The Mechanism Of RSL3 Regulating Glioma Stem Cells Differentiation Via Tgm2/AKT/ID1 Signaling Pathway

Background:Glioma is the most frequently diagnosed primary tumor of the central nervous system,with a high rate of recurrence and high heterogeneous.Current treatment options for glioma patients includes surgery,radiotherapy,and chemotherapy.However,surgery cannot achieve a complete resection effect especially for diffuse invasive glioma,and long-term radiotherapy and chemotherapy may lead to tumor recurrence and drug resistance.Therefore,the median survival time of GBM patients with newly diagnosed glioma is only 14–16 months,and only 5.5%of GBM patients survive for more than 5 years.In recent years,the existence of tumor stem cells has been widely confirmed and is considered to be the main reason for tumor recurrence and chemoresistance.As one of the characteristics of tumor stem cells,the disorder of cell differentiation plays an important role in maintaining the stemness of tumor stem cells.Therefore,deepening the research on the mechanism of tumor stem cells may provide new strategies for the treatment of glioma.Glioma stem cells（GSCs）are a self-renewing population of tumor stem cells which have the capacity to proliferate and generate new stem and daughter cells that undergo differentiation into glioma cells to reduce their tumorigenicity.Studies have found that GSCs and non-GSCs can switch with each other via different mediate mechanism.For example,some glioma cells can dedifferentiate to GSCs after TMZ therapy,while GSCs can inhibit their differentiation ability via depending on different ways,thus maintaining abnormal self-renewal,enabling treated tumors to regain their proliferation ability,and further inducing tumor recurrence and drug resistance.This suggests that the stem cell characteristics of GSCs can be regulated.Once GSCs undergo differentiation,their stemness and pluripotency can be reduced to the extent that current drugs can strongly block their growth which not only improve the sensitivity of chemotherapy but also reduce the tumorigenicity.Therefore,new methods that can target GSCs differentiation may be used as potential therapeutics for glioma.RSL3 is a small-molecule inhibitor of glutathione peroxidase 4（GPX4）.Studies have found that RSL3 not only induce the death of various types of differentiated cancer cells via ferroptosis,but also enhance the sensitivity of tumor cells to chemotherapy.RSL3combined with radiotherapy can exhibit a synergistic therapeutic effect in inducing glioma cell death;RSL3 can enhance the therapeutic sensitivity of TMZ via inhibiting EMT-related pathway;Our results also reported that RSL3 can enhance the sensitivity of prostate cancer cells to cisplatin by inhibiting glycolysis;In addition,RSL3 can induce glycolysis dysfunction of glioma cells.To sum up,RSL3 may play a significant role in anti-tumor therapy,but the effects of RSL3 on stem cells has not yet been elucidated.In the previous work,our research established a mouse spontaneous glioma model through transgenic mice(h GFAP-Cre+;P53^lox/lox;Pten^lox/+),and isolated brain glioma stem cells which were significantly similar to pathological and molecular levels of clinical human primary glioma.Therefore,the experiment on GSCs can help us to further study glioma.We detected the effect of RSL3 on the proliferation,self-renewal and differentiation of GSCs and the results showed that RSL3 could inhibit cell viability,self-renewal capacity,and regulate the differentiation ability of GSCs.Therefore,in-depth study of the underlying mechanism of RSL3 in regulating GSCs differentiation may provide a new theoretical basis for glioma treatment.Objective:To explore the effects of RSL3 on the proliferation,stem cell characteristics and differentiation ability of glioma stem cells,and to further detect its regulatory mechanism in vivo and in vitro.Methods:1.The effects of RSL3 on proliferation,self-renewal and differentiation ability of glioma stem cells in vitro.（1）The mouse glioma stem cell CSC2078 and human glioma stem cell TS576were cultured in neurobasal media,which added epidermal（EGF;20 ng/m L）and basic fibroblast（b FGF;10 ng/m L）growth factors.The effects of RSL3 on the proliferation of glioma stem cells were detected by CCK-8,growth count,and soft agar colony formation assay.The effects of RSL3 on the self-renewal ability of glioma stem cells were detected by limiting dilution analysis and neuro sphere formation test.（2）The mouse glioma stem cells CSC2078 and human glioma stem cells TS576were cultured in differentiation medium which containing 1%heat-inactivated fetal bovine serum without growth factors.To detect the effect of RSL3 on the stem cell characteristics of glioma stem cells,immunofluorescence staining and western blotting were used to evaluate the expression of stem cell markers（Nestin,Sox2）.To ascertain the differentiated phenotypes of RSL3-treated GSC,immunofluorescence staining and western blotting were used to exam the expression of several differentiation markers including S-100β/GFAP（astrocytic）,Olig2（oligodendrocytic）,and Neu N（neuronal）.2.The effects of RSL3 on proliferation and differentiation ability of glioma stem cells in vivo.The mouse glioma stem cell CSC2078 were used to construct the subcutaneous tumor model of nude mice.After tumor formation,the nude mice