Role And Mechanism Of Hypoxic Microenvironment In Promoting The Proliferation Of Breast Tumour Repopulating Cells

Part Ⅰ Effects of hypoxic microenvironment on proliferation of breast tumor-repopulating cellsObjective:Cancer stem cells（CSCs）,also known as cancer-initiating cells or tumor-proliferating cells,are characterized by the capability of self-renewal,differentiation,and tumorigenicity in vivo.CSCs are the key drivers in tumor initiation,invasion,and metastasis after therapy.Hypoxia is a key microenvironmental factor that regulates the self-renewal of cancer stem cells and can enrich tumor stem cells at the same time.In the present study,we used tumor stem cells（defined as tumor-regenerating cells,TRCs）which were separated by 3D soft fibrin gels culture,to explore the effect of hypoxia on the proliferation of cancer stem cells in vitro and in vivo.Methods:（1）Establishment of tumor hypoxia model was performed with sunitinib malate,which is the tyrosine kinase inhibitor of vascular endothelial growth factor receptor;the size of tumor volume,hypoxic areas and vascular distribution in the two groups were detected by immunofluorescence staining at the same time.（2）ALDH1^high or ALDH1^lowcells from breast tumor tissue were sorted by flow cytometry and seeded in the soft 3D fibrin gels,then count of formed colony numbers of the two group were recorded.（3）The expression of stemess genes（ALDH1A1,OCT4,SOX2,KLF4）were analyzed by Real-time PCR;Western blot was used to detect the protein expression of ALDH1A1 and PCK2.（4）The BrdU of stem cells in both groups were detected by immunofluorescence staining.（5）MCF-7/BT474 and human primary breast cancer TRCs were cultured under aerobic（21%O₂）and hypoxic（1%O₂）conditions for 6 days,with a record of the clony volume and clony number every day.（6）The clony volume and number of CoCl2-treated MCF-7 TRCs were determined.（7）The BrdU staining was used to detect the proliferation of the two groups;PI and AnnexinV were used to detect the apoptosis.（8）Real-time PCR was used to detect the expression of stemness genes of MCF-7 TRCs in aerobic and hypoxic microenvironment.（9）Different concentration of aerobic and hypoxic MCF-7 TRCs were inoculated into mammary gland of NOD-SCID mice,to observe the tumorigenic rate and tumorigenesis of the two groups.Results:（1）smaller sizes and less vascularized distribution were observed in tumors of the sunitinib treatment group and the tumors tissues from sunitinib-treated mice displayed multiple areas of intense hypoxia determined by pimonidazole.（2）Most ALDH1^hightcells could grow colonies.By contrast,more than 90%ALDH1^lowow tumor cells could not grow colonies in the soft 3D fibrin gels.（3）Sunitinib treatment increased the expression of ALDH1 as well as other stem cell marker genes such as Sox2,Oct3/4 and KLF4 in the breast tumor tissues.（4）These ALDH1^<sup>+cells in sunitinib-treated hypoxic microenvironment showed much higher BrdU incorporation,compared to the control group.（5）MCF-7 and BT474 TRCs grew much better in hypoxic（1%oxygen）than those in normoxic（21%oxygen）.Similarly,this phenomenon was also observed in TRCs isolated from clinical fresh breast tumor samples.（6）As the concentration of CoCl₂ increased,the clone size of MCF-7 TRCs increased,but after a certain concentration,the growth of the clones was limited.（7）Compared to their normoxic counterparts,MCF-7 TRCs in hypoxia markedly increased DNA synthesis（S phase）;few TRCs underwent apoptosis,similar to the alive TRCs in normoxic.（8）TRCs showed the phenotype of upregulated stemness markers in hypoxic,including Sox2,Oct3/4,KLF4,CD133 and ALDH1.（9）Hypoxic TRCs had stronger tumorigenic ability to form a tumor.Conclusions:Hypoxic microenvironment is favorable for breast cancer TRCs proliferation in vivo and in vitro,and also induces the stem cell phenotype;meanwhile,the tumorigenicity is obviously enhanced.Part Ⅱ Investigation the mechanism of hypoxic microenvironment mediated TCA cycle remodeling and ROS productionObjective: To explore the material basis for hypoxia-induced ROS elevation,by analyzing the changes of intermediate metabolite（s）in the TCA cycle;and further investigate the mechanism of ROS to promote the proliferation of breast cancer Tumor-repopulating cells.Methods:（1）ROS fluorescence probe was used to detect the intracellular ROS levels of MCF-7 and human primary breast cancer TRCs under aerobic and hypoxic conditions;also,the ROS levels of MCF-7 TRCs,treated with NAC,were detected by Cell ROX? green flow cytometry assay.