Mechanism of mitochondrial respiratory dysfunction by oncogenic K-Ras
암유전자 K-ras에 의한 미토콘드리아의 기능손상 유도기전 연구
Among the several features of cancer cells, mitochondrial impairment in relation to altered metabolism has been extensively researched. Although the importance of mitochondrial impairment in cancer development has been emphasized, how and why mitochondrial impairment occurs remains unclear. To study the role of mitochondrial impairment in early stages of carcinogenesis, an experimental model for oncogenic transformation without cell death was required. Already known as a strong oncogene driving transformation, oncogenic K-RasV12 was adopted to transform cultured fibroblast. When cultured Rat-2 fibroblast cells were infected with a retrovirus harboring constitutively active K-RasV12, the expressions of both respiratory and non-respiratory proteins were decreased. Such decreased expression of mitochondrial proteins was functionally reflected in the results of significant mitochondrial dysfunction parallel with the acquisition of the transformed characteristics. During the oncogenic transformation by K-RasV12, acidic vesicles appeared simultaneously with mitochondrial respiration defects. Although the role of Ras family proteins in autophagy mediation has gradually emerged through several reports, the importance of the coincidental occurrence of mitochondrial impairment and the autophagy process during the oncogenic transformation process has not been understood. In a previous study, mitochondrial biogenesis, which is another process that maintains whole mitochondrial homeostasis in cells, was not implicated in the mitochondrial impairment of transformed cells. However, the respiration defects of mitochondria in transformed cells were inversely associated with the increased mitophagy accompanying the induction of autophagy-related proteins, such as autophagy-related gene5 (Atg5), Beclin 1, microtubule-associated protein 1 light chain 3-II (LC3-II), and vacuolar adenosine tri-phosphatases (VoATPase). The respiratory protein expression and respiratory activity were recovered by blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes. Interestingly, cellular ATP level was not changed during the oncogenic transformation process, and the ATP generation occurred mainly through glycolysis without induction of glucose transporter 1 (GLUT1), which has a low Km value and is a well-known glucose transporter induced by oncogenic Ras signaling. Finally, LC3-II formation, which is indicative of autophagy initiation, was modulated by extracellular glucose levels during the oncogenic transformation; through the study with hepatocellular carcinoma tissues, the modulation of LC3-II formation was also observed only in the tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, these observations suggest that oncogenic K-RasV12-induced mitophagy leads to mitochondrial functional loss even in the absence of hypoxia during early tumorigenesis, and that this mitophagic process may be an important strategy for overcoming the cellular energy deficit triggered by insufficient glucose supply.
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