were randomly divided into two groups,vehicle and RSL3 treatment group（50mg/kg）.The RSL3treatment group was injected with drugs around the tumor every other day.The weight and tumor volume of mice were evaluated every three days and draw the growth curve.Observe the mental state,activity and food intake of the mice,and observe and record the nervous system symptoms.After 3 weeks of treatment,the mice were euthanized.Tumor tissues resected from the treated mice were used for IHC analysis to determine the proliferation and differentiation indices.3.To explore the mechanism of RSL3 on GSCs differentiationUnder the condition of differentiation culture,the effect of RSL3 on the expression of glutathione peroxidase 4 in GSCs was detected by Western blotting.Intracellular Fe²⁺levels and reactive oxygen species（ROS）were evaluated by iron colorimetric assay kit and flow cytometry.Further,RNA-Seq and proteomics were used to detect the genes that changed at the transcriptional and protein levels after RSL3 treatment on GSCs,and then conjoint analyze the transcriptomic and proteomic data to explore the key role targets and related pathways of RSL3 in inducing GSCs differentiation.Construction of overexpression plasmids,applied inhibitor of pathway,RT-q PCR,Western blotting and immunofluorescence experiments were used to verify the therapy target and mechanism.Results:1.The effects of RSL3 on proliferation,self-renewal and differentiation ability of glioma stem cells in vitro.Under proliferation medium,CCK-8 assay showed RSL3 inhibited cell viability of GSCs in a dose-dependent manner.The growth curve and soft agar colony formation assay showed that RSL3 inhibited cell proliferation and suppressed colony formation ability.Neural sphere formation assay and limiting dilution analysis showed that the self-renewal capacity of GSC was strongly inhibited by RSL3.Under differentiation medium,western blotting and immunofluorescence staining showed that the levels of Nestin and Sox2 markedly decreased in RSL3-treated GSC.The treated cells were stretched and showed strong adherence when compared to the sham-treatment controls that were round-shaped under light microscopy.Western blotting and immunofluorescence staining results showed that S-100βlevels significantly increased,Olig2 and GFAP levels decreased,and Neu N levels did not significantly alter in RSL3-treated GSCs cells.Therefore,RSL3 triggers GSC differentiation into astrocytes.2.The effects of RSL3 on proliferation and differentiation ability of glioma stem cells in vivo.RSL3 significantly reduced tumor volume and tumor growth rate in compared to those of the vehicle group.Importantly,HE results showed that RSL3 did not cause any significant damage to organs or tissues（heart,liver,spleen,lung,and kidney）or weight loss during the entire treatment period.Therefore,RSL3 inhibits GBM growth without causing side effects.IHC results showed that RSL3 strongly suppressed the expression of Ki-67,Nestin,and Sox2 compared to that of the vehicle-treated control,which means that RSL3 inhibited the stem cell characteristics of GSCs in vivo.The RSL3-treated tumors expressed higher levels of S-100βand lower levels of GFAP,Olig2 and equivalent levels of Neu N compared to those of the vehicle-treated controls,which proved that RSL3 promoted GSCs differentiation into astrocytes in vivo.3.To explore the mechanism of RSL3 on GSCs differentiationUnder differentiation medium,western blotting results showed that GPX4 level was not downregulated by RSL3 treatment.Intracellular Fe²⁺levels were not altered by RSL3 treatment.Similarly,flow cytometry did not show any significant differences in intracellular ROS levels following RSL3 treatment.Therefore,ferroptosis-related mechanisms are not involved in GSC differentiation induced by RSL3.RNA-seq analyses showed that approximately 100 genes were upregulated and139 were downregulated upon RSL3 treatment compared to those of the controls.In proteomic analysis,51 proteins were downregulated and 120 were upregulated.Conjoint analyses of transcriptomic and proteomic data revealed that the expression of12 genes changed concurrently at both m RNA and protein levels.These observations were also confirmed by Western blotting and RT-q PCR analyses which implied that RSL3 induce GSCs differentiation via inhibiting the expression of Tgm2.Western blotting results showed that Tgm2 overexpression rescued the changes in stemness-related markers,and differentiation caused by RSL3.The levels of Tgm2 and ID1significantly decreased in RSL3-treated GSC in comparison to those in the vehicle-control.ID1 overexpression could rescue the differentiation effects caused by RSL3,while did not increase Tgm2 expression which identified ID1 as a downstream signaling target of Tgm2.Blocking the phosphoinositide-3 kinase（PI3K）/AKT pathway with LY294002 suppressed PI3K,p-AKT,and ID1 levels but not Tgm2.Tgm2overexpression abrogated the changes in PI3K,p-AKT,and ID1 levels caused by LY294002.Taken together,RSL3 triggers GSCs differentiation by suppressing the Tgm2/AKT/ID1 signaling axis.Conclusion:1.RSL3 suppresses GSCs proliferation and self-renewal capability.2.RSL3 promotes GSCs differentiation into astrocytes.3.RSL3 inhibits GSCs growth in vivo with no significant side effect.4.RSL3 promotes GSCs differentiation via downregulation of the Tgm2/AKT/ID1 axis.