（2）After treated with different concentrations of NAC,the clony size of hypoxic MCF-7 TRCs was observed;and after treated with different concentrations of H2O2,the clony size of normoxic MCF-7 TRCs was observed as well.（3）The ROS fluorescent probe was used to detected the ROS level of MCF-7 and MCF-7 TRCs in the hypoxic conditions,and detected the proliferation of MCF-7 cells after adding different concentrations of NAC;（4）Western blot was used to detect the protein levels of NF-κB and Akt in MCF-7 and human primary breast cancer TRCs and the level of phosphorylation of NF-κB and Akt at different time after adding 20 m M NAC;（5）After treated with NF-κB and Akt inhibitor,the phosphorylation of NF-κB and Akt of MCF-7 TRCs was detected by Western blot and the changes of the clone volume of MCF-7 TRCs was observed at the same time;（6）The TCA cycle’s carbon flow was tested by 13 C labeled glucose;（7）The expressions of CS,IDH3 G and OGDH,which are the key enzymes in TCA cycle,were detected by Western blot;（8）After silencing MDH2 and IDH3 G with si RNA,observing the volume of MCF-7 TRCs and detected the intracellular ROS level by ROS fluorescent probe;（9）IDH3G was silenced by si RNA,and added different concentrations of H2O2 simultaneously,observed the change of clone volume of MCF-7 TRCs.（10）Using LC/MS/MS to detect the citric acid,alpha-ketoglutaric acid,succinic acid,fumaric acid and malic acid,which are the intermediates of TCA cycle;（11）si RNA silenced the expression of MDH2,and the content of fumarate was detected by LC/MS/MS;（12）Under the normoxic condition,MCF-7 TRCs were treated with different concentration of dimethyl fumarate（DMF）,then the level of intracellular ROS was detected by ROS probe;（13）Under the condition of aerobic and hypoxia,the content of GSF in MCF-7 TRCs was detected by LC/MS/MS;the ratio of NADPH/NADP⁺ and GSH/GSSG in MCF-7 TRCs were measured by NADPH and GSH assay kit;（14）After treating the hypoxic MCF-7 TRCs with GSH-MEE,intracellular NADPH/DNAP⁺ ratio and ROS levels were detected by NADPH assay kit and ROS fluorescent probe respectively.Treating MCF-7 TRCs with different concentrations of GSH-MEE,the change of the clone volume was observed;（15）After silencing IDH3 G by si RNA,the content of GSF and the ratio of NADPH/DNAP⁺ of MCF-7 TRC cells were detected by LC/MS/MS and NADPH kit respectively.Results:（1）hypoxic MCF-7 and primary human breast cancer TRCs exhibited higher levels of ROS than normoxic TRCs;（2）blocking ROS levels with antioxidant N-acetyl-cysteine（NAC）effectively hindered TRC growth in hypoxia.By contrast,addition of hydrogen peroxide to culture medium increased MCF-7 TRCs growth in normoxia;（3）MCF-7 cells cultured in conventional 2D plastic had strikingly high levels of ROS under hypoxic condition and the addition of NAC could relive the hypoxia-induced growth retardation;（4）NF-κB and Akt,were found to be effectively activated in MCF-7 and primary human breast cancer TRCs under hypoxic condition,and the addition of NAC could blunt the above activation of NF-κB and Akt;（5）Blocking the activity of NF-κB or Akt with inhibitors,then the promoting effect of hypoxia on TRCs growth was blocked;（6）The carbon flow of TCA cycle attenuated in hypoxia;（7）The expression of citrate synthase（CS）,isocitrate dehydrogenase 3G（IDH3G）and α-ketoglutarate dehydrogenase was downregulated in hypoxic MCF-7 TRCs;（8）Knocking down MDH2,the MCF-7 TRCs growth was further promoted,concomitant with increased ROS levels;（9）Knocking down IDH3 G,the hypoxic TRCs growth was not promoted but inhibited,concomitant with decreased ROS levels,and replenished hydrogen peroxide rescued the growth of impaired TRCs;（10）Hypoxia result in an increase of intermediates of TCA cycle（citric acid,α-ketoglutaric acid,succinic acid,fumaric acid and malic acid）,and the level of fumarate acid and malic acid were increased obviously;（11）Using Dimethyl 2-oxoglutarate（MOG）,dimethyl fumarate（DMF）,succinic acid dimethyl ester（SAD） and L-malate to treat hypoxic TRCs,and found that only DMF increased ROS levels;（12）Hypoxic breast cancer TRCs showed much higher succinic GSH than normoxic TRCs.Also,the ratios of GSH/GSSG and NAPDH/NADP⁺were lower in hypoxic TRCs.