Energy Metabolism Reprogramming Induced By Twist In Breast Cancer Cells And Their Underlying Mechanisms

Objective:To investigate the role of Twist in energy metabolism reprogramming of human breast cancer cells and the effect of energy metabolism reprogramming on migration and EMT of breast cancer cell, and discuss the relevant molecular mechanisms, which would provide new experimental evidences for the treatment strategy by targeting tumor metabolism.Methods:MCF10A-Vector and MCF10A-Twist cells were cultured in humidified atmosphere of 5% CO2 at 37℃, and then the cells were exposed to 21% O2 or 1% O2 for 1 h,3 h,6 h and 12 h, respectively. Glucose consumption was assayed by Glucose Assay Kit and lactate production was detected using Lactate Assay Kit according to the manufacturer’s instructions. Mitochondrial mass was measured by staining with Mito-Tracker green. Mitochondrial morphology was analyzed by transmission electron microscope and photographed using a CCD camera. The lentivirus vector containing Twist siRNA was constructed, and then MCF10A-Twist and BT549 cells were infected with the recombinant lentivirus and the silencing effect of Twist gene was evaluated by qRT-PCR and western blot. Subsequently, glucose consumption, lactate production and mitochondrial mass of MCF10A-Twist-sh-Twist cells, BT549-sh-Twist cells and their respective control cells were detected using the methods that were described earlier in this article. Our previous mRNA microarray and proteomic data of MCF10A-Twist and MCF10A-Vector cells were re-analyzed by bioinformatics methods to identify differentially expressed genes which were associated with energy metabolism. Among these genes, some differentially expressed genes were selected to be verified by qRT-PCR. Western blot was applied to analyze the expressions of some key genes under normoxic or hypoxia condition for the indicated time. After knockdown of Twist in MCF10A-Twist cells, the expressions of some key genes were also assayed. Moreover, western blot was used to detect the expressions of these key genes in MCF10A and BT549 cells. The changes of the key genes in BT549 cells treated with hypoxia or with Twist gene silence were determined. LY294002 was applied to evaluate the role of PI3K/AKT signaling in energy metabolism of MCF10A-Twist and BT549 cells. Subsequently, the phenotype of energy metabolism and the expressions of key genes were measured. After silencing mTOR by using siRNA in MCF10A-Twist and BT549 cells, the expression of downstream metabolism-related genes including PKM2 and LDHA were investigated. After silencing p53 in MCF10A-Vector cells and overexpressing p53 in MCF10A-Twist cells, the expressions of G6PD were analyzed. The endogenous P53 and G6PD in Twist-low and Twist-high level of breast cancer cells, the representative MCF-7 and BT549 cells were detected. And then siRNA targeting p53 was used to interfere the expression of p53 in BT549 cells. The level of G6PD expression in BT549 cells with p53 silence was detected. To further investigate the role of p53 in modulating G6PD, the wild-type p53 was stably transfected into BT549 cells and then the expression of G6PD was analyzed. Finally, whether EMR could lead to acquire a migration potential was further investigated. Transwell assay was applied to measure the migration abilities of cells treated with LY294002 or with p53 overexpression, and the expressions of biomakers for EMT in the cells treated with LY294002 or with p53 overexpression were analyzed by western blot.Results:The glucose consumption and the lactate production were increased in MCF10A-Twist cells compared to MCF10A-Vector under normoxic or hypoxia conditions, and hypoxia treatment made MCF10A-Twist cells consume more glucose and produce more lactate and the changes showed a time-effect relationship. Compared with the control cells, MCF10A-Twist cells presented weaker fluorescence intensity of Mito-Tracker green, which indicated that MCF10A-Twist cells had lower mitochondria mass than that in MCFIOA-Vector cells. Moreover, the mitochondrial mass of MCF10A-Twist cells was further reduced under hypoxia conditions. Knockdown of Twist in MCFIOA-Twist cells decreased the glucose consumption and the lactate production compared with its control