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Planarians have been long known for their regenerative ability, which hinges on pluripotency. Recently, however, the planarian model has been successfully established for routine toxicological screens aimed to assess overproliferation, mutagenicity and tumorigenesis. In this study, we focused on planarian tumor suppressor genes (TSGs) and their role during chemically induced carcinogenic stress in Schmidtea mediterranea. Combining in silico and proteomic screens with exposure to human carcinogen type 1A agent cadmium (Cd), we showed that many TSGs have a function in stem cells and that, in general, exposure to Cd accelerated the onset and increased the severity of the observed phenotype. This suggested that the interaction between environmental and genetic factors plays an important role in tumor development in S. mediterranea. Therefore, we further focused on the synergistic effects of Cd exposure and p53 knockdown (KD) at the cellular and molecular levels. Cd also produced a specific proteomic landscape in homeostatic animals, with 172 proteins differentially expressed, 43 of which were downregulated. Several of these proteins have tumor suppressor function in human and other animals, namely Wilms Tumor 1 Associated Protein (WT1), Heat Shock Protein 90 (HSP90), Glioma Pathogenesis-Related Protein 1 (GLIPR1) and Matrix Metalloproteinase B (Smed-MMPB). Both Glipr1 and MmpB KD produced large outgrowths, epidermal lesions and epidermal blisters. The epidermal blisters that formed as a consequence of Smed-MmpB KD were populated by smedwi1+ cells, many of which were actively proliferating, while large outgrowths contained ectopically differentiated structures, such as photoreceptors, nervous tissue and a small pharynx. In conclusion, Smed-MmpB is a planarian TSG that prevents stem cell proliferation and differentiation outside the proper milieu. This work was supported by Fonds Wetenschappelijk Onderzoek (R-6411 to A.W.; 1522015N and G0B8317N to A.W., A.V.R., K.S.), Universität des Saarlandes (T.A.T.), Universiteit Hasselt (A.W., A.V.R., K.S.), Bijzonder Onderzoeksfonds (G0B81317 to A.W., A.V.R., K.S.), Deutscher Akademischer Austauschdienst (50753940 to L.G.), Hercules Foundation (R-3986), Egyptian Cultural Affair Mission Sector of the Ministry of Higher Education/National Research Centre (A.Z.A.-Z.B.), Max Planck Institute for Molecular Biomedicine (L.G.) and Fraunhofer Institute for Biomedical Engineering (L.G.).

Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short-(3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 mu M) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed. Bijzonder OnderzoeksFonds of Hasselt University (grant number BOF08G01); Hasselt University tUL-impulsfinanciering (project toxicology) Fonds voor Wetenschappelijk Onderzoek (grant number 1522015N and PhD grant number R6411 to A.W.); Agentschap voor Innovatie door Wetenschap en Technologie (grant number 101442 to A.-S.S. and OZM grant number 100631 to M.W.).

Planarians have been long known for their regenerative ability, which hinges on pluripotency. Recently, however, the planarian model has been successfully established for routine toxicological screens aimed to assess overproliferation, mutagenicity and tumorigenesis. In this study, we focused on planarian tumor suppressor genes (TSGs) and their role during chemically induced carcinogenic stress in Schmidtea mediterranea. Combining in silico and proteomic screens with exposure to human carcinogen type 1A agent cadmium (Cd), we showed that many TSGs have a function in stem cells and that, in general, exposure to Cd accelerated the onset and increased the severity of the observed phenotype. This suggested that the interaction between environmental and genetic factors plays an important role in tumor development in S. mediterranea. Therefore, we further focused on the synergistic effects of Cd exposure and p53 knockdown (KD) at the cellular and molecular levels. Cd also produced a specific proteomic landscape in homeostatic animals, with 172 proteins differentially expressed, 43 of which were downregulated. Several of these proteins have tumor suppressor function in human and other animals, namely Wilms Tumor 1 Associated Protein (WT1), Heat Shock Protein 90 (HSP90), Glioma Pathogenesis-Related Protein 1 (GLIPR1) and Matrix Metalloproteinase B (Smed-MMPB). Both Glipr1 and MmpB KD produced large outgrowths, epidermal lesions and epidermal blisters. The epidermal blisters that formed as a consequence of Smed-MmpB KD were populated by smedwi1+ cells, many of which were actively proliferating, while large outgrowths contained ectopically differentiated structures, such as photoreceptors, nervous tissue and a small pharynx. In conclusion, Smed-MmpB is a planarian TSG that prevents stem cell proliferation and differentiation outside the proper milieu., Summary: Combining exposure to the human carcinogen cadmium with in silico and proteomic top-down approaches, we disclosed the conserved function of two novel tumor suppressor genes in planarian.