Involvement of mitophagy in oncogenic K-Ras-induced transformation: overcoming a cellular energy deficit from glucose deficiency - PubMed (original) (raw)

. 2011 Oct;7(10):1187-98.

doi: 10.4161/auto.7.10.16643. Epub 2011 Oct 1.

Hee Young Kim, Young-Kyoung Lee, Young-Sil Yoon, Wei Guang Xu, Joon-Kee Yoon, Sung-E Choi, Young-Gyu Ko, Min-Jung Kim, Su-Jae Lee, Hee-Jung Wang, Gyesoon Yoon

Affiliations

Involvement of mitophagy in oncogenic K-Ras-induced transformation: overcoming a cellular energy deficit from glucose deficiency

June-Hyung Kim et al. Autophagy. 2011 Oct.

Abstract

Although mitochondrial impairment has often been implicated in carcinogenesis, the mechanisms of its development in cancer remain unknown. We report here that autophagy triggered by oncogenic K-Ras mediates functional loss of mitochondria during cell transformation to overcome an energy deficit resulting from glucose deficiency. When Rat2 cells were infected with a retrovirus harboring constitutively active K-Ras (V12) , mitochondrial respiration significantly declined in parallel with the acquisition of transformation characteristics. Decreased respiration was not related to mitochondrial biogenesis but was inversely associated with the increased formation of acidic vesicles enclosing mitochondria, during which autophagy-related proteins such as Beclin 1, Atg5, LC3-II and vacuolar ATPases were induced. Interestingly, blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes recovered respiratory protein expression and respiratory activity; JNK was involved in these phenomena as an upstream regulator. The cells transformed by K-Ras (V12) maintained cellular ATP level mainly through glycolytic ATP production without induction of GLUT1, the low Km glucose transporter. Finally, K-Ras (V12) -triggered LC3-II formation was modulated by extracellular glucose levels, and LC3-II formation increased only in hepatocellular carcinoma tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, our observations suggest that mitochondrial functional loss may be mediated by oncogenic K-Ras-induced mitophagy during early tumorigenesis even in the absence of hypoxia, and that this mitophagic process may be an important strategy to overcome the cellular energy deficit triggered by insufficient glucose.

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Figures

Figure 1

Figure 1

Mitochondrial respiration was progressively impaired without changes in biogenesis, but with induction of autophagy-related proteins during oncogenic K-Ras-induced transformation. Rat2 cells were infected with retrovirus harboring K-RasV12 for the indicated periods. (A) soft-agar assay was performed as described in ‘Materials and Methods’. Representative images are shown (left part) and colonies greater than 50 µm in diameter were counted (right part). (B) cellular maximum respiration rates were determined as a KCN-sensitive DNP-uncoupled O2 consumption rate and expressed as percent of control. (C) expression levels of the respiratory and nonrespiratory mitochondrial proteins were analyzed by protein gel blot analysis. (D) mRNA levels with RT-PCR. (E) expression levels of autophagy-related proteins with protein gel blot analysis. *p < 0.05; **p < 0.01 vs. MFG control by student's t-test.

Figure 2

Figure 2

K-RasV12 activates autophagic vesicle formation, accompanied by mitochondrial loss. Rat2 cells were infected with retrovirus harboring K-RasV12 for the indicated periods. (A) Mitochondrial mass decrease and lysosomal mass increase were visualized after co-staining the cells with 50 nM LysoTracker Green (LysoG) and 200 nM MitoTracker Red (MitoR) without fixation using a Plan-Apochromat x100, 1.4 NA oil-immersion objective. Green indicates lysosomes and red indicates mitochondria. (B) Flow cytometric analysis was performed for quantification of mitochondrial mass (left part) or lysosomal mass (right part) of the co-stained cells with MitoR and LysoG. (C) To visualize live images, Rat2 cells were transiently transfected with mtRFP and infected with K-RasV12 retrovirus, and further cultured for 3 d on chamlide™ chamber as described in ‘Materials and Methods’. Targeting of mtRFP-labeled mitochondria into autophagic vesicles was visualized without fixation using a Plan-Apochromat x100, 1.4 NA oil-immersion objective. Representative images are presented. (D) Representative electron microscopic images are presented. The open arrows indicate representative mitochondria within autophagosome, thin arrows indicate autophagosome and arrowheads indicate endoplasmic reticulum entrapping mitochondria. (E) Numbers of total vesicles, clear empty vesicles (square), vesicles with debris (circle) and vesicles with organellar remnants (dotted square) were counted from 26 whole cell electron microscopic images (3,000x or 4,400x) of K-Ras infected Rat2 cells (right part). Representative images are shown in the left part. (F) Western blot analysis. After infected by K-RasV12 retrovirus for 24 h, cells were replenished with RPMi media containing 10 µM chloroquine (C6628, Sigma-Aldrich) for 2 d. *p < 0.05; **p < 0.01 vs. MFG control.

