Effect Of Tumor Acidic Microenvironment On Cellular Dryness And Glucose Metabolism In Human Breast Cancer And Its Mechanism

Cancer stem cells(CSCs), a very small subpopulation that are highly resistant to chemotherapy and radiotherapy, capable of self-renewal, self-repairing, maintaining a certain totipotent and pluripotent and forming cloned cell subsets in vitro, are currently considered as one of the root causes of tumor recurrence and metastasis.The stemness maintenance of CSCs needs to be supported by the surrounding microenvironment, which guarantees CSCs in an undifferentiated state and maintain the potential of proliferation and differentiation. The acidic microenvironment is a common character of cancer. Many studies have showed that the pH value in the tumor tissues is between6.9-6.0, even lower at5.8, while the pH value in the normal tissues is between7.35-7.45. It was well known that the acidic tumor microenvironment was caused by the hypoxia; however the unique metabolic phenotype of cancer cells (Warburg effect) was ignored. The cancer cells obtain energy from glycolysis reaction no matter they are in aerobic or in hypoxic environment, thereby causing a large amount of lactic acid and hydrogen ion efflux, which result in an acidic tumor microenvironment.Lin28is a kind of RNA binding protein that is highly conserved from lower to higher organisms. Recent studies have found that Lin28can promote cell reprogramming to maintain stem cell self-renewal and pluripotency. Lin28was identified to suppress some microRNAs in the maturation process, which suggested Lin28as a key molecule in the regulation of cell fate in the post transcription level. However the molecular mechanisms of Lin28regulating stemness and the interaction of stemness and metabolism in breast cancer are still not fully understood.In this study, First, in order to clarify the effects of acidic microenvironment that generated by aerobic glycolysis on stemness properties of CSCs, the stemness properties in human breast cancer cells MDA-MB-231that were cultured with either pH6.8or pH7.4medium were detected by using FCM, Real-time PCR, Western blot, sphere test or wound healing experiment. Moreover, the activation of TCF4/β-catenin signaling pathway was also detected to explore the potential mechanism. The results indicated that all stemness including the percentage of ALDHhigh subpopulation, the expression of sternness gene (SOX2, OCT4and Lin28b), the capability of sphere formation and cell mobility were increased in MDA-MB-231cells cultured with pH6.8culture medium. Lin28b and TCF4/β-catenin signaling pathway were activated in MDA-MB-231cells cultured with low pH medium as well.Next, to explore the relationship between stemness and metabolism, initially, it was constructed the stable cell line that knock down the stemness key molecule Lin28b in MDA-MB-231cells (shLin28b-MDA-MB-231). Then, the expression of aerobic glycolysis related genes and the ATP production were detected in DNA level, protein level or Cell Titer-Blue Cell Viability Assay on shLin28b-MDA-MB-231cells. The microRNAs that related to Lin28b were screened by using shLin28b-MDA-MB-231cells as cell model. The results display that the expression of aerobic glycolysis related genes (GLUT1, PKM2, LDHA,PDK1) were down regulated and the ATP production was reduced through down-regulate miR-34a-5p on shLin28b-MDA-MB-231cells.Furthermore, to define the effects of acidic microenvironment that generated by aerobic glycolysis on metabolism of cancer cells, the expression of aerobic glycolysis related genes, miR-34a-5p and the ATP production were detected by using Real-time PCR, Western blot or Cell Titer-Blue Cell Viability Assay on MDA-MB-231cells cultured with either pH6.8medium or treated with the inhibitors for blockade of the TCF4/β-catenin signaling pathway. The results showed that the expression of aerobic glycolysis related genes were up-regulated and the ATP production was increased on MDA-MB-231cells treated with TCF4/β-catenin inhibitors or cultured with pH6.8medium.Together, we summarized the main findings as follows:the acidic tumor microenvironment up-regulated Lin28b via activating TCF4/β-catenin signaling pathway, which result in enhancement and maintaining of CSCs phenotype in MDA-MB-231cells. On the other hand, acidic tumor microenvironment increased the expression of aerobic glycolysis related genes and reduced the ATP production, promoted glycolysis reaction, accelerated the generation of lactic acid and efflux of hydrogen ions through down-regulated miR-34a-5p, thereby further strengthen the formation of the acidic tumor microenvironment.