Role And Mechanism Of PGAM1 In MTOR-mediated Tumor Aerobic Glycolysis

Glucose metabolism reprograming is considered as one of the hallmarks of cancer. In the 1920 s, German scientist Otto Warburg first discovered that unlike in normal cells, tumor cells preferentially metabolize glucose by glycolysis, even in the presence of oxygen. This process was also called "Warburg effect". However, key questions about why and how cancer cells perform this highly glycolytic phenotype is still not well understood.The mTOR signaling pathway is one of the most frequently altered pathways in tumor. Inactivation of tumor suppressor Tsc2 and Pten lead to hyperactivation of mTOR pathway. Our previous studies have shown that hyperactivation of mTOR pathway can promote the Warburg effect.PGAM1 is an important glycolytic enzyme which was overexpressed in several human cancers and was participated in the regulation of glucose metabolism. To explore the possible mechanism of PGAM1 upregulation in tumor cells, as well as the potential role PGAM1 might play in mTOR-mediated Warburg effect and tumorigenesis, this research is divided into the following three parts:Chapter 1: MEFs with hyperactive mTOR activity due to Tsc2 and Pten knockout were used to observe PGAM1 expression and enzyme activity compared with wild-type MEFs, mTOR inhibitor rapamycin was used to validate the results. ELT3 cells treated with rapamycin or transfected with TSC2 were further detected to observe PGAM1 expression. PGAM1 expression was also measured in kidney tissue of Tsc2 heterozygous mice, and human lung cancer tissue samples were also checked. HIF1α stable knockdown in Tsc2-/-, Pten-/- MEFs were used to observe the molecular mechanism of PGAM1 expression and ChIP was further used to explore whether HIF1α directly promotes the transcription of PGAM1. The results show: PGAM1 expression and activity were both increased in mTOR hyperactivated MEFs. mTOR signaling pathway regulated PGAM1 through HIF1α-mediated transactivation.Chapter 2: PGAM1 knockdown Pten-/- MEFs were established and glucose consumption as well as lactate production of these cells were measured. MTT assay and plate colony formation assay were used to determine the proliferation and colony formation capability of these cells. Moreover, nude mice were injected with PGAM1 knockdown Pten-/- MEFs or control MEFs and tumor-free survival and overall survival of both groups were observed. The results show: Knockdown of PGAM1 in mTOR hyperactive MEFs reduced Warburg effect, cell proliferation and colony formation. Nude mice carried PGAM1 knockdown Pten-/- MEFs had longer tumor-free survival and overall survival.Chapter 3: The study consisted of RNA samples of 28 pairs of non-small cell lung cancer(NSCLC) carcinoma and adjacent tissue, paraffin sections of 227 cases of NSCLC and 30 cases of colorectal cancer from Tianjin Medical University Cancer Institute and Hospital. mRNA expression of PGAM1 was measured. PGAM1 and pS6 protein level was measured by immunohistochemical assay. Correlations between PGAM1 and pS6 expression, as well as patients’ prognosis were analyzed. The results show: In NSCLC, PGAM1 expression was higher in carcinoma tissue than in adjacent tissue. In NSCLC and colorectal cancer samples, patients with high PGAM1 expression would have shorter overall survival(OS) and shorter progression free survival(PFS). PGAM1 and pS6 expression were well correlated.Conclusions:1) Both protein expression level and the enzyme activity level of PGAM1 are regulated by mTOR signaling pathway, mTOR increases PGAM1 expression through HIF1α-mediated transactivation. 2) PGAM1 expression influence the mTOR mediated aerobic glycolysis, proliferation, colony formation and in vivo tumorigenesis. 3) In NSCLC, PGAM1 expression is higher in carcinoma tissue than in adjacent tissue. PGAM1 high expression may indicate poor prognosis for NSCLC or colorectal cancer patients. PGAM1 and mTOR activation were well correlated in cancer samples.