The Role And Mechanism Of Glycogen Metabolism And Its Intermediate Metabolites In Breast Cancer Stem Cells

Objective: Cancer stem cells(CSCs)are a small subgroup of tumor cells,which has the ability of self-renewal,differentiation and tumorigenesis.And they are resistant to conventional chemotherapy and radiotherapy,which is very likely to be the source of tumor metastasis.Therefore,CSCs are considered as an important target for the development of new anticancer drugs.Glycogen is the intracellular storage form of glucose,which mainly exists in skeletal muscle and liver.In the process of tumor research,glycogen metabolism has always been neglected,only some data showed that the increase of glycogen content in tumor cells during hypoxia was beneficial to the survival of tumor cells.However,the specific role of glycogen metabolism in tumors and tumor stem cells is not clear.In this study,three-dimensional soft fibrin matrix gel(3D fibrin)was used to screen and expand tumor stem cells,which were defined as tumor-repopulating cells(TRCs),to explore the level of glycogen metabolism of breast cancer stem cells,as well as the role and mechanism of glycogen metabolism and its intermediate metabolites in breast cancer stem cells.Methods:(1)Detection of glycogen content: The glycogen content of different normal tissues,the glycogen content of tumor and paracancerous tissues in breast cancer patients and the glycogen content of eight kinds human breast cancer cell lines weredetected by periodate-Schiff(PAS)reaction.MCF-7 was seeded in soft 3D fibrin gel,and the content of glycogen in 3D and 2D cells was detected by electron microscope.Glycogen detection kit was used to detect the content of glycogen of MCF-7,T47 D and SUM159 cell lines cultured in 3D and 2D.(2)Detection of glycogen metabolism related enzymes: The expression of glycogen metabolism related enzymes of three breast cancer cell lines cultured in 3D and 2D was detected by real-time PCR and Western Blot.Breast cancer stem cells were screened by cultured on ultra-low adherence plates(mammosphere assay)and ALDH flow sorting,and the expression of glycogen-related enzymes in screened breast cancer stem cells and common cultured breast cancer cell lines were detected by real-time PCR.(3)Knock down the enzymes related to glycogen metabolism and observe the growth of TRCs: After knocking down PYGL,GYS1,UGP2 and PGM1 with si RNA,the colony size of MCF-7,T47 D and SUM159 TRCs was observed.While knocking down UGP2 and PGM1,the colony size of MCF-7,T47 D and SUM159 TRCs was observed with exogenous supplement of UDPG.(4)Knocking down UGP2 and GYS1 with si RNA,the content of UDPG in MCF-7,T47 D and SUM159 TRCs cells was detected by liquid chromatography-mass spectrometry(LC-MS).(5)The expression of P2Y14 in breast cancer stem cells: The overexpression plasmid vector of P2Y14-GFP was constructed and the expression of P2Y14 in MCF-7,T47 D and SUM159 was detected by confocal microscope.Real-time PCR and Western Blot were used to detect P2Y14 expression of 3D cells and 2D cells.Breast cancer stem cells were screened by mammosphere assay and ALDH flow sorting,and the expression of P2RY14 in breast cancer cell lines and common cultured breast cancer cell lines was detected.(6)Analyze the correlation between ALDH1A1 and P2Y14: Analyze the correlation between P2Y14 and ALDH1A1 of breast cancer patients with TCGA database.Immunofluorescence was used to detect the expression and distribution ofP2Y14 and ALDH1A1 in tumor paraffin sections of patients with breast cancer.(7)Detect the influence of P2Y14 on the growth of breast cancer TRCs:Knocking down P2Y14 with si RNA,the colony size of MCF-7,T47 D and SUM159 TRCs was observed with exogenous supplement of UDPG.The colony size of MCF-7,T47 D and SUM159 TRCs which were treated with P2Y14 selective inhibitor PPTN was observed with exogenous supplement of UDPG.Lentivirus expression plasmid p LVX-EF1?-Ac GFP1-P2Y14,was constructed to package lentivirus.After infected with lentivirus,the cells were seeded in soft 3D fibrin gel,and the colony size was observed.While knocking down GYS1,PPTN was used to block the signal of P2Y14,and the colony size of breast cancer TRCs was observed.(8)Determination of the process of UDPG release to extracellular: The transport from Golgi apparatus to endoplasmic reticulum was blocked by Brefeldin A or si GM130 respectively,and the colony size of MCF-7,T47 D and SUM159 TRCs were observed by exogenous supplement of UDPG.Real-time PCR was used to detect MCF-7,T47 D and SUM159 cell lines cultured in 3D or 2D the m RNA expression of SLC35 family.After knocking down SLC35A2,SLC35B2,SLC35C1,SLC35C2,SLC35G2 and SLC35F5 with si RNA,the colony size of MCF-7,T47 D and SUM159 TRCs were observed.While knocking down SLC35A2 or SLC35F5,the colony size of MCF-7,T47 D and SUM159 TRCs was observed with exogenous supplement of UDPG.(9)Detection of downstream signal pathway of P2Y14: MCF-7 TRCs,was treated with PKA and PKC,selective inhibitor H89 and Sotrastaurin,respectively to observe the change of colony size.The phosphorylation levels of MAPK,NF-?B and Akt in TRCs,while TRCs were knocked down and overexpressed P2Y14,were detected by Western Blot.Western Blot was used to detect the change of phosphorylation level of Er K after silencing P2Y14 or UGP2,and then supplementing UDPG,respectively.