Regulation of TGF-ß1-Induced EMT by Autophagy-Dependent Energy Metabolism in Cancer Cells.

Metastasis is associated with poor prognosis and is the major cause of death in cancer patients. The epithelial to mesenchymal transition (EMT) is essential for cancer cells to acquire a highly migratory phenotype. Metabolic reprogramming is required to meet the energy demands during this process. Recent studies have indicated that autophagy is involved in EMT, during which cancer cells depend on autophagy activation for survival. However, accumulating evidence indicates that autophagy's involvement in cancer is context-dependent, acting as either promoter or inhibitor. In this study, we investigated the role of autophagy in supplying energy to support EMT. We induced EMT in Non-small cell lung cancer A549 cells using TGF-ß1 with and without autophagy inhibition. Suppression of autophagy activity by knocking down of BECN1 or chloroquine (CQ) treatment inhibited mesenchymal protein expression. Interestingly, TGF-ß1 promoted the transcription of target mRNAs, SNAI1, VIM, and CDH2, regardless of autophagy status. The imbalance between protein and mRNA levels indicated the possibility of autophagy-dependent translational regulation. Since protein synthesis consumes large amounts of energy, it is tightly regulated via various cellular signaling pathways such as AMPK and mTOR. Our investigation showed inhibition of autophagy decreased ATP production from OXPHOS and led to the suppression of mRNA translation by phosphorylation of eukaryotic elongation factor 2 (eEF2). These results suggest that A549 non-small cell lung cancer required autophagy to maintain mitochondrial homeostasis during TGF-ß1 induced EMT. In conclusion, blocking autophagy decreased energy production and down-regulated proteins synthesis inhibiting TGF-ß1 induced EMT.

Autophagy-mediated degradation of NOTCH1 intracellular domain controls the epithelial to mesenchymal transition and cancer metastasis.

BACKGOUND: Autophagy controls levels of cellular components during normal and stress conditions; thus, it is a pivotal process for the maintenance of cell homeostasis. In cancer, autophagy protects cells from cancerous transformations that can result from genomic instability induced by reactive oxygen species or other damaged components, but it can also promote cancer survival by providing essential nutrients during the metabolic stress condition of cancer progression. However, the molecular mechanism underlying autophagy-dependent regulation of the epithelial to mesenchymal transition (EMT) and metastasis is still elusive. METHODS: The intracellular level of NOTCH1 intracellular domain (NICD) in several cancer cells was studied under starvation, treatment with chloroquine or ATG7-knockdown. The autophagy activity in these cells was assessed by immunocytochemistry and molecular analyses. Cancer cell migration and invasion under modulation of autophagy were determined by in vitro scratch and Matrigel assays. RESULTS: In the study, autophagy activation stimulated degradation of NICD, a key transcriptional regulator of the EMT and cancer metastasis. We also found that NICD binds directly to LC3 and that the NICD/LC3 complex associates with SNAI1 and sequestosome 1 (SQSTM1)/p62 proteins. Furthermore, the ATG7 knockdown significantly inhibited degradation of NICD under starvation independent of SQSTM1-associated proteasomal degradation. In addition, NICD degradation by autophagy associated with the cellular level of SNAI1. Indeed, autophagy inhibited nuclear translocation of NICD protein and consequently decreased the transcriptional activity of its target genes. Autophagy activation substantially suppressed in vitro cancer cell migration and invasion. We also observed that NICD and SNAI1 levels in tissues from human cervical and lung cancer patients correlated inversely with expression of autophagy-related proteins. CONCLUSIONS: These findings suggest that the cellular level of NICD is regulated by autophagy during cancer progression and that targeting autophagy-dependent NICD/SNAI1 degradation could be a strategy for the development of cancer therapeutics.

Chlorogenic acid protects human chondrocyte C28/I2 cells from oxidative stress-induced cell death through activation of autophagy.

AIMS: The development of osteoarthritis (OA), the most common form of arthritis, is commonly associated with oxidative stress. Indeed, the lack of antioxidant responses largely increases OA incidence. OA is a leading cause of disability in the elderly, which reduces the quality of life and places high socioeconomic burdens on them. Several polyphenolic compounds, including chlorogenic acid (CGA), have shown cytoprotective effects via their antioxidant activity, but the exact mechanism (s) remain elusive. In this study, we demonstrated how CGA protects human chondrocytes against H2O2-induced apoptosis. MATERIALS AND METHODS: The cytoprotective effect by CGA in 500 µM hydrogen peroxide-treated C28/I2 cells was evaluated by cell viability, TUNEL assay, and Western blotting analyses, and autophagy assessment was further performed by AO and MDC staining and tandem mRFP-GFP fluorescence analyses. KEY FINDINGS: Treatment of CGA to the human chondrocytes under oxidative stress significantly decreased apoptosis markers, such as cleaved caspase 3 and cleaved PARP, and increased anti-apoptotic marker Bcl-xL and the antioxidant response proteins NRF2 and NF-κB. Furthermore, CGA-dependent activation of antioxidant response proteins NRF2 and NF-κB and its protective effects in chondrocytes depended on autophagy. Indeed, CGA treatment and autophagy induction significantly decreased reactive oxygen species (ROS)-induced apoptosis. SIGNIFICANCE: CGA exhibited the protective effect to human chondrocyte C28/I2 cells against oxidative stress-induced cell death by activating autophagy. These findings indicate that CGA is a potential therapeutic agent for the development of OA drugs.

Cross talk between autophagy and oncogenic signaling pathways and implications for cancer therapy.

Autophagy is a highly conserved metabolic process involved in the degradation of intracellular components including proteins and organelles. Consequently, it plays a critical role in recycling metabolic energy for the maintenance of cellular homeostasis in response to various stressors. In cancer, autophagy either suppresses or promotes cancer progression depending on the stage and cancer type. Epithelial-mesenchymal transition (EMT) and cancer metastasis are directly mediated by oncogenic signal proteins including SNAI1, SLUG, ZEB1/2, and NOTCH1, which are functionally correlated with autophagy. In this report, we discuss the crosstalk between oncogenic signaling pathways and autophagy followed by possible strategies for cancer treatment via regulation of autophagy. Although autophagy affects EMT and cancer metastasis, the overall signaling pathways connecting cancer progression and autophagy are still illusive. In general, autophagy plays a critical role in cancer cell survival by providing a minimum level of energy via self-digestion. Thus, cancer cells face nutrient limitations and challenges under stress during EMT and metastasis. Conversely, autophagy acts as a potential cancer suppressor by degrading oncogenic proteins, which are essential for cancer progression, and by removing damaged components such as mitochondria to enhance genomic stability. Therefore, autophagy activators or inhibitors represent possible cancer therapeutics. We further discuss the regulation of autophagy-dependent degradation of oncogenic proteins and its functional correlation with oncogenic signaling pathways, with potential applications in cancer therapy.

Transcriptional Repression of Raf Kinase Inhibitory Protein Gene by Metadherin during Cancer Progression.

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-κκB. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.