this reason, to contribute to
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the understanding of the dynamics of the infection, we formerly developed an animal model
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of cryptosporidiosis using dexamethasone-treated or untreated adult SCID mice orally
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infected with Cryptosporidium parvum or C. muris oocysts. Unexpectedly, we observed that
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C. parvum-infected SCID mice developed digestive adenocarcinoma (Certad et al., 2007).
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Low or high grade intraepithelial neoplasia and invasive adenocarcinoma associated with
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numerous C. parvum life stages were detected in the digestive tract of SCID mice, including
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stomach, ileo-caecal region and intrahepatic biliary tree (Certad et al., 2012,Certad et al.,
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2010a,Certad et al., 2010b,Certad et al., 2007). A highly significant correlation was found
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between the extension of cryptosporidiosis and the severity of neoplastic lesions (Certad et al.,
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2010b).
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Further analysis allow us to show that different strains of C. parvum isolated from either
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animals or humans, induced digestive neoplasia in this rodent model (Certad et al.,
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2012,Certad et al., 2010a,Certad et al., 2010b,Certad et al., 2007), even in infections induced
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with very low inoculum sizes (1 – 10 oocysts) (Benamrouz et al., 2012). On the other hand, in
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the same experimental model, the species C. muris with gastric tropism (infecting mice and
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Accepted manuscript Disease Models & Mechanisms DMM
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humans) did not induce that type of epithelial cell transformation independently of the grade
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of immunosupression (Certad et al., 2010b).
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Consistently, different evidences have shown direct or indirect association between
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cryptosporidiosis and cancer in different human populations: In a study among HIV-infected
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subjects known to be highly susceptible to Cryptosporidium infections, the incidence of
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colorectal cancer was found to be higher than in the general population (Patel et al., 2008).
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Another study reported that the risk of developing a colon carcinoma is significantly elevated
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among AIDS patients presenting cryptosporidiosis (Shebl et al., 2012). A possible association
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between human cryptosporidiosis and liver cancer was suggested in children with X-linked
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hyper-IgM syndrome (Tomizawa et al., 2004). Two epidemiological studies in Poland
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reported a frequency of 18% and 12.6% of cryptosporidiosis in patients with colorectal cancer
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of recent diagnosis before any immunosuppressive treatment (Sulżyc-Bielicka et al.,
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2012,Sulzyc-Bielicka et al., 2007).
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As well, it has been shown experimentally that C. parvum infection alters gene profile
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expression of the host cell. These altered genes include those associated to apoptosis such as
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BCL2 and the c-Myc proto-oncogene (Liu et al., 2009), proinflammatory signaling cascades
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and cytoskeletal dynamics (Deng et al., 2004). Nevertheless, even if we can hypothesize that
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the acquired transformed phenotype of Cryptosporidium infected epithelial cells is a
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consequence of modulation of cell signaling by the parasite, to our knowledge, no data about
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the mechanism of C. parvum induced neoplasia are available.
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The present work belongs to a series of experiments exploring the ability of C. parvum to
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induce neoplastic changes in the digestive epithelium of the animal model. The aim of this
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study was to further characterize this animal model and to explore metabolic pathways
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potentially involved in the development of ileo-caecal neoplasia induced by C. parvum
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infection, which is to our knowledge the first parasitic protiste able to induce epithelial
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invasive neoplasia in mammals (Benamrouz et al., 2012,Certad et al., 2012,Certad et al.,
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2010a,Certad et al., 2010b,Certad et al., 2007). We searched for alterations in genes or
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proteins commonly involved in cell cycle, differentiation or cell migration, such as β-catenin
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and Apc (components of the Wnt signaling pathway), the Kras oncogene and p53 by
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molecular and immunohistochemical approaches.
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Accepted manuscript Disease Models & Mechanisms DMM
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RESULTS
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Development of intraepithelial neoplasia and adenocarcinoma
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The histopathological study showed the development of neoplasia of different grades of
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severity.
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adenocarcinoma. A total of 27 Dex-treated SCID mice were successfully infected with
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different strains of C. parvum (IOWA: 20 mice, TUM1: 4 mice and IIaA15G2R1 human
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isolate: 3 mice).
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At histological examination of the ileo-caecal region we observed LGIEN in 2/19 mice. The
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detected lesions were characterized by slight modified mucosal architecture, including
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irregular glands lined by cells with slight atypias and containing minimal mucin or depletion.
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In 8/19 animals, we discovered exophytic adenomas showing an increasing architectural
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distortion, glandular crowding and major cellular atypias (HGIEN). In 8/19 mice,
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adenocarcinoma processes invading the submucosae though the muscularis mucosae were
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observed. In 1/19 mouse we observed a well differentiated adenocarcinoma invading the inner
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layer of the muscularis. In general, lesions showed a gradual progression from LGIEN to
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HGIEN and invasive well-differentiated adenocarcinoma progressing into the lamina propria
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(intramucosal carcinoma), into the submucosa and through the muscularis-mucosae into the
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subserosa. The severity of lesions increased steadily according to the delay P.I.. Those
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neoplastic lesions were accompanied by a diffuse inflammatory cell infiltrate, particularly in
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mice infected with TUM1 and II2A15G2R1 strains. In summary, the incidence of ileo- caecal
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neoplasia was present in 19/19 (100%) infected animals euthanatized after 40 days P. I.
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Apc labeling
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In the mucosa of ileo-caecal regions of uninfected control mice, Apc cytoplasmic
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immunoreactivity was detected in all animals. In 100% of infected animals presenting
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LGIEN, HGIEN or adenocarcinoma, gradual decrease of the intensity of cytoplasmic Apc
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labeling after infection with diverse C. parvum strains was recorded in the lesions, while
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contiguous normal mouse tissue showed a staining pattern similar to that seen in normal tissue
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(Fig. 1). The decrease of Apc labeling was observed after 25 days P.I and was found in lesions
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with either intraepithelial neoplasia or invasive adenocarcinoma (Table 1). An association
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between loss of Apc labeling and a longer time P.I. was extremely significant (p