Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon

Schistosomiasis (bilharzia) is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, in sub-Saharan Africa, and particularly also in Europe. The WHO describes an increasing global health burden with more than 290 million people threatened by the disease and a potential to spread into regions with temperate climates like Corsica, France. The aim of our study was to investigate the influence of S. mansoni infection on colorectal carcinogenic signaling pathways in vivo and in vitro. S. mansoni infection, soluble egg antigens (SEA) and the Interleukin-4-inducing principle from S. mansoni eggs induce Wnt/β-catenin signaling and the protooncogene c-Jun as well as downstream factor Cyclin D1 and markers for DNA-damage, such as Parp1 and γH2a.x in enterocytes. The presence of these characteristic hallmarks of colorectal carcinogenesis was confirmed in colon biopsies from S. mansoni-infected patients demonstrating the clinical relevance of our findings. For the first time it was shown that S. mansoni SEA may be involved in the induction of colorectal carcinoma-associated signaling pathways.

Schistosomiasis is one of the most prevalent parasitic diseases worldwide with at least 290 million people requiring preventive treatment in 2018 1 . While the majority of people at risk live in the endemic regions of Africa, Schistosoma species are also prevalent in the Middle East, the Caribbean, South America, and South East Asia 2 . Schistosomiasis is increasingly imported into regions with temperate climates by immigrants and travelers from endemic areas 3,4 . A recent epidemiological case study, investigating an outbreak of urogenital schistosomiasis in Corsica, France, emphasized the potential risk of schistosomiasis spreading into novel areas 5 . It has been suggested that the persistent establishment of the tropical disease in Europe relies on the preadaptation of schistosomes and the adaptation of secondary hosts to overwinter in countries like France, Italy, Portugal, Spain, and Greece 6 . In humans, intestinal schistosomiasis is caused by different species such as Schistosoma mansoni (S. mansoni) and S. japonicum 7 . During infection, the paired adult worms lodge in the mesenteric veins and produce approximately 300 eggs (ova) per day 8 . About half of the eggs migrate through the intestinal wall into the gut lumen and are excreted with the feces, thus completing the parasites life cycle 9 . Without providing any obvious intrinsic motility mechanisms, schistosome eggs are likely to depend on host-driven processes to pass tissues 10 . In the colorectal wall, ova induce an inflammatory response, which can lead to fibrotic granuloma formation and lymphoid hyperplasia, resulting in ova entrapment 7 . Eggs deposited in living host tissues might survive for up to three months 11 . Being damaged by the eggs' extravasation, the overlying mucosa develops small superficial ulcers with hyperplastic changes 7 . During chronification, this process provokes mucosa hypertrophy and subsequent pseudopolyp formation 12 . However, about half of the eggs, instead of reaching the intestinal lumen, are swept into the liver, provoking marked granulomatous inflammation 10 . Typical symptoms are abdominal cramping, diarrhea, and dysentery 13 . Schistosomiasis is diagnosed by the detection of parasite eggs in stool (S. mansoni, S. japonicum) or urine (S. haematobium) samples or, if accessible, by serological and immunological tests 1 .
The global burden of cancer was estimated to be 18.1 million new cases and 9.6 million cancer-related deaths in 2018 14 . Approximately 20% of human cancers are caused by infectious diseases 15 . It was estimated, that 0.4% of the new cancers that are attributable to infections were caused by the trematodes Schistosoma haematobium (0.3%), Opisthorchis viverrini, and Clonorchis sinensis (both liver flukes together: 0.1%), which are considered as group 1 carcinogens by IARC 16 . The infection with the liver flukes O. viverrini and C. increase the risk to develop cholangiocarcinoma, while in endemic areas, 46-75% of all bladder cancers were attributed to S. haematobium 17 . Clinical studies and animal experiments have also indicated that schistosomiasis promotes the development of hepatocellular carcinoma (HCC), prostate cancer, follicular lymphomas, and also colorectal cancer (CRC) [18][19][20][21] . A recent study has suggested that schistosomal colitis is more commonly associated with an earlier onset of multicentric CRC, high percentage of mucinous adenocarcinoma, and presents at an advanced stage 19 . Another recently published report demonstrated that S. japonicum infection facilitates the development of CRC, particularly in the early and intermediate stages, suggesting that schistosomiasis may alter the mechanisms underlying the progression of CRC 22 . Nevertheless patients with schistosomal and nonschistosomal CRC, had the same outcome after 7.5 years 22 . Although numerous studies have demonstrated an association of S. mansoni infection with liver cancer, colorectal carcinoma, and bladder cancer 17 , no reliable quantitative data to substantiate this relationship are available, yet.
Lastly, in contrast to schistosomiasis-related carcinogenesis, the link between inflammatory bowel disease (IBD) and CRC has been investigated extensively. A recent meta-analysis, including 7199 cases of IBD, demonstrated that patients with long lasting ulcerative colitis (UC) and colon-associated Crohn's disease (CD) have an increased risk of CRC 23 . While CD is associated with T-helper type 1 (Th1) immune responses, UC shows a Th2-prone fibroinflammatory phenotype 24 . S. mansoni egg-derived antigens also induce colitis characterized by a Th2-prone fibroinflammatory phenotype which may contribute for increasing the susceptibility to oncogenic provocations 2,10 .
Previous research has, moreover, provided first insights into the molecular mechanisms underlying Schistosoma-associated carcinogenesis. It has been postulated that genotoxic agents, produced by the eggs, may play a role in CRC via alteration of the Bcl-2 protein expression pattern, leading to a dysregulation of apoptosis 25 . The glycoprotein IL-4-inducing principle from Schistosoma mansoni eggs (IPSE) is the major egg secreted product with immunoglobulin-binding properties 26 . The interaction of IgE bound IPSE with the FcεRI receptor can trigger the release of IL-4 and IL-13 from basophils and is therefore involved in immunemodulation during schistosomiasis 27 . Moreover, IPSE/alpha-1 has a C-terminal functional nuclear localization sequence (NLS) for translocation into the nucleus, which suggests a possible function as transcription factor 28 .
Similar to what has been described in various cancer entities, the growth of new blood vessels was enhanced in S. mansoni infection 10 and it was suggested that the eggs might induce angiogenesis-related processes by www.nature.com/scientificreports/ up-regulating vascular endothelial growth factor and creating a proangiogenic environment, characterized by hypoxia, acidic pH, and low glucose concentration, due to vessel occlusion 29 . Based on these findings, we hypothesize that S. mansoni-associated colitis could promote the development of CRC. Therefore, we analyzed CRC-associated signaling pathways and CRC markers in colon samples from S. mansoni-infected patients, from S. mansoni-infected hamsters, and from SEA-stimulated enterocytes.

