1 FGF19/SOCE/NFATc2 signaling circuit facilitates the self- 2 renewal of liver cancer stem cells 3 4 Jingchun Wang 1†, Huakan Zhao2†*, Lu Zheng3†, Yu Zhou2, Lei Wu2, Yanquan Xu1, Xiao 5 Zhang1, Guifang Yan2, Halei Sheng1, Rong Xin1, Lu Jiang1, Juan Lei2, Jiangang Zhang1, Yu 6 Chen2, Jin Peng1, Qian Chen1, Shuai Yang1, Kun Yu1, Dingshan Li1, Qichao Xie4*, 7 Yongsheng Li1,2* 8 9 1Clinical Medicine Research Center, Xinqiao Hospital, Army Medical University, 10 Chongqing 400037, China. 11 2Department of Medical Oncology, Chongqing University Cancer Hospital, Chongqing 12 400030, China. 13 3Department of Hepatobiliary Surgery, Xinqiao Hospital, Army Medical University, 14 Chongqing 400037, China. 15 4Department of Oncology, The Third Affiliated Hospital, Chongqing Medical University, 16 Chongqing 401120, China 17 18 †These authors contributed equal to this work. 19 *Correspondence ([email protected]) (H.Z.), ([email protected]) (Q.X.), 20 ([email protected]) (Y.L.). 21 22 Running title: FGF19-NFATc2 signaling promotes self-renewal of LCSCs. 23 Keywords: FGF19; Self-renewal; SOCE; NFATc2, LCSCs 24 1 25 Abstract 26 Background & Aims: Liver cancer stem cells (LCSCs) mediate therapeutic resistance and 27 correlate with poor outcomes in patients with hepatocellular carcinoma (HCC). Fibroblast 28 growth factor (FGF)-19 is a crucial oncogenic driver gene in HCC and correlates with poor 29 prognosis. However, whether FGF19 signaling regulates the self-renewal of LCSCs is 30 unknown. 31 Methods: LCSCs were enriched by serum-free suspension. Self-renewal of LCSCs were 32 characterized by sphere formation assay, clonogenicity assay, sorafenib resistance assay and 33 tumorigenic potential assays. Ca2+ image was employed to determine the intracellular 34 concentration of Ca2+. Gain- and loss-of function studies were applied to explore the role of 35 FGF19 signaling in the self-renewal of LCSCs. 36 Results: FGF19 was up-regulated in LCSCs, and positively correlated with certain self- 37 renewal related genes in HCC. Silencing FGF19 suppressed self-renewal of LCSCs, whereas 38 overexpressing FGF19 facilitated CSCs-like properties via activation of FGF receptor 39 (FGFR)-4 in none-LCSCs. Mechanistically, FGF19/FGFR4 signaling stimulated store- 40 operated Ca2+ entry (SOCE) through both the PLCγ and ERK1/2 pathways. Subsequently, 41 SOCE-calcineurin signaling promoted the activation and translocation of nuclear factors of 42 activated T cells (NFAT)-c2, which transcriptionally activated the expression of stemness- 43 related genes (e.g., NANOG, OCT4 and SOX2), as well as FGF19. Furthermore, blockade of 44 FGF19/FGFR4-NFATc2 signaling observably suppressed the self-renewal of LCSCs. 45 Conclusions: FGF19/FGFR4 axis promotes the self-renewal of LCSCs via activating 46 SOCE/NFATc2 pathway; in turn, NFATc2 transcriptionally activates FGF19 expression. 47 Targeting this signaling circuit represents a potential strategy for improving the therapeutic 48 efficacy of HCC. 49 2 50 Graphical Abstract 51 52 The pivotal molecules and pathways orchestrating the self-renewal of LCSCs remain elusive, 53 Wang et al. demonstrate that FGF19/SOCE/NFATc2 signaling circuit promotes the self- 54 renewal of LCSCs, implicating its potential as a therapeutic target for HCC therapy. 3 55 Introduction 56 Hepatocellular carcinoma (HCC), the third lethal cancer worldwide, is characterized by a 57 high rate of recurrence and therapy-resistance [1, 2]. Compelling evidence have 58 demonstrated that small heterogeneous population of liver cancer stem cells (LCSCs) with 59 self-renewal properties are critical for the progression and therapy-resistance of HCC [2, 3]. 60 Self-renewal is one of the most important properties employed by the CSCs to sustain the 61 proliferating capacity [4, 5]. Understanding the mechanisms underlying the self-renewal of 62 LCSCs will provide potential strategy to overcome the recurrence and therapy-resistance of 63 HCC. 64 Fibroblast growth factors (FGF) signaling can promote self-renewing proliferation and 65 inhibit cellular senescence in many tissues and organs [6, 7]. As a member of the hormone- 66 like FGF family, FGF19 shows high binding affinity with FGF receptor (FGFR)-4, which is 67 predominantly expressed in liver [8, 9]. Growing evidence indicates aberrant FGF19-FGFR4 68 signaling axis is a key driver in the development of HCC [8, 10, 11]. Our earlier study and 69 others indicated that FGF19 regulates a variety of functions of hepatocytes, such as bile acid 70 synthesis, proliferation, epithelial-mesenchymal transition (EMT) and apoptosis-resistance 71 in a FGFR4-dependent manner [12-15]. In addition, previous study demonstrated that 72 expressions of FGF19 positively correlated with undifferentiated state of human embryonic 73 stem cells (ESCs) [16]. However, whether FGF19 signaling is related to self-renewal 74 characteristics of LCSCs is unknown. 