The Effect Of Defeating Liver Cancer Stem Cells By Targeting PIK3C3 And PI3K In Hepatocellular Carcinoma

Our previous study found that SGK3 plays an important role in the invasive potential of HCC cells and epithelial-mesenchymal transition(EMT).However,the role and mechanism of SGK3 in CSCs has not been reported.PI3K activity is stimulated by diverse oncogenes and growth factor receptors,and elevated PI3K signaling is considered a hallmark of cancer.SGK3 has been reported to be a critical effector of oncogenic PIK3CA mutant breast cancer cells in which Akt is dispensable.The mechanism of PI3K regulating SGK3 is unclear.The Class III phosphoinositide 3-kinase(PIK3C3),also called vacuolar protein sorting 34(Vps34),plays important roles in the control of both autophagic and endocytic trafficking systems,which plays critical roles in a wide range of cellular processes.Furthermore,the lipid kinase activity of PIK3C3 acts as a major source of phosphatidylinositol3-phosphate(PtdIns3P)in cells,which functions as secondary messenger or docking signal for proteins containing PtdIns3P-binding domains,such as FYVE or PX.Although accumulating evidence indicates that Vps34 may play a critical role in the progression and development of cancers,including colon cancer,breast cancer,the role and mechanism of PIK3C3 in CSCs was unknown.Part?The effect of class I PI3K inhibition in liver cancer stem cell self-renewalObjective:To explore the role of SGK3 in liver CSCs regulation and the mechanism underlying PI3K regulation SGK3.Methods:1.To explore the relevance between SGK3 and liver CSCs,we detected expression levels of SGK3 in CSC-enriched hepatoma spheroids and CD133+cells.2.To assess the role of SGK3 in liver CSC regulation,we established a lentivirus-mediated stable SGK3 or negative control(NC)expression cell lines and the expression of stemness genes were detected by Western blot and qPCR.To further elucidate the role of SGK3 in CSCs,we examined spheroid-forming ability.3.To further confirm the involvement of SGK3 in liver CSC expansion,2 lentivirus vectors were designed to express shRNA for SGK3 knockdown(SGK3 shRNA1 and SGK3 shRNA2).The expression of stemness genes were detected by Western blot and qPCR.Spheroid formation assay was used to determine the spheroid formation ability.The proportion of CD133+cells in SGK3 knockdown cells was evaluated by flow cytometric assay.4.We treated HCC cells with PI3K inhibitors(LY294002 and ZSTK474)for 24 h with increasing doses.Western blot and qPCR were used to detected the expression of stemness genes.The proportion of CD133+cells was evaluated by flow cytometric assay.The effect of the Class I PI3K inhibitorZSTK474onthegrowthofMHCC97Hcells.Immunohistochemical staining of CD133 in the xenografted tumor xenografts treated with ZSTK474 or control.5.Huh7 and MHCC97H cells were treated with VPS34-IN1 for 24 h,and cell lysates were subjected to western blot analysis to detect the expression of SGK and stemness genes.Immunofluorescence and flow cytometric were also used to determine the expression of stemness genes.6.Expression of?-catenin,active GSK3?(phosphorylated at Ser9)/total GSK3?and SGK3 in HCC cells stably overexpressing SGK3 or shRNA was detected by western blot.Treatment of MHCC97H cells with AR-A014418 for 24 h and the expression of CD133 was detected by RT-PCR.MHCC97H-SGK3 or MHCC-97H-NC were treated with 20?M of GSK3?inhibitor AR-A014418 and subjected to spheroid formation assay.7.The expression of?-catenin in MHCC97H cells treated with 2.5?M of ZSTK474 for 0 to 5 days was detected by western blot.Western blot analysis of the effect of SGK3 knockdown by siRNA on the induced overexpression of?-catenin by treatment with 2.5?M ZSTK474.Results:1.The results showed that the expression of several stem cell markers and the activation of SGK3 were all upregulated in the spheroids and CD133+cells.2.Notably,SGK3 overexpression significantly enhanced the expression of stemness genes and spheroid formation ability.Conversely,suppression of SGK3 in HCC cells had an opposite effect.3.Prolonged treatment of HCC cells with class I PI3K inhibitors leads to activation of SGK3 and expansion of liver CSCs in in vitro and in vivo experiment.4.Inhibition of hVps34 can block SGK3 activity and suppress liver CSC expansion induced by PI3K inhibitors.5.Mechanistically,SGK3 promoted?-catenin accumulation by suppressing GSK-3?-mediated?-catenin degradation in liver CSCs,and then promoting the expansion of liver CSCs.6.We also found that prolonged treatment of HCC cells with PI3K inhibitors stimulates the?