Investigation Of Metabolic Role Of Phosphoenolpyruvate Carboxykinase In Tumor-repopulating Cells

Part ? Investigation of glucose side-branch metabolic promotion by upregulating PCK1 in melanoma cells that repopulate tumorsObjective:Cancer stem cells (CSCs) are critical for tumor initiation, invasion,metastasis and play important roles in the relapses after therapy. So far, researches about tumor metabolism have been investigated extensively in a mixture of CSCs and non-CSCs and less attention has been directed to metabolic properties of CSCs and relative regulatory mechanisms. This study will assess the expression and potential metabolic roles of cytosolic phosphoenolpyruvate carboxykinase (PCKI), a key gluconeogenic gene, in melanoma cells that repopulate tumors (tumor-repopulating cells [TRC]) which were separated by 3D soft fibrin gels culture.Methods:(1) TRCs were isolated from murine melanoma B16 cells, hepatoma H22 cells, lymphoma EL4 cells via 3D soft fibrin gels culture. The expression of PCK1 in these TRCs and control cells were determined by RT-PCR, Real time PCR and Western blot. Meanwhile, the expression of PCK1 in undifferentiated mMSCs and mESCs were also tested. (2) The correlation of PCK1 expression with commonly used cancer stem cell markers including CD 133, ALDH1A1 and ABCG5 in multiple human cancer cell lines were analyzed with cbioportal database. To further validate it, the CD133+cells were sorted from B16 cells by flow cytometry and the expression of PCK1 in CD133+and CD133- B16 cells were determined with Real time PCR. (3) The expression of PCK1 in nine paraffin-embedded melanoma samples and TRCs isolated from two cases of fresh human melanoma tissues were detected by immunohistochemical staining and Real time PCR respectively. (4) The expression of FBP1 and G6Pase, two enzymes involved in gluconeogenesis were determined by RT-PCR and Western blot. (5) The rate of growth, glucose consumption, lactate production and intermediate metabolite including serine or glycine and glycerol-3-phosphate (G-3-P) in PCK1 knockdown B16 TRCs were detected. (6) The PCK1 siRNA transfected B16 or H22 TRCs were inoculated into mice and then the tumor formation in vivo was monitored. (7) Intermediate metabolites were added into B16 TRCs after PCK1 knockdown and the growth of TRCs in vitro was analyzed. (8)PCK1-overexpressing (PCK1-OE) B16 cells were constructed and seeded into 3D fibrin gels for culture. The growth of PCK1-OE B16 TRCs in vitro was analyzed. (9) PCK1 promoter-controlled EGFP expressing B16 cells were constructed and cultured in 3D fibrin gels for TRCs spheroid formation. Then these TRCs were harvested and seeded onto conventional rigid dish for further culture. The fluorescence was measured with microscope. To further evaluate the effects of stiffness on PCK1 expression in TRCs, B16 TRCs were cultured on rigid dish for several days and the expression of PCK1 were detected by RT-PCR, Real time PCR and Western blot at different time points. (10) Small molecule inhibitors or blocking antibodies were used to block corresponding signal pathways in B16 TRCs and PCK1 expression were analyzed by Real time PCR to explain the regulatory mechanism. (11) Methylation of promoter region of PCK1 gene in B16 TRCs and control cells were analyzed by bisulphite sequence.Results:(1) TRCs from B16 cells, H22 cells or EL4 cells upregulated PCK1 compared with control cells and PCK1 was also found to be highly expressed in undifferentiated mMSCs as well as mESCs. (2) PCK1 was found to be positively co-expressed with commonly used cancer stem cell markers including CD 133, ALDH1A1 and ABCG5. PCK1 expression was much higher in CD133+B16 cells compared with CD133- B16 cells. (3) PCK1 was highly expressed in roughly one-third of paraffin-embedded melanoma samples and PCK1 was upregulated in TRCs isolated from two cases of fresh human melanoma tissues. (4) FBP1 and G6Pase were not expressed by B16 TRCs. (5) Silencing PCK1 slowed the growth of B16 TRCs in vitro, resulted in the decrease of glucose consumption, lactate production rate and the levels of glycine and G-3-P in B16 TRCs. (6) Silencing PCK1 in B16 or H22 TRCs impeded tumorigenesis in vivo. (7) Impaired growth of B16 TRCs in vitro caused by PCK1 knockdown could be partially rescued by adding glycine or G-3-P. (8) PCK1-OE could promote the growth of B16 TRCs in vitro. (9) The expression of PCK1 in B16 