The Metabolic Role Of PCK2 In Melanoma Tumor Repopulating Cells

Objective:Although metabolic abnormality as a hallmark of cancer has been investigated extensively in differentiated tumor cells, much less attention has been directed to the metabolic properties of stem cell-like cancer cells (SCLCC) that repopulate tumors. Tumor repopulating cells (TRCs) as a self-renewing, highly tumorigenic subset of cancer cells not only play a crucial role in tumorigenesis, but also are the major contributor, most likely responsible for treatment failures and cancer recurrences. The expression of PCK2 appears to play a critical role in promoting the growth of TRCs.But how PCK2 regulates the metabolism pathway still remains elusive.Methods:(1)The PCK2 expressions in different TRCs and differential tumor cells were analyzed by Real-time PCR and Western blot. The expression of PCK2 in differential and undifferential mESCs was detected by Real-time PCR and Western blot. The expression of PCK2 was determined in CD133+ B16-F1 cells by Real-time PCR. TRCs from two melanoma patients were used to analyze the expression of PCK2 by Real-time PCR. (2) To confirm the role of PCK2 in cancer patients, the relationship between PCK2 and patient’s overall survival time was analyzed by the Kapla-Meier survival method. (3)The PCK2 overexpression plasmid was constructed and the stable PCK2 Tet-inducible cell was selected. In vivo,5×104 B16-F1 TRCs transfected with PCK2 plasmids or 1×105 B16-F1 cells transfected with PCK2 or scramble siRNA were i.v. injected to C57BL/6 mice (n= 8).24 days later, the lung metastases were recorded and the metastatic nodules were counted. In vitro, the PCK2 Tet-On TRCs were cultured in presence or absence of 2μg/ml doxycycline, the colony size andcolony number of TRCs was recorded and calculated for 5 days. The PCK2 Tet-On TRCs was harvested and treated with different concentration of cisplatin,24 hours later the apoptosis was analyzed with flow cytometry. In vivo, 1×104 Tet-inducible PCK2 TRCs were s.c. injected to mice, fed with doxycycline water. The incidence of tumor in mice was analyzed by Kaplan-Meier analysis. (4) To investigate how PCK2 to modulate the carbon flow of TRCs. Firstly, the glucose consumption and lactate production of TRCs and differential tumor cells was measured. Then glycolytic capacity was measured after forced expression of PCK2 in TRCs. As citrate is the central intermediate of TCA Cycle, and PCK2 downregulation may accelerate citrate formation. We also detected the citrate concentration of TRCs. Then overexpression of PCK2, the citrate concentration of TRCs was also measured. In order to verify citrate is used for lipid synthesis, or is it more involved in the TCA cycle, the growth rate and glucose consumption was recorded and calculated after silencing IDH3A or MDH2 in TRCs and B16-F1 cells. Moreover, the glucose consumption of TRCs was measured after silencing SLC25A1,ALCY, FASN or ME1. The TCA intermediates fumarate concentration was detectedin TRCs and B16-F1 cells. After forced expression of PCK2, the fumarate concentration was measured in PCK2 Tet-On TRCs. As fumarate concentration is capable of inhibiting the activity of enzyme prolyl hydroxylase (PHD) and Ten-Eleven Translocation (TET) enzymes. Therefore, we analyzed the expressions of HIF1α and HIF2α in TRCs by western blot, under hypoxia (1% O2) or normoxia (20% O2). Also, after overexpression or silencing PCK2, HIFα expressions in TRCs or B16-F1 cells were also analyzed by western blot under hypoxia. Moreover, the relative levels of 5hmC and 5mC were analyzed by LC-MS/MS in TRCs. After forced overexpression PCK2, the relative levels of 5hmC and 5mC in TRCs were also analyzed. Considering the OXPHOS coupling with TCA Cycle, we also measured the oxygen consumption rates (OCRs) of TRCs and B16-F1 cells by Seahorse XF24. After forced expression PCK2, OCRs were also measured in TRCs. Moreover, with treatments of different concentration of OXPHOS inhibitor rotenone and oligomycin, the glucose consumption and cell proliferation was measured, respectively.Results:(1)The expression of PCK2 was downregulated in TRCs. And the expression of PCK2 was lower in undifferentiatial mESC. Compared with CD133-B16-F1 cells, CD133+ cells exhibited lower expression of PCK2. In addition, the expression of PCK2 was also downregulated in TRCs derived from two melanoma patient samples. (2) The Kaplan-Meier analysis showed that patients with PCK2 low had a significant lower survival time in melanoma, lung cancer and gastric cancer. (3) In vivo the tumor formation ability of B16-F1 TRCs was remarkably reduced after overexpression of PCK2 by plasmid transfection. Consistently, knockdown of PCK2 with siRNA, the tumorigenicity of B16-F1 cells was enhanced significantly. Moreover, with doxycycline treatment in culture medium, the growth of PCK2 Tet-On TRCs was inhibited, and the drug resistance was also decreased. Furthermore, after s.c. injected PCK2 Tet-On TRCs, the mice administrated with doxycycline showed a reduced tumor incidence. (4) Compared to differential tumor cells, glucose consumption and lactate production of TRCs was increased. However, the glycolytic capacity of TRCs was decreased after overexpression of PCK2. In the meantime, TRCs also showed high citrate concentration, which could be reduced after overexpression of PCK2. Consistently, the glucose consumption of TRCs was remarkably decreased after silencing SLC25A1, ACLY, FASN and ME1.Intriguingly, knockdown of IDH3A or MDH2, the colony size and number of TRCs was not changed, whereas the growth of differential B16-F1 cells was decreased significantly. The glucose consumption of B16-F1 TRCs or B16-F1 cells was as same as their growth rates. Furthermore, the fumarate concentration of TRCs was higher than B16-F1 cells, and forced overexpression of PCK2 decreased the concentration of fumarate in TRCs. Under hypoxia or normoxia, the expression of HIF1αa and HIF2a was enhanced in TRCs. And after overexpression of PCK2, the levels of HIF1α and HIF2a were lower under hypoxia. HIF1α and HIF2a also were upregulated in B16-F1 cells under hypoxia, after silencing PCK2 by siRNA. Moreover, compared to B16-F1 cells, the relative level of 5hmC in TRCs was lower. And the levels of 5hmC became higher after overexpression of PCK2. Finally, we found that TRCs exhibited lower oxygen consumption rates, and were more sensitive to OXPHOS inhibitor rotenone and oligomycin. And the oxygen consumption rates of TRCs would increase if PCK2 was forced expressed.Conclusions:In this study, we found that PCK2 downregulation is necessary for maintenance of B16-F1 TRC growth and tumorigenesis. The PCK2 expression is related to cancer patient’s overall survival. The mechanism maybe:(i)PCK2 downregulation results in the increase of citrate production;(ii) more citrate in the cytosol is decomposed to acetyl-CoA for fatty acid production and OAA; (iii) downregulation of PCK2 results in the accumulation of fumarate, may leading to inhibiting enzymes PHD and TET; (iv)decreased production of NADH and FADH2 by downregulation of PCK2 leads to attenuated oxidative phosphorylation. Taking together, downregulation of PCK2 is a metabolic feature of TRCs which is needed for TRCs growth and tumorigenesis.