www.nature.com/scientificreports OPEN Stimulation of hepatocarcinogenesis by activated cholangiocytes via Il17a/f1 pathway in kras transgenic zebrafsh model Mohamed Helal1,2, Chuan Yan1 & Zhiuyan Gong1* It has been well known that tumor progression is dependent on secreted factors not only from tumor cells but also from other surrounding non-tumor cells. In the current study, we investigated the role of cholangiocytes during hepatocarcinogenesis following induction of oncogenic krasV12 expression in hepatocytes using an inducible transgenic zebrafsh model. Upon induction of carcinogenesis in hepatocytes, a progressive cell proliferation in cholangiocytes was observed. The proliferative response in cholangiocytes was induced by enhanced lipogenesis and bile acids secretion from hepatocytes through activation of Sphingosine 1 phosphate receptor 2 (S1pr2), a known cholangiocyte receptor involving in cholangiocyte proliferation. Enhancement and inhibition of S1pr2 could accelerate or inhibit cholangiocyte proliferation and hepatocarcinogenesis respectively. Gene expression analysis of hepatocytes and cholangiocytes showed that cholangiocytes stimulated carcinogenesis in hepatocytes via an infammatory cytokine, Il17a/f1, which activated its receptor (Il17ra1a) on hepatocytes and enhanced hepatocarcinogenesis via an ERK dependent pathway. Thus, the enhancing efect of cholangiocytes on hepatocarcinogenesis is likely via an infammatory loop. Liver cancer is one of the most prevalent cancers and it is ranked the ffh most common cancer and the second most cause of cancer related mortality with a dismal fve-year survival rate of 10%1–3. Hepatocellular carcinoma (HCC), which is developed from hepatocytes, is the primary liver cancer. Several etiological factors contribute to HCC development, including hepatitis viral infection, cirrhosis, nonalcoholic fatty liver disease, environmental toxin, alcoholism, etc.4–6. Now several gene mutations causing increased susceptibility for liver cancers have also been identifed such as TERT (telomerase reverse transcriptase), TP53, CTNNB1 (β-catenin), etc.7.Tese studies highlights the importance of both genetic and environmental factors in promoting liver cancers. In the liver, hepatocytes and cholangiocytes are two main cell types. Cholangiocytes are small, heterogene- ous and dynamic group of epithelial cells lining the biliary ducts in the liver and have important absorptive and secretory functions8–13. Cholangiocytes are generally quiescent and are activated only under certain liver infam- matory or pathological conditions8–12,14. Several molecular pathways have been demonstrated to be involved in cholangiocyte proliferation15–18 such as Tgf19, Notch20,21 and Hedgehog signalling pathways12. Various receptors are also known to control cholangiocyte proliferation and determine their response upon injury 13,22. Among these receptors, Sphingosine 1 phosphate receptor 2 (S1pr2) appears to be a strong mediator of cholangiocyte proliferation in pathological and oncogenic conditions of cholangiocytes 23,24. In our laboratory, we have previously developed several transgenic zebrafsh models for HCC by inducible expression of a selected oncogene in hepatocytes, such as krasV1225,26, Myc27, xmrk28 and tgf1b29. HCC are induced robustly from all of these transgenic zebrafsh and they show resemblance to human liver cancer at both histologi- cal and molecular levels 30,31. Tese inducible transgenic models allow us to control transgenic oncogene expres- sion at desired time points to initiate tumorigenesis, thus providing valuable tools for investigation of tumour initiation, which may be difcult to achieve in human clinical studies and other animal models. 1Department of Biological Sciences, National University of Singapore, Singapore, Singapore. 2Marine Pollution Lab, Marine Environment Division, National Institute of Oceanography and Fisheries, Alexandria, Egypt. *email: [email protected] Scientifc Reports | (2021) 11:1372 | https://doi.org/10.1038/s41598-020-80621-6 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Increase of cholangiocytes upon induction of oncogenic krasV12 expression in hepatocytes of kras+ transgenic zebrafsh larvae. Cholangiocytes were determined by using two diferent molecular markers: Alcam and Cytokeratin 18 (CK18). 