(10)The effect of glycogen metabolism on the progression of breast cancer was verified by experiments in vivo: With CRISPR-Cas9 gene editing technique,MCF-7cell lines stably knocking down P2Y14 or UGP2 were constructed and seeded in soft3 D fibrin gel and inoculated subcutaneously in nude mice to observe the tumorigenesis.The expression of P2Y14 in tumor tissues of patients with breast cancer was analyzed by immunohistochemistry,and the correlation between the expression of P2Y14 and breast cancer stage was analyzed.Results:(1)PAS results showed that the glycogen contents in liver,muscle,kidney,colon and heart were relatively high,while those in breast,bladder,skin,thyroid and lung were relatively low.The content of glycogen in tumor site of patients with breast cancer was significantly higher than that adjacent to tumor.The content of glycogen was different in different breast cancer cell lines.The results of electron microscope and glycogen detection kit showed that the content of glycogen in 3D breast cancer cells was significantly higher than that in 2D cells.This indicated that the content of glycogen in breast cancer stem cells was increased.(2)The expression of glycogen synthesis and decomposition enzymes in 3D cells of MCF-7,T47 D and SUM159 was higher than that in 2D cells.The expression of glycogen synthase in breast cancer stem cells screened by mammosphere assay and ALDH flow sorting was higher than that in ordinary culture cells.This indicated that the flow of glycogen metabolism in breast cancer stem cells was accelerated.(3)Knocking down PYGL had no significant effect on the growth of TRCs clones in breast cancer.After knocking down GYS1,the clone growth increased significantly.After knocking down UGP2,the clone growth was significantly inhibited,and the clone growth was significantly restored with exogenous supplement of UDPG.After knocking down PGM1,the clone growth was significantly inhibited,and the clone growth was significantly restored with exogenous supplement of UDPG.It was suggested that UDPG,an intermediate of glycogen metabolism,promoted the growth of TRCs in breast cancer.(4)The results of liquid chromatography-mass spectrometry showed that the intracellular UDPG content of TRCs decreased after knocking down UGP2,while theintracellular UDPG content of TRCs increased after knocking down GYS1.This result was consistent with the trend of clonal growth after knockout of UGP2 and GYS1,which further confirmed the role of UDPG in promoting the growth of breast cancer TRCs.(5)Confocal results showed that P2Y14 was expressed on the cell membrane of MCF-7,T47 D and SUM159.The results of real-time PCR and Western Blot showed that the expression of P2Y14 in 3D cells was higher than that in 2D cells.The P2RY14 gene expression of breast cancer stem cells obtained by mammosphere assay and ALDH flow sorting was significantly higher than that of breast cancer cell lines cultured in common culture.This indicated that P2Y14 receptor was highly expressed on the membrane of breast cancer stem cell.(6)The results of database and immunofluorescence showed that there was a significant correlation between the distribution and expression of P2Y14 and ALDH1A1 in breast cancer patients.It was suggested that P2Y14 might be related to the stemness of breast cancer.(7)After blocking the signal of P2Y14 with si RNA or inhibitor PPTN respectively,the clone growth was significantly inhibited,and it did not recover with exogenous supplement of UDPG.However,the clone growth was significantly accelerated by overexpression of P2Y14.At the same time,PPTN could reverse the promoting effect of UDPG-mediated on breast cancer TRCs growth,and the clone of si GYS1+PPTN group was even smaller than that of si NC group.This suggested that UDPG could promote the growth of TRCs in breast cancer by activating P2Y14 receptor.(8)UDPG could partially reverse the growth arrest of TRCs induced by Brefeldin A and knocking down MG130.Real-time PCR results showed that the m RNA expression of SLC35 family in MCF-7,T47 D and SUM159 3D cells was higher than that in 2D cells.Only knocking down SLC35A2 and SLC35F5,the clone growth was significantly inhibited,and the clone growth was significantly restored by exogenous supplement of UDPG.This suggested that UDPG produced by glycogenmetabolism was secreted out of the cell depending on SLC35A2,SLC35F5 and Golgi.(9)Clonal growth was significantly inhibited with H89 treatment,but there was no significant change with Sotrastaurin treatment.After knocking down P2Y14,only the phosphorylation level of Er K was significantly inhibited,and the phosphorylation level of Er K was enhanced after overexpression of P2Y14.Knockdown P2Y14 alone and exogenous supplement of UDPG,there was no significant change in the phosphorylation level of Er K.However,after knocking down UGP2,alone and supplementing UDPG,the phosphorylation level of Er K was significantly restored.This indicated that UDPG promoted the growth of TRCs in breast cancer through P2Y14-PKA-Er K signal pathway.(10)The ability of subcutaneous tumorigenesis of breast cancer TRCs with P2Y14 and UGP2 knockdown was decreased in nude mice.At the same time,the expression of P2Y14 in tumor tissues of patients with advanced breast cancer was high,and it was positively correlated with the expression of Ki67 in tumor tissues.This suggested that UDPG,an intermediate metabolite of glycogen metabolism,could affect the formation and development of breast cancer in vivo.Conclusion: Compared with differentiated breast cancer cells,the content of glycogen in breast cancer stem cells increased,the expression of enzymes related to glycogen metabolism also increased,and glycogen metabolism was more active.UDPG is not only involved in glycogen metabolism,but also an important extracellular signal molecule and an activator of P2Y14 receptor.It could affect the growth of breast cancer TRCs in vitro and affect the formation and development of breast cancer in vivo.The specific mechanism was that UDPG produced by glycogen metabolism of breast cancer stem cells could enter the Golgi matrix and endoplasmic reticulum through SLC35A2 and SLC35F5 transporters,and then be transported outside the cells through Golgi-dependent secretory pathways,activating P2Y14 receptors on the cell membrane and activating P2Y14-PKA-Er K signaling pathways,thus promoting the growth of breast cancer stem cells.In addition,the up-regulated expression of P2Y14 in TRCs further promoted this process.