Arsenic Trioxide Effectively Disrupts Small Cell Lung Cancer Stem Cells By Blocking Hedgehog Signaling Pathway

?Background and Objective?Small cell lung cancer?SCLC?is a type of highly malignant neuroendocrine tumors and accounts for about 15%to 20%of lung cancer.SCLC has aggressive biological behavior such as rapid growth and early invasion of blood vessels or lymphatic vessels.When diagnosed,SCLC is usually widely metastatic.The treatment of SCLC is mainly based on chemotherapy combined with radiotherapy.Although the initial remission rate is high,the long-term survival has not been significantly improved because of the high resistance and recurrence rate.For a long time,there has been no significant progress in the treatment of SCLC.Therefore,the clinicians need to find a more effective strategy for the treatment of SCLC.The theory of cancer stem cells?CSCs?provides a new breakthrough for cancer research.CSCs just like seeds in tumors.They are small subpopulations of malignant cells with unlimited proliferation capacity and multipotency.They generate heterogeneous cell populations to constitute the tumors,so they are also called tumor initiating cells?TICs?.CSCs have abilities of migration and movement.After reaching the metastatic site by vascular dissemination,they differentiate into heterogeneous tumor cells,maintain a certain proportion through self-renewal,and generate tumors at the metastatic sites consistent with the primary tumor type.Multiple signaling pathways,such as Hedgehog,Wnt/?-catenin and Notch pathway,play important roles in the self-renewal,differentiation and metastasis of CSCs.These signaling pathways are involved in the organ formation during embryonic development and are inactivated in normal tissues after birth.If they are abnormally activated,tumors may occur.Arsenic trioxide?As₂O₃?,also known as arsenic acid,has been approved by FDA for the treatment of acute promyelocytic leukemia?APL?.As₂O₃ caused a degradation of PML-RAR?fusion protein,induced cell differentiation at low dose?0.1-0.5?M?and led to apoptosis at high dose?0.5-2.0?M?.Since then,As₂O₃ has been found to have inhibitory effects on a variety of solid tumors.As₂O₃ could inhibit the growth of CSCs in gliomas,liver cancer and pancreatic cancer by blocking Hedgehog or Notch pathway.However,most of these studies were performed in vitro.At present,there have been few reports investigating the therapeutic effects of As₂O₃on SCLC.In vitro,As₂O₃ caused cell apoptosis of SCLC through mitochondrial dysfunction,resulting from reactive oxygen species?ROS?production,GSH depletion and Bcl-2 down-regulation.In nude mice xenografts,As₂O₃ inhibited the growth of SCLC and increased the expression of neuroendocrine markers.We have previously demonstrated that As₂O₃ significantly inhibited the growth of SCLC-derived xenograft tumors,which may be related to the antiangiogenic effects of As₂O₃.Our preliminary studies demonstrated that As₂O₃ inhibited the formation of tumor spheres,down-regulated the stem cell biomarker CD133 and stem cell transcription factors such as SOX2 and OCT4,and decreased the expression of Hedgehog signaling molecules in SCLC cell line H446.However,it is still unclear whether As₂O₃ can inhibit SCLC stem cells in vivo.Besides,the mechanism of As₂O₃ acting on SCLC stem cells remains to be further investigated.Firstly,we will enrich tumor spheres from SCLC cell lines?H446 and H209?under serum-free condition and further identify whether tumor spheres have the characteristics of CSCs.Then,through a series of in vitro and in vivo experiments,we will investigate the effects of As₂O₃ on the growth of SCLC stem cells and on the expression of stem cell transcription factors.By constructing SOX2 knockdown and control lentivirus,we will study the effect of SOX2 knockdown on the self-renewal of SCLC stem cells.Finally,whether the Hedgehog signaling pathway is involved in the inhibitory effects of As₂O₃ on SCLC stem cells will be studied.This study investigates the effects of As₂O₃ on SCLC stem cells and its mechanism in detail.It is expected to provide more experimental evidence for the clinical application of As₂O₃ in the treatment of SCLC.?Methods?Part 1 Culture and identification of small cell lung cancer stem cellsFirstly,we enrich CSCs from the SCLC cell lines H446 and H209.The SCLC celllines H446 and H209 were cultured as floating tumor spheres in stem cell culture medium in ultra low-adherent plates.Then,several experiments were performed to study if the H446 spheres and H209spheres have the characteristics of stem cells.We performed qPCR and western blot analysis to detect the expression levels of stem cell transcription factors SOX2,NANOG,OCT4 and c-Myc in spheres and their parental cells.Equal numbers?1×10⁴,1×10⁵,1×10⁶cells?of sphere cells and their parental cells were subcutaneously implanted in the right and left flanks of nude mice,respectively.The tumorigenic ability of the two groups was compared.We performed Hematoxylin and eosin?HE?stain to analyze the tumor histological features.Part 2 Inhibitory effects of As₂O₃ on growth of SCLC stem cells in vitro and in vivoTo study