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.

Cave Microbes as a Potential Source of Drugs Development in the Modern Era.

The world is constantly facing threats, including the emergence of new pathogens and antibiotic resistance among extant pathogens, which is a matter of concern. Therefore, the need for natural and effective sources of drugs is inevitable. The ancient and pristine ecosystems of caves contain a unique microbial world and could provide a possible source of antimicrobial metabolites. The association between humans and caves is as old as human history itself. Historically, cave environments have been used to treat patients with respiratory tract infections, which is referred to as speleotherapy. Today, the pristine environment of caves that comprise a poorly explored microbial world is a potential source of antimicrobial and anticancer drugs. Oligotrophic conditions in caves enhance the competition among microbial communities, and unique antimicrobial agents may be used in this competition. This review suggests that the world needs a novel and effective source of drug discovery. Therefore, being the emerging spot of modern human civilization, caves could play a crucial role in the current medical crisis, and cave microorganisms may have the potential to produce novel antimicrobial and anticancer drugs.

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.

Dissolution of Cu and Zn-bearing ore by indigenous iron-oxidizing bacterial consortia supplemented with dried bamboo sawdust and variations in bacterial structural dynamics: A new concept in bioleaching.

Disposing of low-grade ores involves numerous environmental issues. Bioleaching with acidophilic bacteria is the preferred solution to process these ores for metals recovery. In this study, indigenous iron-oxidizing bacteria Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, and Leptospirillum ferrooxidans were used in consortia supplemented with acid-treated bamboo sawdust (BSD) for copper and zinc recovery. Findings showed the extreme catalytic response of BSD with the best recovery of metals. Maximum of 92.2 ± 4.0% copper (0.35%) and 90.0 ± 5.4% zinc (0.33%) were recovered after 8 days of processing in the presence of 2 g/L BSD. Significant variations were reported in physicochemical parameters during bioleaching in the presence of a different concentration of BSD. Fourier Transform Infrared spectroscopy results of bioleached residues showed significant variations in spectral pattern and maximum variations were reported in 2.0 g/L BSD, which indicates maximum metals dissolutions. The impact of bacterial consortia and BSD on iron speciation of bioleached ores was analyzed by using Mössbauer spectroscopy and clear variations in iron speciation were reported. Furthermore, the bacterial community structure dynamics revealed significant variations in the individual bacterial proportion in each experiment. This finding shows that the dosage concentration of BSD influenced the microenvironment, which effect the bacterial abundance and these variations in the bacterial structural communities were not associated with the initial proportion of bacterial cells inoculated in the bioleaching process. Moreover, the mechanism of chemical reactions was proposed by explaining the possible role of BSD as a reductant under micro-aerophilic conditions that facilitates the bacterial reduction of ferric iron. This type of bioleaching process with indigenous iron-oxidizing bacteria and BSD has significant potential to further upscale the bioleaching process for recalcitrant ore bodies in an environment friendly and cost-effective way.

Protein kinase A activation by ß­Lapachone is associated with apoptotic cell death in NQO1­overexpressing breast cancer cells.

One million females are diagnosed worldwide every year with breast cancer, and the mortality rate of these patients remains high. Several treatments, including surgery, are available for breast cancer. ß­Lapachone (ß­Lap), a natural quinone compound, has been developed for cancer treatment due to its strong cytotoxic effect through its action on NAD(P)H:quinone oxidoreductase 1 (NQO1)­dependent activity. However, the mechanism in regards to how ß­Lap induces cytotoxicity in breast cancer cells is still elusive. In the present study, we showed that ß­Lap induced apoptotic cell death via activation of protein kinase A (PKA) in NQO1­overexpressing MDA­MB­231 human breast cancer cells. This PKA­dependent cell death was observed solely in NQO1­overexpressing 231 cells via the high production of reactive oxygen species (ROS). Cell survival of antioxidant [N­acetylcysteine (NAC)]­treated NQO1­overexpressing 231 cells was significantly recovered, and NQO1­negative 231 cells did not respond to ß­Lap. Antiapoptotic proteins such as Bcl2 and Bcl­xL were decreased, while proapoptotic proteins, including cytochrome c, activation of caspase­3, and cleavage of PARP were increased after ß­Lap treatment of NQO1­overexpressing 231 cells. Furthermore, PKA activators, forskolin or dibutyryl­cAMP, an analog of cAMP, aggravated the ß­Lap­induced apoptotic cell death by decreasing antiapoptotic proteins and further activating proapoptotic proteins in NQO1­positive 231 cells. Treatment with a PKA inhibiter, H89, significantly increased cell viability even in NQO1­overexpressing cells treated with ß­Lap. These data showed that ß­Lap activated PKA via ROS accumulation, subsequently leading to apoptotic cell death in NQO1­positive breast cancer cells.

Control of the Epithelial-to-Mesenchymal Transition and Cancer Metastasis by Autophagy-Dependent SNAI1 Degradation.

Autophagy, an intracellular degradation process, is essential for maintaining cell homeostasis by removing damaged organelles and proteins under various conditions of stress. In cancer, autophagy has conflicting functions. It plays a key role in protecting against cancerous transformation by maintaining genomic stability against genotoxic components, leading to cancerous transformation. It can also promote cancer cell survival by supplying minimal amounts of nutrients during cancer progression. However, the molecular mechanisms underlying how autophagy regulates the epithelial-to-mesenchymal transition (EMT) and cancer metastasis are unknown. Here, we show that starvation-induced autophagy promotes Snail (SNAI1) degradation and inhibits EMT and metastasis in cancer cells. Interestingly, SNAI1 proteins were physically associated and colocalized with LC3 and SQSTM1 in cancer cells. We also found a significant decrease in the levels of EMT and metastatic proteins under starvation conditions. Furthermore, ATG7 knockdown inhibited autophagy-induced SNAI1 degradation in the cytoplasm, which was associated with a decrease in SNAI1 nuclear translocation. Moreover, cancer cell invasion and migration were significantly inhibited by starvation-induced autophagy. These findings suggest that autophagy-dependent SNAI1 degradation could specifically regulate EMT and cancer metastasis during tumorigenesis.

Functional Linkage of RKIP to the Epithelial to Mesenchymal Transition and Autophagy during the Development of Prostate Cancer.

Raf kinase inhibitor protein (RKIP) plays a critical role in many signaling pathways as a multi-functional adapter protein. In particular, the loss of RKIP's function in certain types of cancer cells results in epithelial to mesenchymal transition (EMT) and the promotion of cancer metastasis. In addition, RKIP inhibits autophagy by modulating LC3-lipidation and mTORC1. How the RKIP-dependent inhibition of autophagy is linked to EMT and cancer progression is still under investigation. In this study, we investigated the ways by which RKIP interacts with key gene products in EMT and autophagy during the progression of prostate cancer. We first identified the gene products of interest using the corresponding gene ontology terms. The weighted-gene co-expression network analysis (WGCNA) was applied on a gene expression dataset from three groups of prostate tissues; benign prostate hyperplasia, primary and metastatic cancer. We found two modules of highly co-expressed genes, which were preserved in other independent datasets of prostate cancer tissues. RKIP showed potentially novel interactions with one EMT and seven autophagy gene products (TGFBR1; PIK3C3, PIK3CB, TBC1D25, TBC1D5, TOLLIP, WDR45 and WIPI1). In addition, we identified several upstream transcription modulators that could regulate the expression of these gene products. Finally, we verified some RKIP novel interactions by co-localization using the confocal microscopy analysis in a prostate cancer cell line. To summarize, RKIP interacts with EMT and autophagy as part of the same functional unit in developing prostate cancer.

Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity.

Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment.