Hypoxia Promotes Human Breast Tumorigenic Cell Growth By Resetting Glycogen Metabolic Program And The Investigation Of Mechanism

Part Ⅰ Abundant glycogen is present in hypoxic breast cancer stem cellsObjective:Hypoxia is known to be commonly present in breast tumor microenvironments.In our previous study,breast cancer stem cells（BCSCs）thrive under hypoxic conditions,but the underlying mechanism remains unclear.Given that glycogen synthesis is elevated in hypoxic tumor microenvironments and BCSCs are apt to reside in more hypoxic tumor microenvironments,we wondered whether BCSCs had a distinctive glycogen metabolic mode.We used cancer stem cells（defined as tumor-regenerating cells,TRCs）which were separated by 3D soft fibrin gels culture to explore the glycogen level in hypoxic breast TRCs.Methods:（1）The correlation between the expression of glycogenesis-related enzymes and the prognosis of breast cancer was analyzed by using the TCGA（https://tcga-data.nci.nih.gov/）database of RNA sequencing（RNA-seq）;（2）Glycogen levels in MCF7,T47D,or SUM159 cells cultured in rigid dish（Bulk）or 90 Pa soft3D fibrin gel（TRCs）were detected by periodic acid-Schiff（PAS）staining;（3）Glycogen levels in MCF7,T47D,or SUM159 cells cultured in rigid dish（Bulk）or 90Pa soft 3D fibrin gel（TRCs）were detected by glycogen detection kit;（4）Glycogen levels in MCF7,T47D,or SUM159 cells cultured in rigid dish（Bulk）or 90 Pa soft3D fibrin gel（TRCs）were detected by transmission electron microscopy;（5）Glycogen levels in the breast ALDH1⁺TRCs in vivo were detected by using the above three methods.Results:（1）The higher the expression of glycogenesis-related enzymes including phosphoglucomutase 1（PGM1）,UDP-glucose pyrophosphorylase 2（UGP2）and glycogen synthase 1（GYS1）,the worse the prognosis of breast cancer,the shorter the survival period of breast cancer patients;（2）In vitro experiments have shown that abundant glycogen is present in hypoxic breast TRCs,instead of differentiated tumor cells;（3）In vivo experiments have shown that glycogen levels in breast ALDH1⁺TRCs were very high,and abundant glycogen is present in hypoxic breast TRCs.Conclusion:3 glycogenesis-related enzymes had a strong correlation with a worsen prognosis of breast cancer.Compared with differentiated breast cancer cells,the glycogen levels in hypoxic breast cancer stem cells is increased.Part Ⅱ Effects and mechanism of hypoxia activated gluconeogenesis-mediated glycogen metabolism pathway on the growth of breast tumor repopulating cellsObjective: Glucose-6-phosphate（G6P）is the core metabolite of glycogen metabolism pathway.What is the source and pathway of G6 P in breast TRCs? The part mains to explore the source and flow of G6 P from glycogenesis and glycogenesis in hypoxic breast TRCs and the effect and mechanism of hypoxia-activated gluconeogenesis-glycogenesis-glycogenolysis-PPP metabolic chain on the growth of breast TRCs.Methods:（1）The changes of M+6 glycogen in Bulk and TRCs under hypoxia were detected by ¹³C-glucose tracing;（2）The expression of hexokinase 2（HK2）in Bulk and TRCs under hypoxia was detected using Real-time PCR and Western blot;（3）The expression of gluconeogenesis key enzymes PCK1,FBP1 and G6 pase in Bulk and TRCs under normoxia and hypoxia was detected using Real-time PCR and Western blot;（4）Glycogen concentration using glycogen detection kit in hypoxic MCF7 TRCs treated with 3-MPA（a PCK1 inhibitor）or transfected with PCK1 siRNA;（5）Hypoxic breast TRCs were incubated with ¹³C-pyruvate,¹³C-acetate,¹³C-glucose,or ¹³C-glutamine,and LC-MS was performed for M + 2-or M + 3 PEP;（6）Different concentrations of 3-MPA or PCK1 siRNA are used to block gluconeogenesis,hypoxic breast TRCs were incubated with ¹³C-glucose or ¹³C-glutamine,and LC-MS was performed for M + 2-or M + 3 PEP;（7）Hypoxic breast TRCs were incubated with ¹³C-pyruvate,¹³C-acetate,or ¹³C-glutamine,and LC-MS was performed for M + 2 G6P/G1 P or M + 3 G6P/G1P;（8）Expression of glycogen phosphorylase（PYGL）,glycogen-debranching enzyme（GDE）,and phosphoglucomutase（PGM1）in bulk cells or TRCs under normoxia and hypoxia was analyzed by Real-time PCR and Western blot;（9）MCF7 or SUM159 cells were treated with GPI or transfected with PYGL siRNA and then cultured in soft 3D fibrin gels under hypoxia and colony size were analyzed;（10）Different concentrations of GPI or PYGL siRNA were used to block glycogenolysis,hypoxic breast TRCs were incubated with ¹³C-glucose,and LC-MS was performed for M + 2 G6P/G1 P or M + 2 R5P;（11）Glycogen concentration using glycogen detection kit in hypoxic MCF7 TRCs treated with 6AN or transfected with G6 PD siRNA;（12）Block PPP with different concentrations of 6AN or G6 PD siRNA,hypoxic breast TRCs were incubated with ¹³C-glucose or ¹³C-glutamine,and LC-MS was performed for M + 2 R5 P or M + 3 R5P;（13）The expression of PPP key enzyme G6 PD and PGD in MCF-7 or SUM159 Bulk and TRCs under hypoxia was detected using Real-time PCR and Western blot;（14）MCF7 TRCs cultured in ¹³C-pyruvate medium in hypoxia were treated with GPI for 5 days,and LC-MS was performed for M + 2 glycogen or M + 2 R5P;（15）ROS levels and the colony size were analyzed in hypoxic TRCs treated with DNCB or BSO;（16）Use NADPH kit and LC/MS to detect the ratio of NADPH/NADP⁺ and GSH/GSSG in hypoxic MCF-7 or SUM159 Bulk or TRCs;（17）MCF7 or SUM159 cells were transfected with G6 PD siRNA and.NADPH/NADP⁺,GSH/GSSG ratio,ROS levels,and the colony size were analyzed;（18）MCF7 cells were transfected with G6 PD siRNA and ethyl-GSH in hypoxia,and the colony size was analyzed;（19）Hypoxic MCF7 TRCs were transfected with PYGL siRNA and.NADPH/NADP⁺,GSH/GSSG ratio,ROS levels,and the colony size were analyzed;（20）Hypoxic MCF7 TRCs were transfected with PCK1 siRNA and.NADPH/NADP⁺,GSH/GSSG ratio,ROS levels,and the colony size were analyzed;（21）MCF7 TRCs transfected with PCK1-overexpressing plasmid were cultured in hypoxia,and colony size,NADPH/NADP⁺ ratio,GSH/GSSG ratio,and ROS levels were analyzed.Results:（1）There was no difference in M+6 glycogen between hypoxic breast Bulk and TRCs;（2）HK2 expression was down-regulated in hypoxic breast TRCs;（3）PCK1 and FBP1 expression was significantly up-regulated in hypoxic breast TRCs,but G6 pase was not expressed;（4）Blocking the gluconeogenesis pathway leads to a significant reduction in glycogen content in hypoxic TRCs;（5）¹³C-pyruvate,13 Cacetate,¹³C-glucose or ¹³C-glutamine labeled M + 2 or M + 3 PEP in hypoxic breast TRCs increased;（6）Blocking the gluconeogenesis pathway leads to a decrease in the proportion of M + 2 or M + 3 PEP in hypoxic TRCs;（7）¹³C-pyruvate,¹³C-acetate or ¹³C-glutamine labeled M + 2 or M + 3 G6P/G1 P in hypoxic breast TRCs increased;（8）The expression of PYGL,GDE and PGM1 in hypoxic breast TRCs was significantly up-regulated;（9）Blocking the glycogenolysis pathway leads to a significant decrease in the volume of hypoxic breast TRCs;（10）Blocking the gluconeogenesis pathway leads to a decrease in the proportion of ¹³C-glucose labeled M + 2 G6P/G1 P or M + 2 R5P;（11）Blocking PPP pathway can cause glycogen accumulation and a significant decrease in the proportion of M + 2 R5 P in hypoxic breast TRCs;（12）Blocking PPP leads to a significant decrease in the proportion of ¹³C-glucose or ¹³C-glutamine labeled M + 2 R5 P or M + 3 R5P;（13）The expression of PPP key enzymes G6 PD and PGD in hypoxic breast TRCs increased;（14）We used ¹³C-pyruvate medium to culture breast TRCs for 5 days under hypoxia.After GPI treatment the ¹³C-labeled M + 2 glucose increased from 3% to 13%,and the M + 2 R5 P decreased from 35% to 25%;（15）MCF-7 TRCs treated with DNCB showed little difference in clone size,and ROS increased slightly.MCF-7 TRCs treated with BSO resulted in a significantly smaller clone size and a significant increase in ROS;（16）The ratio of NADPH/NADP⁺ and GSH/GSSG in hypoxic MCF-7 or SUM159 TRCs increased;（17）After blocking the PPP pathway,the clonal volume of hypoxic breast TRCs decreased significantly,the ratio of NADPH/NADP⁺ and GSH/GSSG decreased,and the ROS level increased;（18）After blocking the PPP pathway,the clonal volume of hypoxic breast TRCs decreased,and the clonal volume recovered treated with ethanol-GSH;（19）After Blocking the glycogenolysis