75 Calcium (Ca2+)-mediated signaling pathways are involved in various cellular biological 76 processes including proliferation, apoptosis, metastasis and self-renewal [17]. Store-operated 77 Ca2+ entry (SOCE), triggered by depletion of endoplasmic reticulum (ER) Ca2+, is the major 78 route of Ca2+ influx for non-excitable cells including HCC cells [17, 18]. Stromal interaction 79 molecule (STIM)-1, as an ER Ca2+ sensor, is a key mediator of SOCE activation [19, 20]. 80 We previously reported that STIM1-mediated SOCE orchestrates HCC tumorigenesis under 81 hypoxic conditions [21], whereas how SOCE is mobilized in LCSCs and regulates the HCC 82 self-renewal remain vague. Herein, we sought to assess the effect of FGF19/FGFR4 signaling 4 83 on STIM1-mediated SOCE and the self-renewal of LCSCs in vitro and in vivo. 84 Results 85 FGF19 is up-regulated in LCSCs and correlates with self-renewal related genes in HCC. 86 We firstly enriched LCSCs of Huh-7, RFP/PLC/5 and MHCC97H cells via serum-free 87 suspension [22]. After cultured for 2 weeks, Huh-7 and RFP/PLC/5 cells formed compact 88 round multicellular aggregates, while MHCC97H cells formed branching spheroids (Figure 89 S1A). The high self-renewal capacity of spheroids cells was confirmed by sphere formation 90 assay, clonogenicity assay, sorafenib resistance assay and tumorigenic potential assay (Figure 91 S1B-E). Furthermore, comparing with the parental cells, spheroids exhibited increased 92 mRNA levels of several stem cell markers, e.g., pluripotency transcription factor Nanog, 93 octamer-binding protein 4 (Oct-4), SRY-box transcription factor 2 (Sox2), CD133, c-Myc 94 and aldehyde dehydrogenase1 (ALDH1), but showed decreased mature hepatocyte markers, 95 such as albumin (ALB) and glucose-6-phosphatase (G6P) (Figure S1F). These results 96 demonstrate that the in vitro enriched LCSC populations of Huh-7, RFP/PLC/5 and 97 MHCC97H cells were successfully established. 98 Interestingly, compared with corresponding non-CSCs (NCSCs), both FGF19 expression and 99 secretion remarkably up-regulated in LCSCs (Figure 1A-C). Immunohistochemistry (IHC) 100 analysis revealed high levels of FGF19 in xenograft tumors derived from CSCs compared 101 with tumors from corresponding NCSCs (Figure 1D). During the differentiation of LCSCs, 102 we observed a gradual decrease of FGF19 expression accompanied with the decrease of 103 endogenous Nanog and the increase of ALB (Figure 1E). Next, IHC analysis of human HCC 104 samples showed that FGF19 expression was positively correlated with expressions of Nanog 105 and Oct-4, but negatively correlated with expressions of ALB (Figure 1F). These data 106 indicate that FGF19 is highly expressed in LCSCs, and correlates with stemness-related 107 genes in HCC. 108 Knockdown of FGF19 attenuates self-renewal features of LCSCs. 5 109 To investigate the role of FGF19 on self-renewal of LCSCs, we knocked-down FGF19 via 110 lentiviral introducing shRNA in the CSCs of Huh-7 and RFP/PLC/5 (Figure 2A-B). We 111 found that stemness-associated genes including NANOG, OCT4 and SOX2 were significantly 112 down-regulated, whereas mature hepatocyte markers, such as ALB and G6P were markedly 113 up-regulated, when compared with control (Figure 2A-B). Consistently, FGF19 silencing 114 significantly reduced the sphere and clone formation ability, as well as the sorafenib 115 resistance of LCSCs (Figure 2C-E). Moreover, the growth of xenograft tumors generated 116 from FGF19-deficient LCSCs was attenuated compared with control LCSCs (Figure 2F). 117 These results indicate that FGF19 is required for self-renewal in LCSCs. 118 FGF19 facilitates cancer stem cell-like properties in liver NCSCs. 119 To further validate the role of FGF19 in maintaining the stemness of HCC cells, we 120 overexpressed FGF19 in the NCSCs of Huh-7 and RFP/PLC/5 by lentiviral vector. Ectopic 121 expression of FGF19 upregulated stemness-associated genes (e.g., NANOG, OCT4 and 122 SOX2), while reduced ALB and G6P levels (Figure 3A-B). Over-expression of FGF19 123 significantly promoted clone and sphere formation, as well as the resistance to sorafenib 124 (Figure 3C-E). Furthermore, compared with the control group, FGF19 overexpression 125 significantly enhanced the tumorigenic ability of NCSCs (Figure 3F). Our previous studies 126 have shown that FGF19 promotes EMT and facilitates a survival response to ER stress via 127 FGFR4 in HCC cells [12, 13]. To determine the possible involvement of FGFR4 in FGF19- 128 mediated self-renewal of LCSCs, we generated genomic FGFR4 knockout Huh-7 and 129 RFP/PLC/5 cells by CRISPR-Cas9 system (Figure S2A-B). FGFR4 deficiency dramatically 130 suppressed self-renew properties trigged by FGF19 in NCSCs (Figure 3A-F). Moreover, 131 BLU9931, a specific FGFR4 inhibitor, also attenuated the FGF19-induced self-renewal 132 properties in NCSCs of Huh-7 and RFP/PLC/5 (Figure S3A-E). These results demonstrate 133 that FGF19 facilitates self-renewal properties via FGFR4 activation in HCC cells. 134 Enhanced SOCE is involved in FGF19-promoted self-renewal of HCC cells. 135 Recent evidence indicated that Ca2+ oscillation frequency positively correlated with the self- 6 136 renewal potential in Hep-12 cells [23], we next wondered whether SOCE-mediated Ca2+ 137 signal was involved in FGF19-mediated self-renewal activity.