Metabolic Remodeling In Colorectal Cancer Cells Adapted To Acidic Microenvironment

Colorectal cancer(CRC)is still one of the malignant tumors that endanger human health.Approximately 600,000 people die of colorectal cancer every year all over the world.According to the latest statistics,the new cancer cases of CRC ranks second and the cancer death ranks among top five in China.The urban incidence rate is increasing year by year.At present,early-stage colorectal cancer is mostly treated with endoscopic minimally invasive methods.The treatment of advanced-stage colorectal cancer is based on surgical resection,supplemented by chemotherapy(5-fluorouracil,capecitabine,oxaliplatin),immunotherapy,Traditional Chinese Medicine and other comprehensive programs of supportive treatment.However,due to the large trauma and easy recurrence,the radical cure of CRC is greatly affected.Therefore,it is of great significance to explore the mechanism of colorectal cancer development and to search new drug targets.During tumorigenesis,glycolysis is enhanced due to vascular dysfunction,which cause poor tissue perfusion and reduced oxygen availability in the local microenvironment.Then,a large amount of lactic acid or H+produced via glycolysis cannot be transported to extracellular through the Na+/H+exchanger or proton pump in time,leading to extracellular acidic microenvironment.Although acidic microenvironment is not conducive to the proliferation of tumor cells,it promotes invasion and migration,drug resistance and immunoevasion.In recent years,tumor acidic microenvironment has been gradually recognized as a key regulator of metabolic reprogramming.Currently,existing reports mainly focus on glutamine metabolism,fatty acid synthesis,iron death,autophagy and lipid droplets in tumor cells under acidic microenvironment.Mitochondria,as a center of intracellular energy metabolism,do not only produce ATP,but also participate in the regulation of cell signaling and cell death.Mitochondria also provide intermediates as substrates for the de novo synthesis of lipids and non-essential amino acids,which guarantee the growth and survival of tumor cells under stressful environment(nutrient deficiency,hypoxia,or cancer treatment).However,the characteristics of metabolic patterns in tumor cells under chronic acidic microenvironment are still unclear,especially the mechanism governing mitochondrial homeostasis.In this study,we conducted gene enrichment analysis of RNA sequencing data of colorectal cancer cells adapted to acidic microenvironment(CRC-AA)and their parental cells.We found that mitochondrial-related oxidative phosphorylation pathways and fatty acid pathways were significantly upregulated.2-DG could significantly inhibit the survival of CRC cells but had no effect on CRC-AA cells.On the contrary,the viability of CRC-AA cells treated with Oligomycin was significantly inhibited.Acetyl-CoA,as an important substrate for energy metabolism and epigenetic modification,also had distinct sources in CRC and CRC-AA cells.Compared with parental cells,etomoxir significantly inhibited the production of acetyl-CoA in CRC-AA cells.We further found that mitochondria content was accumulated,mitochondrial membrane potential and the intracellular ATP level were increased in CRC-AA cells detected by western blot,energy analysis and flow cytometry analysis.Next,we found that lipid droplets were accumulated in CRC-AA cells.Autophagy degrades membranous organelles to release fatty acids(FAs)channeled by DGAT1 into new lipid droplets,which provides an "on-demand" source of FAs for mitochondria.At the same time,lipid droplets were crucial to maintaining mitochondrial homeostasis.Excessive fatty acids produce lipotoxic substances accumulation in a short time through fatty acid β-oxidation in mitochondria,indicating that a lipotoxic dysfunction of mitochondria.In addition,we found that fatty acids released by lipid droplets supported autophagosome biogenesis through ACSL4(long-chain fatty acid coenzyme A ligase family)in CRC-AA cells.Inhibition of ACSL4 expression reduced the accumulation of LC3B on autophagosome membrane.Although mitochondria content was increased,the level of intracellular ATP was decreased in CRC-AA cells.This means that blocking autophagy in CRC-AA cells results in the inability to remove dysfunctional mitochondria and disrupts mitochondrial homeostasis.We also analyzed that elevated levels of mtROS accompanied by hyper-activation of AMPK in CRC-AA cells is key molecular signal pathway for maintaining mitochondrial biogenesis and homeostasis.Finally,we explored that ammonia could induce increased autophagy levels in CRC-AA cells.It is also proposed that different concentrations of ammonia have different effects on autophagic flux,clarifying the important role of pH on autophagosome function.In summary,our study confirms that the chronic acidic microenvironment induces the metabolism of colorectal cancer cells to be biased towards oxidative phosphorylation and fatty acid oxidation.On the one hand,autophagy-derived lipid droplets provide mitochondrial acetyl-CoA with fatty acids,and the formation of lipid droplets reduces mitochondrial lipotoxicity.On the other hand,lipid droplets provide fatty acids for autophagosome membrane formation.Increased level of autophagy ensures the clearance of dysfunctional mitochondria.Lipid droplets and autophagy positive feedback loop play a central role in maintaining mitochondrial function and homeostasis.Enhanced mitochondrial function is critical for colorectal cancer cell survival and acquisition of malignant characteristics under chronic acidic microenvironment.Targeting the autophagy-lipid droplets-mitochondria loop in tumor cells may provide new ideas for the clinical treatment of colorectal cancer.