（13）Provision of ethyl-GSH could rescue NAPDH levels to the extent of normoxic TRCs,decrease ROS levels,and hinder hypoxia-promoted TRCs growth;（14）The increased of succinic GSH and decreased of NADPH/NADP⁺ could be remedied by IDH3 G.Conclusions: Hypoxia leads to a slowed flow of carbon in the TCA cycle,resulting in the accumulation of fumarate,an intermediate metabolite.Fumarate accumulation causes the formation of succinic glutathione,leading to increased ROS levels in hypoxic breast cancer TRCs.Increased ROS by hypoxia acts as signal molecules to activate NF-κB and Akt,promoting human breast cancer TRCs growth.Part Ⅲ The role of HIF-1α mediated downregulation of PCK2 in the remodeling TCA of TRCs from breast cancerObjective: To explore the effect of the down-regulation expression of phosphoenolpyruvate carboxykinase（PCK2）on intermediates of the carbon flow in the TCA cycle in tumor regeneration cells using MCF-7 TRCs model,especially for influence of oxaloacetic acid and fumaric acid,and to investigate the cause of the accumulation of fumaric acid in TRCs in hypoxia and the molecular mechanism of down-regulation of PCK2 expression under hypoxic conditions at the same time.Methods:（1）The expression of PCK2 in MCF-7 TRCs under hypoxia were detected by Western blot.（2）Using tetracycline-induced PCK2 overexpression system to detect the levels of fumarate,succinic GSH,ROS and the ratio of NADPH/NADP⁺ and GSH/GSSG were also determined in MCF-7 TRCs.（3）OAA assay kit was used to detect the changes of OAA in aerobic and hypoxic MCF-7 TRCs.（4）Overexpression of PCK2,the flow of carbon in the TCA cycle was detected by isotope-labeled ¹³C-glucose;（5）Western blot was used to detect the expression of HIF in MCF-7 TRCs,and the PCK2 protein was detected after silencing or over-expressing HIF-1α.（6）Isotope labeled 13 C was used to detect the flow of circulating carbon in TCA after silenced HIF-1α;（7）UCSC genome browser and JASPAR were used to analyze the existence of HIF-1 α binding site in the promoter region of PCK2,and the results were further verified by chromatin immunoprecipitation technique.（8）Luciferase expression was determined by PCK2 promoter luciferase in order to determine the regulatory relationship between HIF-1α and PCK2.（9）Western blot was used to detect the expression of HIF-1α and PCK2 in hypoxic breast cancer TRCs,which were treated with Dimethyl fumarate（DMF）.Results:（1）PCK2 was highly expressed in normoxic breast cancer TRCs but much lowly expressed under hypoxic condition;（2）PCK2 overexpression led to the decreased fumarate,succinic GSH and ROS levels and the increased NADPH/NADP⁺and GSH/GSSG ratio in hypoxic breast cancer TRCs;（3）OAA levels were low in normoxic TRCs but high in hypoxic TRCs,while was reduced by PCK2 overexpression;（4）Using ¹³C-glucose to trace carbon flow of TCA cycle.As expected,m⁺2 and m⁺4 forms of citrate,α-ketoglutarate,fumarate,malate and were elevated in hypoxic TRCs with PCK2 overexpression;（5）Both normoxic and hypoxic breast cancer TRCs highly expressed HIF-2α,however,HIF-1α was strikingly upregulated only in hypoxic TRCs.When we used si RNAs to knock down HIF-1α,we found that PCK2 expression was upregulated in hypoxic breast cancer TRCs;（6）¹³C-glucose tracing assay showed that the knockdown of HIF-1α promoted the carbon flow of TCA cycle;（7）The analysis with the UCSC Genome Browser and JASPAR revealed the presence of multiple consensus cis-elements for HIF-1α binding on the promoter of PCK2,and the Chip-PCR assay indicated that HIF-1α directly bind to PCK2 promoter;（8）We then conducted PCK2 promoter luciferase assay,showing that the knockdown of HIF-1α increased luciferase activity,However,HIF-1α overexpression decreased the luciferase activity;（9）we additionally used dimethyl fumarate（DMF）to treat hypoxic breast cancer TRCs,HIF-1a expression was found enhanced and PCK2 expression was further decreased.Conclusions: PCK2 downregulation-caused OAA accumulation hinders the carbon flow of TCA cycle and leads to fumarate accumulation in hypoxic breast cancer TRCs.Hypoxia-induced HIF-1α negatively regulates PCK2 expression and fumarate accumulation further strengthens this feedback in breast cancer TRCs.