cells. This was further confirmed in the BT549 cells with Twist silence and its parent cells. The mitochondria mass were partly increased by knockdown of Twist in MCF10A-Twist cells and BT549 cells. And hypoxia treatment exacerbated these changes. A set of energy metabolism-associated genes were dysregulated in MCF10A-Twist cells compared with MCF10A-Vector cells by reanalyzing our previous mRNA microarray and proteomic data. Some of the genes governing the process of glucose metabolism were validated using qRT-PCR analysis, and the genes such as G6PD, PKM2, LDHA, PGK1, ENO1 and TPI1 were up-regulated in MCFIOA-Twist cells. The high levels of PKM2, LDHA, and G6PD, all those are the critical genes linked to energy metabolism, were further confirmed by western blot analysis. In addition, some genes of interest, such as pAKT and p53, were also detected in this work. It was shown that the protein expression of pAKT was up-regulated and p53 was down-regulated in MCF10A-Twist cells and Twist-positive BT549 cells. Interestingly, the protein levels of G6PD, PKM2, LDHA and pAKT in MCF10A-Twist cells and BT549 cells were increased under hypoxic conditions. Moreover, loss of Twist by siRNA led to decreased expression of G6PD, PKM2, LDHA, pAKT and increased expression of P53 in MCF10A-Twist cells and BT549 cells as expected. After inhibition of PI3K/AKT activity by LY294002, the glucose consumption and the lactate production of MCF10A-Twist cells and BT549 cells were decreased. Mitochondrial mass in the LY294002-treated MCF10A-Twist and BT549 cells were higher than those in their control cells. Correspondingly, the expressions of PKM2 and LDHA in MCF10A-Twist cells and BT549 cells were down-regulated accompanied with inactivation of PI3K/AKT signaling in the presence of LY294002. However, there was no remarkable change of P53 and G6PD expression after LY294002 treatment. Silencing mTOR with siRNA decreased PKM2 and LDHA at protein level in MCF10A-Twist and BT549 cells. To confirm the negative correlation between P53 and G6PD in MCF10A-Twist cells, the wild-type p53 gene was transfected into MCF10A-Twist cells. As expected, restoration of p53 expression in MCF10A-Twist cells suppressed G6PD expression. To extend this finding, the endogenous P53 and G6PD in Twist-low and Twist-high level of breast cancer cells, the representative MCF-7 and BT549 were detected. A faint expression of wild-type p53 and strong expression of G6PD were deteted in MCF-7 cells, and strong mutant p53 expression and weak G6PD expression were identified in BT549 cells, however, the mild expression of P53 and weaker G6PD were in MCF10A cells. Unlike wild-type p53, the p53 mutants showed minimal or no activity in inhibiting G6PD. As a result, there were no obvious changes in the level of G6PD expression in BT549 cells after knockdown of p53 expression with siRNA. To further investigate the role of p53 in modulating G6PD, the wild-type p53 was stably transfected into BT549 cells. Consistent with our expectation, G6PD expression was repressed by wild-type p53 in BT549 cells. Finally, we asked whether EMR could lead to acquire a migration potential. Compared with MCF10A-Vector, MCF10A-Twist showed stronger migratory capacity. Treatment with LY294002 or restoration of p53 expression in MCF10A-Twist cells resulted in a significant decrease in cell migration ability. Similar results were further confirmed in BT549 cells. Our data showed that silence of endogenous mutant p53 in BT549 cells using its siRNA presented a decreased migration and overexpression of wild-type p53 in BT549 cells also decreased cell migration. In addition, after treatment with LY294002 or restoration of p53 expression in MCF10A-Twist and BT549 cells, the expressions of E-cadherin were upregulated and the expressions of N-cadherin and Vimentin were downregulated.Conclusion:In summary, our results indicate that Twist can arouse EMR by activating the PI3K/AKT/mTOR pathway and repressing the p53 pathway, thus linking with a series of biological processes, such as migration and EMT, in Twist-positive breast cancer cells. This study sheds light on the mechanisms of Twist-mediated EMR in breast cancer cells and provides novel insight into the treatment of breast cancer by targeting tumor cell metabolism.