-catenin signaling pathway via activation of SGK3.Conclusion:Prolonged inhibition of class I PI3K promotes liver CSC expansion by augmenting SGK3-dependent?-catenin stabilisation,and effective inhibition of SGK3 signalling may be useful in eliminating liver CSCs and in PI3K pathway-targeted cancer therapies.Part?The effect of PIK3C3 inhibition in liver cancer stem cell self-renewalObjective:To further explore the important role of PIK3C3 in the maintenance of HCC cancer stem cells,and to analyze the efficacy and mechanism of inhibiting PIK3C3 on HCC cancer stem cells elimination.Methods:1.To determine PIK3C3 expression in human HCC,IHC was conducted on commercial HCC tissue microarrays of 163 paired HCC tumor and peritumor tissues.To explore the relevance between PIK3C3 and liver CSCs,we analyzed the expression of PIK3C3 in spheroids and CD133-cells.2.In attempt to explore the role of PIK3C3 in liver CSCs self-renewal,two siRNAs against PIK3C3 were synthesized to silence the expression of PIK3C3.The expression levels of stemness genes after PIK3C3 knockdown in HCC cells were detected by Western blot and qPCR.We next established a lentivirus-mediated stable PIK3C3 overexpression cell lines using MHCC97H and Huh7 cells.Spheroid formation assay was used to examine spheroid formation ability.3.Expression levels of stemness genes in HCC cells treated with DMSO or VPS34-IN-1 detected by qPCR,flow cytometric and Western blot.To further measure the effect of inhibition PIK3C3 on liver CSCs,a tumor initiation capacity assay was conducted in mice.To evaluate the effect of VPS34-IN-1 on tumor growth,we using a subcutaneous mouse xenograft model.After the tumors formed about 400 mm3,the mice in groups of three were orally administered respectively with 0(control),40,80,and 160mg/kg of VPS34-IN-1 for 13 days.The expression of CD133 in tumors was detected by IHC.4.We treated HCC cells with VPS34-IN-1 for 24 h and detected the autophagy markers expression.To investigate whether VPS34-IN-1eliminates liver CSCs via the inhibition of autophagy,we treated HCC cells with VPS34-IN-1 plus Rapamycin,a mTOR signaling inhibitor to induce autophagy.Western blot,qPCR and Flow cytometric analysis were used to detect the expression of stemness genes.5.AMPK total protein levels and activating phosphorylation levels in HCC cells treated with VPS34-IN-1 24 h were detected by Western blot.Expression levels of stemness genes in Huh7 and MHCC97H cells treated with AICAR or Metformin were detected by Western blot.Expression levels of stemness genes in Huh7 and MHCC97H cells after AMPK knockdown or inhibition were detected by Western blot.Western blot was used to detected the expression levels of stemness genes in Huh7 and MHCC97H cells treated with VPS34-IN-1,Compound C and both for 24 h.6.Expression levels of stemness genes in Huh7 and MHCC97H cells treated with VPS34-IN-1,ZSTK474 and both for 24 h were detected by Western blot,qPCR and Flow cytometric analysis.We further evaluate the effect of this combination strategy in vivo using subcutaneous mouse xenograft model.MHCC97H cells were subcutaneously injected into6-week-old female athymic nude mice and allowed to form tumors.Once the tumors reached 400 mm ~3,twelve mice were randomly divided into four groups:the VPS34-IN-1,the ZSTK474 group,the combination group and the control group.Tumors were measured throughout the treatment period.The expression of CD133 in tumors was detected by IHC.Results:1.A remarkable increase of PIK3C3 was detected in HCC tissues and liver CSCs.2.Up-regulated PIK3C3 facilitated liver CSCs expansion in HCC cells,RNA interference–mediated silencing of PIK3C3 had an opposite effect.3.PIK3C3 inhibition by inhibitors effectively eliminates liver CSCs and inhibits the growth of tumors in vivo.4.Although PIK3C3 plays a critical role in autophagy,we find PIK3C3regulates liver CSCs independent of autophagy process.5.We show that PIK3C3 inhibitor suppresses liver CSCs by activation of the AMPK.6.More importantly,treatment with PIK3C3 inhibitor and PI3K inhibitor together dramatically suppress liver CSCs expansion in vitro and in vivo.Conclusion:These findings uncover the effective suppression of liver CSCs by targeting PIK3C3.We have demonstrated that PIK3C3 play a vital role in stemness maintenance of liver CSCs independent of autophagy.PIK3inhibition suppress the expansion of liver CSCs through the activation of AMPK.More importantly,treatment with PIK3C3 inhibitor and PI3K inhibitor together dramatically suppress liver CSCs expansion and tumors growth.