Upregulated Glycolysis Regulates The Stemness Phenotypes Of Prostate Cancer Cell

Background Prostate cancer continues to be the most common cancer diagnosed in male patients and the second leading malignancy of cancer-related deaths in Europe and the United States. In China, the incidence of prostate cancer has been getting higher since the past decades. Most patients with advanced stages of the disease respond to the current treatment(hormonal therapy, radiotherapy, or chemotherapy) only in the beginning, mostly developing progression and subsequently widespread metastasis, resistant to traditionally prostate cancer therapeutic methods. Extensive basic research studies in the mechanisms of carcinogenesis do expand our knowledge of prostate cancer, but the therapeutic effect of the patients with prostate cancer is still challenging. Therefore, further investigations are definitely required in this field.A growing number of studies have repeatedly confirmed the existence of CSCs. CSC populations have already been identified in several solid tumors including prostate cancer prostate cancer. CSCs are a subpopulation of tumor cells, which are not recognized by current chemotherapy or radiotherapy. They play key roles in relapse and metastasis and are thought to be one of the promising targets for cancer treatments.The Warburg effect or named as aerobic glycolysis wherein glycolysis is unregulated even in the presence of oxygen is now recognized as an important hallmark of cancers. Research done in the past 15 years has confirmed the prevalence of Warburg effect. Aerobic glycolysis is also the metabolic property of embryonic stem cells(ESCs), hematopoietic stem cells(HSCs) and induced pluripotent stem cells(i PSCs). In these stem cells, oxidative capacity is reduced and glycolysis are enriched. This enriched glycolysis-dependent pathway along with reduced oxidative capacity and lower mitochondrial activity have been proposed as markers of cell stemness.Pyruvate, a critical molecule for mitochondrial OXPHOS, links the glycolytic pathway with the mitochondrial tricarboxylic acid cycle and plays an important role in this metabolic reprogramming. Pyruvate enters mitochondria through a recently identified mitochondrial pyruvate carrier(MPC), then was catalyzed to acetyl-Co A irreversibly by pyruvate dehydrogenase complex(PDHc). In most differentiated mammalian cells, pyruvate was processed from glucose in the cytosol and thereafter was directed into mitochondria and oxidized for efficient ATP production. While in some cancer cells and stem cells, pyruvate is converted to lactate by aerobic glycolysis in most cases, the so-called Warburg effect, which although is inefficient but seems to enable cancer cells proliferate and maintain the cell stemness.Given the above, we asked whether reduced pyruvate metabolism to acetyl-Co A resulted in aerobic glycolysis and was correlated with higher stemness phenotype of prostatic cancer cells. We immunohistochemcally examined the expression of PDHA1 in human prostate cancer tissues and explored its relationship with conventional clinic pathological characteristics in a series of 88 prostate cancer patients with long-term follow-up. We interfere the oxidation of glucose-derived pyruvate to acetyl-Co A by knockout the E1α subunit of PDHc(PDHA1) gene. Lastly, we use UK5099 to block MPC in prostate cancer cells, in consideration of its effect on cell stemness. We assessed its effect on metabolic phenotypes and stemness-like features. The metabolism switch and stemness were investigatedIn a word, in this study, we analyzed the relationship between metabolism switch and the stemness phenotype of prostate cancer through interfering the oxidation of glucose-derived pyruvate to acetyl-Co A by MPC blocker or by knockout PDHA1 gene.Part Ⅰ: Decreased Expression of PDHE1α Predicts Worse Clinical Outcome in Prostate cancerMethods 1. PDHA1 protein in a series of 88 prostate cancer samples was immunohistochemically investigated and analyzed. 2 Statistical analyses of the data were processed using the SPSS17.0 statistical software package. The Chi-square tests(Pearson and linear-by-linear as appropriate) were performed for analyzing the associations of PDHE1α subunit expression and the clinicopathological variables. Survival curve was plotted using the Kaplan-Meier method and compared with the use of the two-sided log-rank test.Results 1. It was discovered that 34(38.64%) samples were positive for PDHA1 protein expression while all the other 54 tumors(61.36%) were negative. PDHA1 expression was significantly positively associated with the grades of tumor differentiation which are classified by Gleason scores of prostate cancer. PDHA1 protein expression was positive in 15/27(55.6%) samples with Gleason score less than7, but in 14/41(34.15%) samples of Gleason score 7 and only 5/20(25%) samples of Gleason score between 7 and 10(p<0.05). 