Materials and methods
Human material. Pseudonymized human colon samples were kindly provided by cooperation partners (TL, MO, UD, MA, LR, KB, and PW) after approval by the local ethics committee (Ethics vote ID AZ 05/19, Justus Liebig University Giessen). According to the ethics vote, an informed consent was not required for our retrospective analyses of archived tissues. It was not appropriate or possible to involve patients or the public in the design, or conduct, or reporting, or dissemination plans of our research. Isolation of soluble egg antigens. Schistosoma mansoni eggs were obtained from livers of bisex-infected hamsters at day 46 post infection and soluble egg antigens (SEA) were isolated as described earlier 27 . SEA protein concentration was determined colorimetrically, utilizing the Advanced Protein Assay (Cytoskeleton, Inc., Denver, USA) according to the manufacturer's instructions.
Purification of nIPSE/alpha-1 and HEK-IPSE. Natural (n)IPSE/alpha-1 (Interleukin-4 inducing principle from S. mansoni eggs) was purified from SEA in a two-step chromatography procedure including a cation exchange chromatography followed by an affinity chromatography via binding to monoclonal anti-IPSE/alpha-1 antibodies coupled to NHS-activated Sepharose as described before 27 . The eluted neutralized fractions were controlled by SDS-PAGE, silver staining, and western blot for content and purity of IPSE/alpha-1, dialyzed against PBS (pH 7.5), concentrated to a suitable concentration, and stored in aliquots at − 80 °C. Human embryonic kidney (HEK)-IPSE was purified from 293 HEK cells transfected with the expression vector pSecTag2-IPSE. Secreted HEK-IPSE (His-tagged) was sequentially purified from the culture medium (10% FCS / RPMI, Capricorn) by immobilized metal affinity chromatography and antibody affinity chromatography. Eluted fractions were treated as described above for the purification of nIPSE.
Cell culture experiments. SW620 cells (ATCC Cat# CCL-227, RRID:CVCL_0547) were stimulated in vitro at 80% confluency with 1-15 µg/ml SEA or IPSE with denoted concentrations and for the indicated time. SEA contains around 1-2% IPSE (personal communication by G. Schramm). The concentrations of SEA and IPSE were titrated to demonstrate similar levels of activation in blots showing both, SEA and IPSE. Inhibition experiments were conducted after starving the cells overnight in cell culture medium without FCS. The cells were pretreated with SP600125 or U0126 (both 10 µM, 30 min) or XAV939 (11 nM, overnight) before stimulation.
Phospho-kinase proteome profiler array. Lysates were prepared according to the manufacturer's protocol from SEA-and mock-stimulated SW620 cells. The human Phospho-Kinase Proteome Profiler Array (R&D Systems, Minneapolis, MN, USA) were carried out as indicated in the manual. Densitometric analysis was performed as described previously 31