pathway,the clonal volume of hypoxic breast TRCs decreased significantly,the ratio of NADPH/NADP⁺ and GSH/GSSG decreased,and the ROS level increased;（20）After blocking the gluconeogenesis pathway,the clonal volume of hypoxic breast TRCs is significantly reduced,and the intracellular NADPH/NADP⁺ and GSH/GSSG decreased,and the level of ROS increased;（21）After PCK1 overexpression,the clonal volume of hypoxic breast TRCs increased significantly,the ratio of NADPH/NADP⁺ and GSH/GSSG increased,and the level of ROS decreased.Conclusion: Hypoxic breast TRCs use the gluconeogenesis pathway for glycogenesis.The glycogenesis-glycogenolysis-PPP pathway regulates the level of ROS and maintains a moderate level of ROS to promote the growth of hypoxic TRCs.Part Ⅲ The mechanism of hypoxia-activated gluconeogenesis key enzyme PCK1 upregulatedObjective: Explore the mechanism of hypoxia-activated gluconeogenesis key enzyme PCK1 upregulated.Methods:（1）MCF7 TRCs transfected with HIF1 a siRNA or overexpressing plasmid were cultured in hypoxia or normoxia,PCK1 expression was analyzed by Western blot;（2）Chromatin immunoprecipitation（Ch IP）-PCR assay showed that HIF-1α or FoxO1 was bound to the promoter of PCK1;（3）The luciferase reporter assay showed that regulatory relationship between HIF-1α or FoxO1 and PCK1;（4）Real-time PCR,Western blot and immunofluorescence analyzed expression of FoxO1 in hypoxic TRCs;（5）MCF7 TRCs transfected with FoxO1 siRNA or overexpressing plasmid were cultured in hypoxia or normoxia,FoxO1 expression was analyzed by Western blot;（6）Detect the methylation of the PCK1 promoter region under normoxia and hypoxia conditions;（7）Western blot detected the expression of H3K27me3 and H3K4me3 in Bulk and TRCs under normoxia and hypoxia;（8）Ch IP-PCR detected the binding of PCK1 promoter region and H3K27me3 or H3K4me3 in Bulk and TRCs under hypoxia.Results:（1）Use of siRNA to knock down HIF-1α led to the downregulation of PCK1 in MCF-7 TRCs,forced overexpression of HIF-1α resulted in the upregulation of PCK1;（2）HIF-1α and FoxO1 directly binds to the PCK1 promoter region;（3）PCK1 promoter luciferase assay showed that knockout of HIF-1α or FoxO1 reduced luciferase activity,and overexpression of HIF-1α or FoxO1 increased luciferase activity;（4）FoxO1 was strikingly upregulated in hypoxic TRCs and mainly located in the nucleus;（5）Use of siRNA to knock down FoxO1 led to the downregulation of PCK1 in MCF-7 TRCs,forced overexpression of FoxO1 resulted in the upregulation of PCK1;（6）There was no difference in the methylation status of the PCK1 promoter region during normoxia and hypoxia;（7）H3K27me3 expression was downregulated and H3K4me3 expression upregulated in hypoxic breast TRCs;（8）In the PCK1 promoter region of hypoxic breast TRCs,H3K27me3 binding decreased and H3K4me3 binding increased.Conclusion: HIF-1α and FoxO1 positively regulate the expression of PCK1 in breast TRCs,and epigenetic histone methylation modification can also regulate the expression of PCK1.Part Ⅳ PCK1 can be used as a target to inhibit the growth of breast tumor in vivo and enhance the efficacy of paclitaxel in the treatment of TNBCObjective: In order to further explore hypoxia-activated glycogen metabolic pathway in breast TRCs and the effect of PCK1 as a target on breast tumor growth in vivo;triple-negative breast cancer（TNBC）is not sensitive to treatment because of its special phenotype,we explore whether PCK1 can be used as a target in combination with paclitaxel in the treatment of TNBC.Methods:（1）MCF7 tumor cells were inoculated into the mammary gland of nude mice,followed by intragastric administration of sunitinib and then ALDH1⁺ and ALDH1-tumor cells were sorted from breast tumor tissue.The expression of PCK1,FBP1,G6 Pase,UGP2,GYS1,PYGL,G6 PD,and PGD was analyzed by Real-time PCR;（2）NADPH/NADP⁺ ratios was detected by NADPH kit and GSH/GSSG ratios in ALDH1⁺ TRCs was detected by LC/MS;（3）Immunofluorescence staining was used to detect the proliferation of Ki67 in tumor tissues;（4）Gluconeogenesis,glycogen metabolism,and PPP intermediates including G6 P,PEP and R5 P were detected