The Molecular Mechanisms Of Non-CSCs To Maintain Self-renewal Of CSCs In Human Hepatocellular Carcinoma

Background: The poor progression of Hepatocellular carcinoma(HCC) is mainly due to recurrence and metastasis,which are driven by cancer stem cells(CSCs). To explore the mechanisms maintaining the stemness and shaping the fate of CSCs as well as the relationship between CSCs and microenvironment is the key to cure HCC. With the further and profound research on C SCs, scientists have paid more attention to the cancer-stem-cell-niche which plays an important role in mediating the stemness of CSCs. Like embryonic stem cells, the biological characteristics of CSCs regulated both by intrinsic genes and extrinsic signaling stem from the milieu. However, as a part of cancer stem cell niche or tumor microenvironment, the functional role of differentiated cancer cells in maintaining CSCs self-renewal still remains to be explored.Using the previously established Nanog-GFP Report system stem cell model, we have demonstrated that Nanog positive tumor cells have the characteristics of cancer stem cells. Based on these results we observed that the Nanog negative tumor cells play a supporting role in retaining the self-renewal of Nanog positive CSCs. Moreover, cytokines–array analysis showed that Interleukin-17 E might be the mediator to regulate the crosstalking between Nanog positive CSCs and Nanog negative non-CSCs. This work aims to uncover the role and mechanisms of non-CSCs maintaining and supporting the stemness of CSCs, which will enrich the concept of CSCs and provide the new therapeutic strategy of liver cancer treatment.Materials and Methods:1. Isolation of CSCs according to the GFP expression intensity through a Nanog promoter-driven green fluorescent protein(GFP) reporter system. NanogPos CSCs and NanogNeg non-CSCs are sorted from HCC cells by fluorescence activated cell sorting(FACS). Then their distinct biological characteristics were confirmed in vitro and in vivo.2. Cultivation of the cells as NanogPos group, NanogNeg group and their mixture with different proportions groups. All groups were cultured for 9 days in vitro in order that their proliferation rate can be analyzed, and NOD-SCID mice were used to detect the tumor-initiating capability of these groups in vivo.3. Cytokines-array analysis was performed to investigate the supernatant from 24 h cultured NanogPos CSCs and NanogNeg non-CSCs. ELISA and RT-PCR were conducted to confirm the results of cytokines-array analysis. Immunofluorescence, Western Blot, and Immunohistochemistry analysis were performed to detect the receptor RB expression on NanogPos CSCs and NanogNeg non-CSCs.4. Knocking-down Interleukin-IL-17 RB by Lentivirus-based sh RNA knocking-down systems. The p LKO.1–TRC Cloning Vector is used, and the p LKO.1-scramble sh RNA vector was used as control. Lentiviral plasmids were transfected into HEK-293 T cells using calcium phosphate precipitation as our previously study reported for optimization of lentiviral infection conditions. HCC NanogPos cells were infected with sh RNA-IL-17 RB, while HCC NanogNeg cells were infected with shRNA-IL-17 E. Both HCC cells were infected with scramble as control.5. After dealing with recombinant human IL-17 E or knocking-down IL-17 RB of the cells, Cell Titer-Blue Cell Viability Assay was performed to detect the cell proliferation rate, and clone-formation and sphere-formation capability were investigated.6. RT-PCR and Western Blot are conducted to investigate the possible pathways which are involved in IL-17E/RB axis, and then the activators and inhibitors were used to dynamically mediate the pathways signaling.7. Bioinformatics analysis of IL-17 Rs and stimulation of the crystal structure of IL-17 RB on computer. Immunohistochemical stain was used to detect the IL-17 RB expression in tumor and adjacent non-tumor patient tissues.Results:1. Co-cultured NanogNeg non-CSCs and NanogPos CSCs groups grow much faster than the corresponding control cells, and the proliferation rates of co-cultured groups are significantly higher than the theoretical value. Mixing a small amount of NanogNeg cells into NanogPos cells could significantly promote the global cells growth rate, although non-CSCs generally proliferate more slowly than CSCs counterparts do. Moreover, the cell proportion of 1:9(NanogNeg:NanogPos= 1:9) displayed the greatest difference among all these groups.2. NOD-SCID mice tumor-initiating model showed that tumors derived from NanogNeg/Pos=1:9 or NanogPos/Neg=1:9 groups were significantly larger and heavier than NanogPos or NanogNeg groups, respectively. Importantly, the tumor derived from NanogNeg/Pos=1:9 grou initiated the earliest(17 days after injection) among these groups.3. Cytokines-array analysis on supernatant showed that TGF-β2, IL-17 E and MMP-13 expressed higher levels in NanogNeg non-CSC supernatant, while IGFBP-7, GDF-15, WIF-1, u PAR, VEGFR2, CXCL16, and TSP expressed more highly in NanogPos CSC supernatant. Quantitative real-time PCR(RT-PCR) further verified the results in HCC cells and the data showed that among these cytokines, IL-17 E was significantly highly expressed in NanogNeg non-CSCs. ELISA analysis of supernatant from 24 h cultured HCC cells further confirmed that IL-17 E was expressed at a significantly higher level in NanogNeg non-CSCs.4. RT-PCR test showed that IL-17 RB was significantly highly expressed in NanogPos cell compared with NanogNeg cells. Western Blotting tests further confirmed the increased expression of IL-17 RB in NanogPos cells, and patient-derived primary HCC cells. Similarly, an immunofluorescence assay and immunohistochemical analysis demonstrated that IL-17 RB was predominately localized to NanogPos CSCs.5. Exogenous IL-17 E could significantly promote CSC cells proliferation, clone-formation and sphere-formation both in the 100ng/m L groups and 200ng/m L groups. Knocking down of either IL-17 RB or IL-17 E could significantly reduce cell proliferation rate and clone-formation and sphere-formation capabilities.6. Both NF-κB and IκB phosphorylation decreased by silencing IL-17 RB, and exposure to IL-17 E could up-regulate phosphorylation of NF-κB(p65) and IκB, which reproduced the stimulate effect of TNF-α, an activator of NF-k B(p65). In addition, the NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC) blocked these effects of IL-17 E in NanogPos cells. Meanwhile, knocking-down of IL-17 RB remarkably down-regulated phosphorylation Stat3(p-Stat3) in HCC cells and Stat3 inhibitor S31-201, Jak pan-inhibitor INCB, and Jak2 inhibitor AG490 could all effectively block Stat3 phosphorylation following IL-17 E treatment.7. Western blotting and immunohistochemical analysis displayed that the majority of IL-17 RB staining was confined to carcinoma tissue compared with adjacent normal tissue. Simulating the crystal structure of IL-17 RB on computer shows that IL-17 RB as well as other IL-17 Rs family member contains the Box1 and Box2 regions, which enable IL-17 E to activate Stat3 pathway through binding to its upstream activator JAKs directly.Conclusion: In this study we demonstrates that there is cross talk between NanogNeg non-CSCs and NanogPos CSCs in HCC. Non-CSCs nourish and back-feed CSCs as the component of CSC niche through secreting IL-17 E to activate NF-kB and JAK/Stat3 pathways by preventing apoptosis and maintaining stemness, respectively. We provide novel insights into a supporting role of non-CSCs to CSCs, which enrich the concept of CSC-niche and also propose the potential therapeutic strategy for targeting of the IL-17E/IL-17 RB axis in HCC.