ER stress-enhanced HMGA2 plays an important role in Cr (VI)-induced glycolysis and inhibited oxidative phosphorylation by targeting the transcription of ATF4.

Hexavalent chromium [Cr (VI)] is a proven human carcinogen which is widely used in steel manufacturing and painting. Here, the involvement of high mobility group A2 (HMGA2) in Cr (VI)-mediated glycolysis and oxidative phosphorylation (OXPHOS) was investigated. First, Cr (VI) treatment induced aerobic glycolysis by increasing the expression of GLUT1, HK II, PKM2 and LDHA enzymes, and reduced OXPHOS by decreasing mitochondrial mass, the expression of COX IV and ND1, and increasing Ca2+ content in mitochondria in A549 and HELF cells. And overexpression of HMGA2 induced aerobic glycolysis and decreased OXPHOS. Secondly, using endoplasmic reticulum (ER) stress inhibitor, 4-phenylbutyric acid (4-PBA) and knockdown of activating transcription factor 4 (ATF4) gene by siRNA, we demonstrated that ER stress and ATF4 elevation mediated Cr (VI)-induced glycolysis and inhibited OXPHOS. Furthermore, using tunicamycin (Tm), siHMGA2, transfection of HMGA2 and siATF4, we demonstrated that ER stress-enhanced interaction of HMGA2 and ATF4 resulted in Cr (VI)-induced glycolysis and inhibited OXPHOS. Additionally, ChIP assay revealed that HMGA2 protein could directly bind to the promoter sequence of ATF4 gene, which modulated Cr (VI)-induced ATF4 elevation. Finally, in lung tissues of BALB/c mice injected with HMGA2 plasmids, it is verified that HMGA2 involved in regulation of ATF4, glycolysis and OXPHOS in vivo. Combining, our data discovered that ER stress-enhanced the interaction of HMGA2 and ATF4 played an important role in Cr (VI)-mediated glycolysis and OXPHOS. These results imply a root cause for the carcinogenicity of Cr (VI), and could guide development of novel therapeutics for cancers.

Curcumin attenuates Cr (VI)-induced cell growth and migration by targeting autophagy-dependent reprogrammed metabolism.

Hexavalent chromium [Cr (VI)] is a well-established carcinogen. Cr (VI)-treated cells are phenotypically characterized by aberrant levels of growth and migration. Curcumin, a polyphenolic compound from the plant turmeric, has been found to possess antiproliferation, anti-inflammation, and antioxidant properties. In this study, the effect of curcumin on Cr (VI)-induced cell survival and migration and the underlying mechanism were investigated. Cell viability assay on A549 and human embryonic lung fibroblast cells showed that curcumin at the concentration of 10 µM could significantly attenuate Cr (VI)-induced viability in both cell lines. Following Western blot assay and metabolomics assays, cotreatment with curcumin and Cr (VI) resulted in the suppression of Cr (VI)-induced glycolysis-, autophagy-, and migration-related proteins. Meanwhile, curcumin increased Cr (VI)-reduced oxidative phosphorylation (OXPHOS)-related proteins, COXIV and ND1. Moreover, curcumin suppressed Cr (VI)-induced mitochondrial dysfunction, mitochondrial mass decrease, and mitochondrial membrane potential loss. Treatment with curcumin for 24 h significantly attenuated pcATG4B-induced autophagy and the subsequent expression of glucose transporter 1, hexokinase II, and pyruvate kinase M2. Wound healing and transwell assay demonstrated that curcumin reduced Cr (VI)-induced cell migration. Taken together, these results showed that curcumin was able to attenuate Cr (VI)-induced cell viability and migration by targeting autophagy-dependent reprogrammed metabolism from OXPHOS to glycolysis.

A requirement for autophagy in HMGA2-induced metabolic reprogramming to support Cd-induced migration.