TRCs was gradually downregulated along time when those cells were cultured in 2D rigid dish. (10) Blockade of integrin ?V?3 or PI3K, downstream of aV(33, resulted in downregulation of PCK1 in B16 TRCs. Demethylation of H3K9 by G9a or SUV39hl knockdown induced upregulation of PCK1 in B16 TRCs. (11) High methylated sequence of PCK1 promoter were shown in B16 TRCs and control cells and no difference was found between them.Conclusions:Melanoma tumorigenic cells (TRCs) reprogram glucose metabolism by upregulating PCK1, a key player in gluconeogenesis. However, upregulated PCK1 did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including in the serine and glycerol-3-phosphate pathways for lack of FBP1 and G6Pase expression both of which are essential for gluconeogenesis. Knockdown of PCK1 resulted in decreased growth of TRCs in vitro and impaired tumorigenesis in vivo. All in all, these findings disclose that upregulation of PCK1 is a critical metabolic feature of tumorigenic TRCs, thus providing a potential target for melanoma treatment.Part ? Investigation of PCK1 and PCK2 expression in tumor repopulating cells from breast cancer cellsObjective:This study focus on the expression of PCK1, PCK2 in tumor repopulating cells (TRCs) from breast cancer.Methods:(1) TRCs were isolated from MCF-7, T47D and MDA-MB-231 breast cancer cell lines. PCK1 expression in those TRCs and control cells were detected by RT-PCR. (2) The expression of PCK1 in MCF-7, T47D or MDA-MB-231 TRCs and control cells were analyzed by western blot.Results:(1) There were no significant amounts of PCK1 expression to be detected in MCF-7, T47D and MDA-MB-231 TRCs and control cells. (2) PCK2 was upregulated by MCF-7, T47D or MDA-MB-231 TRCs and control cells compared with control cells.Conclusions:TRCs from breast cancer cells upregulated PCK2 instead of PCK1 isoenzyme. Upregulation of PCK2 would be a critical metabolic feature of tumorigenic breast cancer cells and thus have implications for targeting a unique aspect of breast cancer. However, the detailed metabolic function of PCK2 in TRCs remains to be further elucidated.Part ? Investigation of metabolic regulatory effects of PCK2 upregulation coupled with glutaminolysis on the growth of tumor repopulating cells within breast cancerObjective:To study the metabolic function of pyruvate carboxylase (PC) in TRCs PCK2 upregulated and investigate the coupling between glucose metabolism mediated by PCK2 upregulation and glutaminolysis in tumor repopulating cells of breast cancer cells using MCF-7 model. Meanwhile, the inhibition effects of combined interference of PCK2 and PC or glutaminolysis on the growth of TRCs was also investigated to lay good foundation for further lipid metabolism in TRCs.Methods:(1) TRCs were isolated from MCF-7 breast cancer cells via 3D soft fibrin gels culture. The glucose and glutamine consumption rate of MCF-7 TRCs or control cells were determined by enzymatic assay kit and HPLC to study the metabolic coupling between glucose and glutamine. (2) The growth of MCF-7 TRCs in vitro under glutamine-deprived culture medium was analyzed. (3) Western blot were used to detect the expression of PC, SLC1A5, GLS and IDH2 in MCF-7 TRCs and control cells. (4) The growth of MCF-7 TRCs in vitro was analyzed when combined silencing of PCK2 with PC, GLS or IDH2 using siRNAs.Results:(1) MCF-7 TRCs exhibited much higher glucose and glutamine consumption rate compared with control cells. (2) Deprivation of glutamine from medium resulted in significant decrease of MCF-7 TRCs growth in vitro. (3) PC, SLC1A5 and IDH2 were also upregulated in MCF-7 TRCs compared with control cells except for PCK2. (4) Combined interference of PCK2 with PC, GLS or IDH2 exhibited better inhibition effects on growth of MCF-7 TRCs in vitro than single knockdown.Conclusions:TRCs from MCF-7 breast cancer upregulated pyruvate carboxylase to compensate oxaloacetate which would be largely consumed by enhanced expression of PCK2. Moreover, TRCs also could adopt glutaminolysis pathway to directly supplement citrate. TRCs could strengthen the synthesis of lipid through enhanced glycerol backbone metabolism mediated by PCK2 upregulation and fatty acid synthesis via glutaminolysis to meet the demand for accelerated anabolic metabolism. Combined interference of PCK2 with PC or glutaminolysis provide a novel strategy for breast cancer therapy against tumorigenic cells.