Figure 2

Figure 2

K-RasV12 activates autophagic vesicle formation, accompanied by mitochondrial loss. Rat2 cells were infected with retrovirus harboring K-RasV12 for the indicated periods. (A) Mitochondrial mass decrease and lysosomal mass increase were visualized after co-staining the cells with 50 nM LysoTracker Green (LysoG) and 200 nM MitoTracker Red (MitoR) without fixation using a Plan-Apochromat x100, 1.4 NA oil-immersion objective. Green indicates lysosomes and red indicates mitochondria. (B) Flow cytometric analysis was performed for quantification of mitochondrial mass (left part) or lysosomal mass (right part) of the co-stained cells with MitoR and LysoG. (C) To visualize live images, Rat2 cells were transiently transfected with mtRFP and infected with K-RasV12 retrovirus, and further cultured for 3 d on chamlide™ chamber as described in ‘Materials and Methods’. Targeting of mtRFP-labeled mitochondria into autophagic vesicles was visualized without fixation using a Plan-Apochromat x100, 1.4 NA oil-immersion objective. Representative images are presented. (D) Representative electron microscopic images are presented. The open arrows indicate representative mitochondria within autophagosome, thin arrows indicate autophagosome and arrowheads indicate endoplasmic reticulum entrapping mitochondria. (E) Numbers of total vesicles, clear empty vesicles (square), vesicles with debris (circle) and vesicles with organellar remnants (dotted square) were counted from 26 whole cell electron microscopic images (3,000x or 4,400x) of K-Ras infected Rat2 cells (right part). Representative images are shown in the left part. (F) Western blot analysis. After infected by K-RasV12 retrovirus for 24 h, cells were replenished with RPMi media containing 10 µM chloroquine (C6628, Sigma-Aldrich) for 2 d. *p < 0.05; **p < 0.01 vs. MFG control.

Figure 3

Figure 3

Recovery of mitochondriyal mass and function by blocking autophagy. (A–C) Rat2 cells were exposed to bafilomycin A (0.5 and 1 nM) or 3MA (1 to 3 mM) 1 h prior to K-RasV12 infection and further incubated for 3 d. (A) Mitochondrial (left part) and lysosomal (right part) mass were quantitated by flow cytometric analysis after co-staining cells with MitoR and LysoG. (B) western blot analyses for the recovery of respiratory proteins by pretreatment of bafilomycin A (left part) and 3MA (right part). (C) Maximum cellular oxygen consumption rates recovered by pretreatment of bafilomycin A (1 nM) and 3MA (3 mM). (D) Cellular oxygen consumption rates was estimated after Rat2 cells were transfected with si-ATG5, si-VATPaseE and si-Beclin 1 15 h prior to K-RasV12 infection and further incubated for 3 d. **p < 0.01 vs. MFG control and ##p < 0.01 vs. K-Ras-infected cells by one-way ANOVA.

Figure 4

Figure 4

K-RasV12-induced autophagy is mediated through JNK. Rat2 cells were exposed to pharmacological inhibitors (15 µM PD98059, 15 µM SP600125 or 15 µM PD169316) or transfection of si-RNAs (si-NC, si-beclin-1 or si-JNK) prior to K-RasV12 infection and further incubated for 3 d as indicated. (A) Mitochondrial and lysosomal mass were estimated by flow cytometric analysis after co-staining cells with MitoR (left part) and LysoG (right part). (B) Cellular oxygen consumption rates. (C) Western blot analysis. (D) Soft-agar assay was performed as described in ‘Materials and Methods’. **p < 0.01 vs. MFG control and ##p < 0.01 vs. K-Ras-infected cells by one-way ANOVA.

Figure 5

Figure 5

K-RasV12-induced autophagy is dependent on extracellular glucose levels. (A, B and F) Rat2 cells were infected with retrovirus harboring K-RasV12 for 3 d unless indicated. (A) Cell growth rates were measured by counting trypan blue-negative cells (left part) and phase contrast cell images are shown (right part). (B) Total cellular ATP levels were estimated by using luciferin/luciferase ATP assay kit (left part) and LDH activity to oxidize NADH was monitored at 340 nm using spectrophotometry (right part). (C and D) After infected by K-RasV12 retrovirus for 3 d, Rat2 cells were treated with 2 µM oligomycin (Oli) or 25 mM 2-deoxyglucose (DOG) for 3 h. (C) Extracellular acidification rate and cellular oxygen consumption rate were simultaneously measured by Seahorse XF analyzer as described in ‘Materials and Methods’. Dotted arrows indicate lactate increase (a) and respiratory defect (b) by K-Ras. (D) Intracellular ATP levels. DOG-sensitive (glycolysis dependent) and Oli-sensitive (mitochondrial-respiration dependent) ATP production were indicated as percentage of total cellular ATP levels. (E) Intracellular ATP levels. Rat2 cells were transfected with si-ATG5 or si-Beclin 1 (Bec-1) 15 h prior to K-RasV12 infection and further incubated for 3 d, then replenished with 2 µM oligomycin containing medium for 3 h. (F) Expression levels of GLUT proteins were monitored by protein gel blot analysis (left part) and cellular glucose uptake activity was measured as described in ‘Materials and Methods’ (right part). (G) LC3-II formation of Rat2 clone stably expressing K-RasV12 in response to different concentrations of glucose in media for 3 d was monitored by protein gel blot analysis (left part) and phase contrast cell images are shown (right part).

Figure 6

Figure 6

LC3-II formation increased only in the human hepatocellular carcinoma tissues with low glucose uptake. (A) Representative PET/CT images of human hepatocellular carcinoma patients. (B) LC3-II formation and K-Ras expression were monitored by protein gel blot analysis. The expression ratios for tumor/surrounding tissue (T/S) of the normalized values (LC3-II, total LC3, K-Ras) against actin are shown in the lower part.

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