by LC-MS;（5）PCK1-SGs-MCF7 TRCs were inoculated into mammary gland of nude mice and mice were treated with sunitinib and the NADPH/NADP⁺ ratios were analyzed by NADPH kit.The GSH/GSSG ratios were detected by LC-MS.ROS levels were detected by flow cytometry;（6）MCF7 TRCs were inoculated into mammary gland of nude mice and mice were administered intragastrically with sunitinib combined with 3-MPA.The NADPH/NADP⁺ ratios,GSH/GSSG ratios,and ROS levels were detected;（7）PCK1-overexpressing MCF7 TRCs were inoculated into mammary gland of nude mice and mice were treated with sunitinib and the NADPH/NADP⁺ ratios were analyzed by NADPH kit.The GSH/GSSG ratios were detected by LC-MS.ROS levels were detected by flow cytometry;（8）Immunohistochemical staining showed PCK1 expression in 54 patients with breast cancer at different stages and correlation between PCK1 and Ki67;（9）Overall survival compared with the PCK1 level in patients with breast cancer;（10）MCF7 and SUM159 tumors were imaged using PET/CT with ¹⁸F-FMISO after paclitaxel（PTX）treatment;（11）MCF7 and SUM159 tumor vascular endothelium were observed by H&E staining after paclitaxel（PTX）treatment;（12）SUM159 TRCs were treated with paclitaxel in combination with 3-MPA,GPI,or 6AN under normoxia and hypoxia.Cell apoptosis and ROS levels were analyzed by flow cytometry;（13）SUM159 TRCs cells were inoculated into the mammary gland of nude mice.Then,mice were intraperitoneally injected with paclitaxel,or intragastric administered 3-MPA for 4 weeks.Tumor growth was monitored.The lung metastasiswas analyzed by H&E staining and ¹⁸F-FDG PET-CT.The survival of mice was monitored;（14）Cells isolated from tumor were reseeded into soft 3D fibrin gels in hypoxia,and the colony size and the number of TRCs were analyzed;（15）Stem gene m RNA levels were analyzed by real-time PCR.Results:（1）Breast ALDH1⁺ TRCs in vivo highly expressed enzymes involved in gluconeogenesis,glycogen metabolism,and PPP;（2）Breast ALDH1⁺ TRCs in vivo had much higher NADPH/NADP⁺ and GSH/GSSG ratios;（3）Breast ALDH1⁺ TRCs in vivo were in a proliferating state;（4）Breast ALDH1⁺ TRCs in vivo had much higher proportion of gluconeogenesis,glycogen metabolism and PPP intermediate metabolites G6 P,PEP and R5P;（5）PCK1 knockdown retarded the tumor growth and decreased NADPH/NADP⁺ and GSH/GSSG ratios but increased ROS levels;（6）3-MPA treatment retarded the tumor growth and decreased NADPH/NADP⁺ and GSH/GSSG ratios but increased ROS levels;（7）PCK1 overexpression accelerated tumor growth,and led to increased NADPH/NADP 628 t and GSH/GSSG ratios but decreased ROS levels;（8）PCK1 expression was elevated in high stage tumors,correlating with an active tumor cell proliferation,and predicted a worse prognosis;（9）SUM159 TNBC tumors were more hypoxic than MCF7 tumors,and paclitaxel aggravated the hypoxia in SUM159 tumors;（10）Endothelial damage caused by paclitaxel was overt in SUM159 tumors;（11）The SUM159 TRCs death was induced combined with PTX and 3-MPA,GPI,or 6AN,concomitant with elevated ROS levels;（12）The combined treatment strongly inhibited tumor growth,reduced tumor metastasis and prolonged the long-term survival of the mice;（13）The colony size and the number in the gels were reduced in the combined treatment group;（14）The combined treatment downregulated stemness gene expression.Conclusion: In vivo breast ALDH1⁺ TRCs had stronger gluconeogenesis,glycogen metabolism and PPP pathway,enhanced metabolic flow,and stronger antioxidant system,which is consistent with the results in vitro.The growth of breast tumor in mice was significantly inhibited by 3-MPA and knockout of PCK1,and the growth of breast tumor in mice with overexpression of PCK1 became faster.In vitro and in vivo experiments have proved that the combination of 3-MPA and paclitaxel can effectively kill hypoxic breast cancer TRCs,and delay tumor growth,reduce lung metastasis and prolong the survival time of mice.It is suggested that PCK1 can be used as a target to inhibit the growth of breast tumor in vivo and enhance the efficacy of paclitaxel in the treatment of triple negative breast cancer.