the inhibitory effects of As₂O₃ on the growth of SCLC stem cells in vitro,H446 CSCs and H209 CSCs were treated with different concentrations of As₂O₃.Normal saline was used as the control.Cell proliferation of each group was determined by the CCK8 assay.The number of clones was detected by colony formation assay.The sphere formation ability was detected by sphere formation test.The cell apoptosis and cell cycle were detected by flow cytometry.The expression of stem cell transcription factors SOX2and c-Myc was detected by western blot.To study the effects of SOX2 knockdown on the self-renewal capacity of SCLC stem cells,H446 CSCs and H209 CSCs were transiently infected with control lentivirus?shNT?or SOX2 knockdown lentivirus?shSOX2?.After 72 hours of infection,the expression of green fluorescence was observed under a fluorescence microscope.Western blot was performed to detect the expression levels of apoptotic markers cleaved Caspase-3 and cleaved PARP.The sphere formation ability of each group was compared by sphere formation test.To investigate the inhibitory effects of As₂O₃ on the growth of subcutaneousxenografts of SCLC stem cells.We constructed H446 CSCs and H209 CSCs-derived subcutaneous xenografts.When the average tumor volume reached 60-70 mm³,mice were treated with either 2.5 mg/kg or 5.0 mg/kg As₂O₃ or vehicle control?NS?once daily for 14days.The tumor volume and weight of each group were compared.TUNEL stain was performed to detect the proportion of apoptotic cells.Immunofluorescence stain was performed to detect the proportion of SOX2⁺or c-Myc⁺cell population in the tumor tissues.Part 3 Hedgehog pathway mediated the inhibitory effects of As₂O₃ on SCLC stem cellsThe Hedgehog pathway is involved in maintaining the function of CSCs.GLI1 is a key transcription factor of the Hedgehog pathway.Firstly,the effect of As₂O₃ on the expression of GLI1 and its down stream genes PTCH1 and n-Myc were investigated.H446CSCs and H209 CSCs were treated with different concentrations of As₂O₃.Western Blot was performed to determine the expression level of GLI1 protein.qPCR was performed to determine the expression levels of PTCH1 and n-Myc.Then,the effects of As₂O₃ on the expression of GLI1 protein and its target genes PTCH1 and n-Myc in tumor tissues was studied.Western blot was performed to detect the expression of GLI1 protein in the control group,2.5 mg/kg As₂O₃ group and 5.0 mg/kg As₂O₃ group.qPCR was performed to determine the expression levels of PTCH1 and n-Myc in the control group,2.5 mg/kg As₂O₃ group and 5.0 mg/kg As₂O₃ group.GANT-61 is a type of GLI inhibitor.To investigate whether blocking Hedgehogpathway inhibits the growth of SCLC stem cells and enhances the effects of As₂O₃.H446CSCs and H209 CSCs were treated with control,As₂O₃,GANT and combined treatment.The sphere formation ability of each group was detected by sphere formation test.Apoptotic rate was assessed by flow cytometer and western blot analysis.Western Blot analysis was performed to determine the expression levels of GLI1,SOX2 and c-Myc.To investigate whether blocking Hedgehog pathway inhibits the growth of SCLC stem cells in nude mice.We constructed H446 CSCs and H209 CSCs-derived subcutaneous xenografts.Mice were treated with either 50 mg/kg GANT-61 or equivalent solvent?Corn oil:ethanol=4:1?once daily for 14 days.The tumor volume and weight of each group were compared.TUNEL stain was performed to detect the proportion of apoptotic cells.Immunofluorescence stain was performed to detect the proportion of SOX2⁺or c-Myc⁺cell population in the tumor tissues.?Results?Part 1 Culture and identification of SCLC stem cells1.We grew the SCLC cell lines H446 and H209 in defined stem cell medium.After 4to 6 days of culture,we observed that cells were able to form floating spheres.These spheres could be passaged serially more than 30 times,suggesting that these populations are capable of potent self-renewal.2.qPCR and Western blot analysis showed that H446 spheres expressed higher levels of SOX2,OCT4 and c-Myc,compared with H446 parental cells.Similarly,the expression levels of SOX2,NANOG and c-Myc were higher in H209 spheres than in H209 parental cells.3.The tumor formation latency of spheres was shorter than that of their parental cells,the tumor growth rate of the former was faster than the latter,and the tumor volume was larger than the latter.The tumor formation rates of H446 spheres or H209 spheres were significantly higher than those of parental H446 cells or H209 cells.In summary,sphere cells cultured in serum-free medium have the chacteristics of CSCs.Therefore,in the following text,H446 and H209 sphere cells will be referred to as H446 CSCs and H209 CSCs,respectively.Part 2 Inhibitory effects of As₂O₃ on the growth of SCLC stem cells in vitro and in vivo1.As₂O₃ could inhibit the proliferation and clonogenic capacity of H446 CSCs and H209 CSCs in a concentration-and time-dependent manner.As₂O₃ could significantly decrease the number and the size of tumor spheres in H446 CSCs and H209 CSCs.The number of spheres formed in the high concentration group was significantly lower than that in the low