High mobility group A2 (HMGA2) is closely related to the occurrence, development and prognosis of tumors. But the mechanism is unclear. Metabolic reprogramming is a dominant way to meet anabolic and energy requirements of tumor cells for their survival, growth and proliferation. Here, we investigated the role of metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis mediated by HMGA2/autophagy axis in cadmium (Cd, CdCl2)-induced migration. First, we found that Cd induced glycolysis and reduced OXPHOS in vivo (0.5 and 1 mg/kg, i.p. or 0.8 and 1.6 µM, i.t.) and in vitro (2 µM in A549 cells and 0.05 µM in HELF cells). Then, genetic knockdown of HMGA2 restored Cd-reduced mitochondrial mass and OXPHOS and inhibited Cd-increased glycolysis, indicating that HMGA2 was involved in Cd-induced metabolic reprogramming. 2-Deoxy-d-glucose (2DG, 5 mM), the inhibitor of glycolysis decreased Cd/HMGA2-induced cell migration and restored Cd/HMGA2-decreased OXPHOS and mitochondrial mass. Inhibition of autophagy by 3-Methyladenine (3MA, 3 mM) elucidated an essential role of autophagy in HMGA2-induced glycolysis, migration, and HMGA2-reduced OXPHOS. Overall, our study demonstrated that autophagy was required for HMGA2-mediated metabolic reprogramming, which was critical for Cd-induced migration. Targeting HMGA2 and autophagy-dependent reprogrammed metabolism may be an effective way to inhibit Cd-induced cell migration.

The crosstalk between mitochondrial dysfunction and endoplasmic reticulum stress promoted ATF4-mediated mitophagy induced by hexavalent chromium.

Chromium (Cr) compounds are markedly toxic and carcinogenic. Previously, we found that Cr (VI) induced autophagy in A549 cells. Here, the effect of mitochondrial dysfunction and endoplasmic reticulum (ER) stress on inducing mitophagy was investigated in both A549 and H1299 cells. Exposure to Cr (VI) for 6 h significantly enhanced reactive oxygen species (ROS) production and reduced mitochondrial membrane potential (MMP). Transmission electron microscopy showed that Cr (VI) induced mitochondrial morphological changes, such as, mitochondrial swelling and vacuolization. The elevated expression of GRP78 and p-PERK suggested that Cr (VI) resulted in ER stress. Both mitochondrial dysfunction and ER stress played an important role in Cr (VI)-induced mitophagy, as the mitochondrial function inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) induced PINK1 and PARK2 and increased the expression of GRP78 and p-PERK while the levels of Cr (VI)-induced PINK1, PARK2, LC3-II were reduced after ER stress inhibitor, phenylbutyric acid (4PBA) pretreatment. When A549 cells were treated with CCCP and 4-PBA simultaneously, CCCP-induced expressions of PINK1, PARK2 and LC3-II decreased significantly compared with that of only CCCP-treated cells, indicating that there was a crosstalk between mitochondria and ER in inducing mitophagy. Additionally, the crosstalk between mitochondrial dysfunction and ER stress modulated the expression of Cr (VI)-induced ATF4, which resulted in mitophagy. Collectively, our data demonstrated that Cr (VI)-induced mitophagy mediated by ATF4 via the crosstalk between ER stress and mitochondrial dysfunction.

Cr (VI) induced mitophagy via the interaction of HMGA2 and PARK2.

Chromium (Cr) (VI) is a proven toxin, mutagen and carcinogen. Here, the role of high mobility group A2 (HMGA2) mediating Cr (VI)-induced mitophagy was investigated. Cr (VI)-treatment caused the formation of double membrane autophagic vesicles (AVs) engulfing mitochondria and increased the expression of PINK1, PARK2, LC3 as well as HMGA2 particularly in mitochondria in A549 cells. Silencing of HMGA2 by siRNA decreased expression of PINK1, PARK2 and LC3 II especially in mitochondria, while over-expression of HMGA2 increased the expression of them in A549 cells. It indicated that HMGA2 played a critical role in Cr (VI)-induced mitophagy. Most importantly, the results of co-immunoprecipitation showed for the first time that HMGA2 could bind to PARK2 in mitochondria to activate the mitophagy pathway. In BALB/c mice, Cr (VI) increased the expression of PINK1 and PARK2 in lung tissues. Furthermore, over-expression of HMGA2 in BALB/c mice by transfection of plasmid HMGA2 significantly increased the levels of PINK1, PARK2 and LC3 II in lung tissues. Collectively, our data demonstrated that HMGA2 plays an important role in Cr (VI)-induced mitophagy through direct interaction with PARK2 in A549 cells and lung tissue.