2. It was demonstrated that patients with negative expression of PDHA1 in prostate cancer tissues had worse overall survival than the patients with PDHA1 expression positive tumor(p<0.05).Part Ⅱ: PDHA1 gene knockout up-regulates the stemness of prostatic cancer cell in vitroMethods 1. TALEN plasmid was constructed and transferred into Ln Cap cell with liposome. Stable knockout of PDHA1 cell line was established and named PDHA1 KO. 2 The metabolic profiles of PDHA1KO: ATP production, lactate acid production, oxygen consumption rate(OCR), ECAR, mitochondrial membrane potential(ΔΨm) were investigated by ATP determination kit, lactate assay kit, seahorse XF24 extracellular flux analyzer, and a unique cationic dye JC-1. 3. Proliferation was examined by cell growth curve. Chemo-sensitivity to Docetaxel was examined. Side population and stemness markers were examined by flow cytometry and Western blot.Results 1. The TALEN vectors we constructed in this study, successfully replaced some sequence in the exon 1 of PDHA1 gene with a new sequence containing an early terminator code, which terminated PDHA1 protein expression in the middle of exon 1. And a stable knockout of PDHA1 gene cell line was established. 2. OCR, ATP production and membrane potential were significantly decreased and the ECAR, extracellular lactate acid efflux and ROS production were significantly increased in PDHA1 KO cell. 3. Proliferation of PDHA1 KO cell was inhibited. Meantime, the PDHA1 KO cell exhibited resistant to docetaxel, higher migration ability, and higher levels of stemness markers(CD44, ABCG2, Oct3/4, Nanog) expression.Part Ⅲ:Application of UK5099 creates metabolic reprogram and greater stem-like properties in prostate cancer cellsMethods 1. UK5099 was used to block pyruvate transportation into mitochondrial matrix. The mitochondrial pyruvate concentrations were examined by pyruvate assay kit. 2 The metabolism profiles: ATP production, lactate acid production, Oxygen consumption rate(OCR), ECAR, mitochondrial membrane potential(ΔΨm) were investigated by ATP determination kit, lactate assay kit, seahorse XF24 extracellular flux analyzer, and a unique cationic dye JC-1. 3. Proliferation was examined by cell growth curve. Cell cycle, side population and stemness markers were examined by flow cytometry and Wester blotting.Results 1. We confirmed that pyruvate transportation was blocked by UK5099 in Ln Cap prostate cancer cell line. 2. Upon UK5099 application, OCR, ATP production and membrane potential were significantly decreased and the extracellular lactate acid efflux and ROS production were significantly increased in the UK5099 treated cells. 3. Upon UK5099 application, cell proliferation was inhibited. This was accompanied by increased proportion of side population(SP) fraction, higher level of stemness markers(Oct3/4, Nanog) expression.Conclusions 1. We are probably the first to use UK5099 to block MPC or gene knockout technology to knock out PDHA1 gene in prostate cancer cells to interfere pyruvate metabolism to acetyl-Co A, in consideration of its effect on cell stemness. We assessed its effect on metabolic phenotypes and stemness-like features. 2. It was found that interfering pyruvate metabolism enhanced aerobic glycolysis and decreased mitochondrial OXPHOS in Ln Cap cells in vitro concomitant with upregulated cell stemness features. We propose that MPC blocker(UK5099) application or PDHA1 KO cell may be an ideal model for Warburg effect/cancer stem cell studies in prostatic cancer cells.3. By immunhistochemically examined PDHA1 protein expression in prostate cancer samples, it was revealed that negative PDHA1 protein expression was related with poor clinical outcome in patients with prostate cancer. This hinted that aerobic glycolysis-dependent pathway plays important role in the development of prostate cancer. 4. These findings provide new insights into the metabolic regulation involved in prostate cancer stem cells and further studies on this direction may lead to the development of new approaches for better diagnosis and effective therapy. Although still in an exploratory stage, targeting the metabolic reprograming has already produced promising results. With improved knowledge and better understanding of the metabolism swift, metabolic reprograming–based cancer stem cell targeting therapy in combination with oncogenic and metabolic probably become a very promising prostate cancer treatment option.