Schistosoma mansoni eggs induced Wnt/β-catenin-signaling in colon epithelial cells.
To date, the effects of S. mansoni egg-induced colitis on enterocytes remain poorly understood. Therefore, we performed a phospho-kinase array to detect possible impact of S. mansoni egg-secreted antigens on distinct intracellular signaling pathways in colon epithelial cells. The Human Phospho-Kinase Array is a rapid and sensitive tool to simultaneously detect the relative levels of phosphorylation of kinases and two related total proteins, which is essential for understanding how cells recognize and respond to changes in their environment. cAMP-response element binding protein (CREB S133), AMP-dependent Kinase (AMPKa1 T183), Heat shock protein 27 (Hsp27 S78/S82), Epidermal Growth Factor Receptor (EGFR Y1086), Gsk3β S21/S9, β-catenin (unphosphorylated), and other factors were induced by stimulation of the human epithelial cell line SW620 with SEA (Fig. 1a,b). Given its role as a potential therapeutic target in CRC, we further focused on the Wnt/β-catenin pathway. Both SEA and the stimulation of SW620 with raising concentrations of HEK-IPSE induced the phosphorylation of the inhibitory phosphorylation site Ser9 of Gsk3β (Fig. 1c, quantification in Suppl. Fig. 1). While we observed a trend of β-catenin expression following SEA stimulation (Fig. 1c, Suppl. Fig. 1), the inhibition of Wnt-signaling by the tankyrase inhibitor XAV939 led to a clear reduction of β-catenin after SEA stimulation (Fig. 1d, Suppl. Fig. 2). In order to visualize the cellular activation pattern of the Wnt/β-catenin-signaling pathway in vivo, immunohistochemical stainings of β-catenin were performed on colon sections of S. mansoni-infected hamsters. It appears noteworthy that especially enterocytes in crypts adjacent to extravasating eggs stained positive for β-catenin (Fig. 1e). Higher magnifications of β-catenin-positive crypts revealed both an enhanced cytoplasmatic and nuclear staining (Fig. 1f, quantification in Suppl. Fig. 3). Taken together, these results suggest that S. mansoni

Schistosoma mansoni infection provoked fibrogranulomatous colitis in patients and hamsters.
To assure life cycle progression, S. mansoni eggs need to egress from the human body. Arising in the mesenteric veins, S. mansoni eggs pass through the intestinal wall into the gut lumen 10 . The histological examination of human colon biopsies showed multiple parasite eggs along with pronounced multifocal granulomatous inflammation in the mucosa and submucosa (Suppl. Fig. 4a,b). Colon samples of S. mansoni-infected hamsters presented a comparable phenotype (Suppl. Fig. 4c-f). The submucosa appeared thickened at the sites of eggextravasation, and polyp-like structures were identified (dashed line, Suppl. Fig. 4c,e). Granulomatous inflammation was accompanied by marked fibrosis, which was visualized by Masson's trichrome staining (arrowheads, Suppl. Fig. 4e,f) 5). In addition to the enhanced nuclear translocation of c-Jun in the enterocytes at the sites of egg extravasation (Suppl. Fig. 5), the expression, and phosphorylation of c-Jun was globally enhanced in the colon of bisex-infected animals (Fig. 2d). Interestingly, we detected a comparable, but much weaker activation pattern of c-Jun in the small bowel of S. mansoni-infected hamsters (Suppl. Fig. 6). However, nuclear translocation of c-Jun was less prominent in the small bowel, and the assessment of protein expression and activation by western blot yielded no differences in ileal c-Jun activation between bisex-infected hamsters and controls.