3-dpf kras+ and WT zebrafsh larvae were treated with 20 µg/ml Dox till 7 dpf. Samples were collected at diferent time points afer Dox induction for immunohistochemistry. All liver sections were counter stained with DAPI. (A–D) Representative images of Alcam antibody (A) and Cytokeratin 18 (Ck18) (C) and quantifcation of cholangiocyte density (B,D) in kras + and WT larvae at 96 h following Dox induction. (E,F) Time course of increase of cholangiocytes from 8 to 96 h following Dox induction. Representative images of Alcam staining of kras+ and WT larvae from 8 to 72 h are shown in (E) and 96 h in (A). Quantifcation of cholangiocyte density in kras+ and WT larvae is shown in (F). N = 10 at each time point. Scale bar: 20 μm. Statistical signifcance: *P˂0.05. Te importance of tumor microenvironment for tumor initiation and progression has been increasingly recognized32,33. In liver tumorigenesis, it is interesting to investigate the interaction between hepatocytes and surrounding cellular/non-cellular components. In zebrafsh, several previous studies have shown that non- tumour cells can contribute to HCC progression through diferent secreted mediators, e.g. hepatic stellate cells via serotonin secretion34, immune cells neutrophils and macrophages via transforming growth factor beta (Tgf)35 and cortisol36. As cholangiocytes are the second largest cell population in the liver, it is tempting to investigate the roles of cholangiocytes to development of HCC. In particular, cholangiocytes have been shown to have a strong connection to several liver diseases such as liver fbrosis11, cholestatic liver injury37, primary scleros- ing cholangitis38 and also ethionine-induced HCC39. Hence, in this study, we attempted to examine the role of cholangiocytes during HCC initiation and progression using krasV12 transgenic zebrafsh model we earlier established26. Our experiments demonstrated that cholangiocytes play an important role in promoting HCC development through an infammatory pathway. Results Increase of cholangiocytes upon induction of oncogenic krasV12 expression in hepatocytes of krasV12 transgenic zebrafsh larvae. To visualize the response of cholangiocytes upon krasV12 induc- tion in hepatocytes. 3-dpf (day postfertilization) krasV12 transgenic (shorted as kras + in this report) larvae were induced by doxycycline (Dox) for 5 days to initiate hepatocarcinogenesis. Two specifc cholangiocyte markers, Scientifc Reports | (2021) 11:1372 | https://doi.org/10.1038/s41598-020-80621-6 2 Vol:.(1234567890) www.nature.com/scientificreports/ Alcam and Cytokeratin 18 (Ck18), were used for identifying cholangiocytes in the liver sections. As shown in Fig. 1A–D, both Alcam and Ck18 stained cholangiocytes showed signifcant increases in the kras + group in comparison with the wildtype (WT) control group upon induction of oncogenic krasV12 expression by Dox. Cholangiocyte density was determined following Dox induction from 8 to 96 h. A shown in Fig. 1E,F, Alcam staining revealed signifcant increases in cholangiocyte density in the kras + group compared to the WT group at all time points and a signifcant increase was observed as early as 8 h afer the initiation of Dox treatment. Acceleration of tumor growth via S1pr2 induction. Several cholangiocyte receptors have been reported to control cholangiocyte proliferation under diferent physiological and pathological conditions 8,9,11,40. Tese receptors include secretin receptor (Sctr)41, estrogen receptor (Esr), insulin receptor (Insra and Insrb), glucagon-like-peptide receptor 2 (Glpr2), nerve growth factor receptor (Ngfr) and sphingosine 1 phosphate receptor 2 (S1pr2)40,42–45, etc. Expression of these cholangiocyte receptor genes in cholangiocytes and hepato- cytes of WT and kras + adult zebrafsh afer Dox induction were analyzed by RT-qPCR. As shown in Supplemen- tary Fig. S1, majority of these receptor genes showed little changes in expression in cholangiocytes and hepato- cytes following oncogenic krasV12 expression in hepatocytes, but s1pr2 expression had striking increases in both hepatocytes (28 fold) and cholangiocytes (> 120 fold). Tis is reminiscence of several previous reports that s1pr2 is highly expressed in cholangiocarcinoma and bile duct cell diseases43,46. Tus, in view of high s1pr2 expression in cholangiocytes and its correlation with cholangiocarcinoma and cholestatic liver disease, we chose s1pr2 as a promising candidate receptor to study its role during HCC progression in our zebrafsh model. To analyze the role of S1pr2 during hepatocarcinogenesis, both an agonist, taurocholate (TCA), and an antagonist, JTE-013, were used 42. kras + and WT larvae were treated with TCA or JTE-013 in conjunction with Dox from 3 to 8 dpf. TCA led to a further increase in liver size compared to the group treated with Dox alone (Fig. 2A,B). JTE-013 had an opposite efect, as the liver size became signifcantly smaller than that in the Dox alone group (Fig. 2A,B). To confrm the link between S1pr2 and cholangiocytes, Alcam staining was used to determine cholangiocyte density in the same treatment groups. As shown in Fig. 2C and quantifed in Fig. 2D, cholangiocyte density in kras + larvae was signifcantly increased in the TCA/Dox group compared