ATF4-mediated autophagy-dependent glycolysis plays an important role in attenuating apoptosis induced by Cr (VI) in A549 cells.

Chromium (Cr) (VI) compounds are known to be serious toxic and carcinogenic, but the mechanism is not clear. In our previous study, we found that Cr (VI)-induced ER stress plays an important role in the crosstalk between apoptosis and autophagy, while autophagy was apoptosis-dependent and subsequently prevents apoptosis cell death to keep A549 cells resistant to Cr (VI)-induced toxicity. In this study, we found that Cr (VI) could induce aerobic glycolysis in A549 cells. Both ER stress inhibitor, phenylbutyric acid (4-PBA) and the inhibitor of autophagy, 3-MA, repressed Cr (VI)-induced glycolysis, indicating that both ER stress and autophagy were involved in Cr (VI)-induced glycolysis in A549 cells. Co-treatment of the inhibitor of aerobic glycolysis, 2-DG and Cr (VI) for 24 h increased Cr (VI)-induced cleaved caspase-3, caspase-9 and the number of apoptotic cells, demonstrating that aerobic glycolysis played an important role in attenuating Cr (VI)-induced apoptosis. Furthermore, knockdown of ATF4 by siATF4 significantly decreased Cr (VI)-induced aerobic glycolysis and apoptosis, suggesting that ATF4 was involved in Cr (VI)-induced aerobic glycolysis and its effect of attenuating apoptosis in A549 cells. Taken together, our results demonstrated that autophagy-dependent glycolysis played a role in attenuating Cr (VI)-induced apoptosis. ER stress was involved in facilitating glycolysis, whose induction was mediated by ATF4. These findings open a window for the development of therapeutic interventions to prevent Cr (VI)-induced toxicity.

Cadmium induces cell growth in A549 and HELF cells via autophagy-dependent glycolysis.

Cadmium (Cd) is a pervasive harmful metal in the environment. It is a well-known inducer of tumorigenesis, but its mechanism is still unclear. We have previously reported that Cd-induced autophagy was apoptosis-dependent and prevents apoptotic cell death to ensure the growth of A549 cells. In this study, the mechanism was further investigated. Cd treatment increased glucose uptake and lactate release significantly. Meanwhile, the protein level of GLUT1，HKII，PKM2 and LDHA increased in a time-dependent manner, indicating that Cd induced aerobic glycolysis in A549 and HELF cells. The inhibitors of autophagy, 3MA, and CQ, repressed Cd-induced glycolysis-related proteins, indicating that autophagy was involved in Cd-induced glycolysis in A549 and HELF cells. Knockdown of ATG4B or ATG5 by siATG4B and siATG5 decreased Cd-induced glycolysis, while overexpression of ATG4B enhanced glycolysis. These results demonstrated that Cd-induced glycolysis was autophagy-dependent. Then, glycolysis inhibitor, 2DG and siPKM2 could inhibit Cd-induced cell viability and cell cycle progression compared to only Cd treatment, indicating that glycolysis played an important role in Cd-induced cell growth. Finally, co-treatment of transfection of ATG4B-DNA plasmids with 2DG or siPKM2 further demonstrated that the autophagy-glycolysis axis played an important role in Cd-induced cell cycle progression. Taken together, our results suggested that Cd-induced glycolysis is autophagy-dependent and the autophagy-glycolysis axis underlies the mechanism of Cd-induced cell growth in A549 and HELF cells.