SEA and IPSE lead to c-Jun activation in vitro.
Our results suggested a mechanism of c-Jun activation that depends on substances released from S. mansoni eggs. To further substantiate these findings, we next performed in vitro experiments, again utilizing human SW620 cells. A reporter gene assay demonstrated, that SEA and IPSE stimulation lead to the functional activation of the AP1 promotor (Fig. 2e). The induction of c-Jun protein expression and phosphorylation in consequence of the SEA stimulation was demonstrated by western blot (Fig. 3a,b). Moreover, the activation of c-Jun was attenuated using a selective and cell permeable inhibitor of c-Jun N-terminal kinase (JNK, SP600125) and the MEK1/2-inhibitor U0126 (Fig. 3a,b). To assess, whether secreted products from S. mansoni eggs were responsible for the activation of c-Jun, we repeated the experiments with natural IPSE (nIPSE), purified from S. mansoni eggs, and recombinant IPSE produced from HEK cells (HEK-IPSE). Western blot analysis revealed a concentration-dependent activation of c-Jun (Fig. 3c,d) which was reversed by the addition of SP600125 (Fig. 3e).

Schistosoma mansoni eggs induced the expression of proliferation markers. The Wnt-path-
way and the transcription factor c-Jun are well characterized key regulators of epithelial cell proliferation in carcinogenesis 36,37 . To monitor proliferation, we analyzed the expression patterns of the cellular proliferation markers Mcm2, Cyclin D1, and Ki67, which have been described as prognostic markers in the context of CRC 38,39 . Enhanced nuclear staining of Cyclin D1 and Ki67 adjacent to S. mansoni eggs indicated increased enterocyte proliferation in human (Suppl. Fig. 7) and hamster colon samples (Fig. 4a,b, Suppl. Fig. 8). Immunostain- www.nature.com/scientificreports/ ing of Cyclin D1 in cross sections, displaying the distinct layers of the colon mucosa, demonstrated that the enhanced staining surrounding the eggs (Fig. 4b) differentiates from the staining pattern in unaffected areas, resulting from physiological enterocyte proliferation in basal crypts (Fig. 4a). The global Cyclin D1 and Mcm2 expression was increased in the colon of bisex-infected hamsters (Fig. 4c). Furthermore, SEA were capable of inducing Cyclin D1 expression in SW620 cells, which was inhibited by SP600125 and U0126 (Fig. 4d). Also HEK-IPSE induced expression of Cyclin D1 in SW620 cells was inhibited by SP600125 (Suppl. Fig. 9). This indicated that Cyclin D1 induction is a downstream event of JNK-and MEK1/2-activation. Additionally, IPSE stimulation led to Cyclin D1 expression in a concentration-dependent manner (Fig. 4e). The inhibition of Wntsignaling by XAV939 reduced the SEA-but not the IPSE-induced expression of Cyclin D1 (Fig. 4f).