concentration group.After the withdrawal,the inhibitory effects of As₂O₃on sphere formation still exists.2.In H446 CSCs and H209 CSCs,the total apoptotic rates of the As₂O₃-treated groups were significantly higher than that of the control group.The total apoptotic rate of the high concentration group was significantly higher than that of the low concentration group.The apoptotic marker cleaved PARP was increased in response to As₂O₃ treatment.However,cell cycle analysis showed that the proportions of G0/G1,S and G2/M phase cells in the As₂O₃-treated groups were not significantly different from those in the control group.3.As₂O₃ treatment significantly down-regulated the expression of SOX2 and c-Myc in H446 CSCs and H209 CSCs compared with the control.Besides,the down-regulation effects of the high concentration group were more obvious than the low concentration group.4.Green fluorescence could be observed in H446 CSCs or in H209 CSCs infected with shNT or shSOX2.The mRNA level of SOX2 in the shSOX2 groups was significantly lower than that in the shNT group.The number of tumor spheres formed in the shSOX2groups was significantly lower than that in the shNT group.The volume of tumor spheres was also significantly reduced by the shSOX2 groups.shSOX2 groups showed higher expression levels of Caspase3 cleavage and PARP cleavage when compared with the shNT group.5.In H446 and H209 CSCs xenograft models,the average tumor volumes of the 2.5and 5.0 mg/kg As₂O₃ groups were significantly smaller than that of the control group.At the end of the intervention,the mean tumor weight of the As₂O₃-treated groups was significantly lower than that of the control group,and the mean tumor weight of the 5.0mg/kg As₂O₃ group was lower than that of the 2.5 mg/kg As₂O₃ group.TUNEL stain showed that the proportions of apoptotic cells in the 2.5 and 5.0 mg/kg As₂O₃ groups were significantly higher than that in the control group.The proportion of TUNEL⁺cells in the5.0 mg/kg As₂O₃ group was the highest.Immunofluorescence staining showed that the proportions of SOX2⁺or c-Myc⁺cells in the 2.5 and 5.0 mg/kg As₂O₃ groups were significantly lower than that in the control group.The proportion of SOX2⁺or c-Myc⁺cells in the 5.0 mg/kg As₂O₃ group was the lowest.Part 3 Hedgehog pathway mediated the inhibitory effects of As₂O₃ on SCLC stem cells1.In vitro,As₂O₃ treatment could significantly decrease the expression levels of GLI1protein and its target genes PTCH1 and n-Myc in H446 CSCs and H209 CSCs.The down-regulation effect of the high concentration group was more obvious than the low concentration group.2.In vivo,2.5mg/kg or 5mg/kg As₂O₃ intervention could down-regulate the expression levels of GLI1 protein and its target genes PTCH1 and n-Myc in SCLC xenograft tissues.The effect of the 5mg/kg group was more obvious than the 2.5mg/kg group.3.As₂O₃,GANT-61 and the combined treatment significantly decreased the number and the size of the tumor spheres in H446 CSCs and H209 CSCs when compared with the control.The sphere number of the combined group was the least,which was significantly less than that of the As₂O₃ group or GANT-61 group.As₂O₃,GANT-61 and the combined treatment significantly increased the total apoptotic rate of H446 CSCs and H209 CSCs when compared with the control.The total apoptotic rate of the combined group was the highest.As₂O₃,GANT-61 and the combined treatment significantly decreased the expression of GLI1,SOX2 and c-Myc protein in H446 CSCs and H209 CSCs when compared with the control.The down-regulation effect of the combined group was the most significant.4.In H446 and H209 CSCs xenograft models,the average tumor volumes of GANT-61group were significantly smaller than that of the control group.At the end of the intervention,the mean tumor weight of the GANT-61 group was significantly lower than that of the control group.TUNEL stain showed that the proportion of apoptotic cells in the GANT-61 group wa significantly higher than that in the control group.Immunofluorescence staining showed that the proportions of SOX2⁺or c-Myc⁺cells in the GANT-61 group were significantly lower than that in the control group.?Conclusion?1.CSCs could be enriched from the SCLC cell lines H446 and H209 by their ability to form tumor spheres in serum-free conditioned culture.2.As₂O₃ could effectively inhibit the growth of SCLC stem cells,induced significant apoptosis and down-regulated the expression of stem cell transcription factors SOX2 and c-Myc in vitro and in xenografts.SOX2 knockdown could inhibit the growth and induce apoptosis of SCLC stem cells.3.As₂O₃ significantly down-regulated GLI1 protein?a key transcription factor of the Hedgehog pathway?and its target genes PTCH1 and n-Myc in vitro and in vivo.The GLI inhibitor?GANT-61?mimicked and enhanced the inhibitory effects of As₂O₃ on SCLC stem cells?including induction of apoptosis and down-regulation of SOX2 and c-Myc?.These results suggest that As₂O₃ may down-regulate SOX2 and c-Myc proteins by inhibiting the Hedgehog pathway transcription factor GLI1,thereby inducing apoptosis and supressing the growth of SCLC stem cells in vitro and in nude mice.