Schistosoma mansoni eggs induced the expression of markers for DNA repair. Genetic instabil-
ity and DNA damage are hallmarks of the Fearon and Vogelstein model of colorectal carcinognesis 40 . Hence, we analyzed the expression patterns of specific markers for DNA damage and/or DNA repair, i.e. Parp-1 and γH2a.x. The global expression of γH2a.x and Parp-1 was increased in the colon of bisex-infected hamsters (Fig. 5a). SEAand HEK-IPSE-treated SW620 cells showed an enhanced expression of γH2a.x and Parp-1 (Fig. 5b). Enhanced nuclear staining of γH2a.x indicated increased DNA double strand breaks in the nuclei of enterocytes adjacent to S. mansoni eggs in hamster colon (Fig. 5c,d, Suppl. Fig. 10).
The genotoxic potential of SEA on SW620 cells was analyzed directly by a comet assay (Fig. 5e). In order to quantify DNA damage, the ratio of nuclei with DNA damage (comet tail) in relation to visualized nuclei without DNA damage was assessed in a blinded manner by two persons individually in at least 10 randomly photographed fields of view.  It has been proposed that processes like DNA damage, mutagenesis, and oncogene activation could modify parenchymal cell proliferation and survival. These processes enhance carcinogenicity upon infection with pathogens like Clonorchis, Opisthorchis, and Schistosoma 17 . Being one of the key cascades regulating development and stemness, the Wnt-signaling pathway is tightly associated with tumorigenesis, most prominently with CRC 37 . Loss of APC is the main driver of Wnt-signaling in CRC and its pivotal role has been highlighted by recent studies underlining the importance of continuous Wnt-signaling for tumor maintenance 37,41 . Cyclin D1 is a typical downstream target of the Wnt-pathway 42 . The induction of Cyclin D1 is associated with β-catenin expression in colorectal adenocarcinoma 43,44 . Herein, the inhibition experiments demonstrate the reduction of β-catenin and Cyclin D1 45 by inhibition of the Wnt-pathway, thus providing mechanistic evidence for the stimulation of Wnt-signaling by S. mansoni SEA (Fig. 1d, Suppl. Figs. 2 and 4f). Upon infection also the group 1 carcinogen O. viverrini activates Wnt/β-catenin signaling pathways in a model of cholangiocarcinogenesis 46 . Interestingly, IPSE-stimulated Cyclin D1-induction is not reduced by the inhibition of the Wnt-pathway (Fig. 4F). As SEA is a complex mixture of egg-secreted factors, we assume that complex reciprocal effects might be responsible for this discrepancy. Furthermore, the concomitant activation of distinct signaling pathways might exaggerate or mask downstream effects. Since S. haematobium IPSE is also an egg-secreted protein, and promoting proliferation of bladder cancer cells and angiogenesis, it is tempting to speculate that similar mechanisms are in play as indicated in the present article 47 . Furthermore, it is noteworthy to underline that the Clonorchis and Opisthorchis genomes do not encode IPSE, which may suggest that different carcinogenic mechanisms are involved in cholangiocarcinogenesis induced by these liver flukes.
Our data suggest the induction of two markers for DNA repair, γH2a.x and Parp-1, in the colon of S. mansoniinfected hamsters and in SEA-or IPSE stimulated SW620 cells. The induction of phospho-H2a.x indicates DNA double strand breaks and predicts a poor prognosis in CRC 48 . On the other hand Parp-1, which is also causally linked to DNA repair, protects against colorectal tumor induction. But it promotes inflammation-driven colorectal tumor progression 49 . In addition, SEA inducing genotoxic stress has also been demonstrated (Fig. 5e), which strengthens our hypothesis on schistosome eggs inducing DNA damage and genome instability. Taken together our results provide strong evidence, that DNA damage is another egg-induced cancer-promoting mechanism, which refers to the Fearon and Vogelstein concept. The main findings of the current study are summarized in Fig. 6. Precision therapy concepts, focusing on a pharmacologic antagonization of the distinct associated signaling pathways or downstream cellular functions, could be implemented in the therapy of Schistosoma-associated CRC. Currently, the control of neglected tropical diseases and the improvement of the basal medical infrastructure in endemic areas, however, remain the primary goal 1 . Figure 6. Schematic summary of the main findings of the current study. Granuloma formation around S. mansoni eggs trapped within colon tissue is a hallmark of schistosomal colitis. The eggs are metabolically active and highly antigenic. Egg secreted factors (red circles) activate CRC-associated signaling pathways (JNK/c-Jun, Wnt/β-catenin), which initiate proliferation and DNA-damage. These biomolecular processes upon the exposure to schistosomiasis may characterize underlying mechanisms for the predisposition to the development of colorectal cancer as suggested recently 19 .

Scientific Reports
| (2020) 10:22373 | https://doi.org/10.1038/s41598-020-79450-4 www.nature.com/scientificreports/ It is striking that IBD and other immunopathologies are less prevalent in areas where helminth parasites are endemic 50 . The rising number of studies showing anti-inflammatory effects of helminths, call for the development of translational therapeutic approaches. So far, S. mansoni infections, but not egg antigens, have been used to promote recovery from colitis in outbred NMRI mice 51 . Furthermore, it has been shown that helminth-derived compounds induced a Th1/Th2 switch that ameliorated experimental colitis 52,53 . Other studies reported that the coinfection with S. mansoni attenuated Crohn's like inflammatory bowel disease, preserving the epithelial barrier function by downregulating the inflammatory response 54 . In a recently published study, the therapeutic effect of autoclaved S. mansoni antigens on toxin induced colon cancer was demonstrated in a murine model 55 . An immunomodulatory effect, characterized by reduced circulatory IL-17 and induced IL-10, as well as the rise of splenic CD4 + T-cells and intestinal FoxP3 + Treg cells were reported 55 .
Yet, the findings of our study and the aforementioned reports, describing colorectal carcinogenesis in patients with S. mansoni infection 19,25,[56][57][58] , should be taken into consideration when designing translational approaches using immunomodulatory properties of helminths and helminth-based vaccination. To this end, it is important to consider our findings in concepts of utilizing immunomodulatory effects of helminth infections as therapeutic approaches in line with the "IBD hygiene hypothesis" 59 . In this context, it should also be reflected that S. mansoni infection is associated with alterations to the mammalian intestinal microbiome [60][61][62] . Finally, detailed insights into the interplay of Th2-prone co-infections or pathologies in the gut and the inflammatory environment established by S. mansoni eggs are needed.
In conclusion, we have demonstrated that S. mansoni egg-secreted factors induce the Wnt/β-catenin pathway and the protooncogene c-Jun, thus verifying a notable role of S. mansoni in intestinal carcinogenesis-associated signaling pathways.