In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field
Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.
High-grade serous ovarian cancer (HGSOC) is hallmarked by early onset of peritoneal dissemination, which distinguishes it from low-grade serous ovarian cancer (LGSOC). Here, we describe the aggressive nature of HGSOC ascitic tumor cells (ATCs) characterized by integrin α5high (ITGA5high) ATCs, which are prone to forming heterotypic spheroids with fibroblasts. We term these aggregates as metastatic units (MUs) in HGSOC for their advantageous metastatic capacity and active involvement in early peritoneal dissemination. Intriguingly, fibroblasts inside MUs support ATC survival and guide their peritoneal invasion before becoming essential components of the tumor stroma in newly formed metastases. Cancer-associated fibroblasts (CAFs) recruit ITGA5high ATCs to form MUs, which further sustain ATC ITGA5 expression by EGF secretion. Notably, LGSOC is largely devoid of CAFs and the resultant MUs, which might explain its metastatic delay. These findings identify a specialized MU architecture that amplifies the tumor–stroma interaction and promotes transcoelomic metastasis in HGSOC, providing the basis for stromal fibroblast-oriented interventions in hampering OC peritoneal propagation.
The mevalonate pathway provides metabolites for post-translational modifications such as farnesylation, which are critical for the activity of RAS downstream signaling. Subsequently occurring regulatory processes can induce an aberrant stimulation of DNA methyltransferase (DNMT1) as well as changes in histone deacetylases (HDACs) and microRNAs in many cancer cell lines. Inhibitors of the mevalonate pathway are increasingly recognized as anticancer drugs. Extensive evidence indicates an intense cross-talk between signaling pathways, which affect growth, differentiation, and apoptosis either directly or indirectly via epigenetic mechanisms. Herein, we show data obtained by novel transcriptomic and corresponding methylomic or proteomic analyses from cell lines treated with pharmacologic doses of respective inhibitors (i.e., simvastatin, ibandronate). Metabolic pathways and their epigenetic consequences appear to be affected by a changed concentration of NADPH. Moreover, since the mevalonate metabolism is part of a signaling network, including vitamin D metabolism or fatty acid synthesis, the epigenetic activity of associated pathways is also presented. This emphasizes the far-reaching epigenetic impact of metabolic therapies on cancer cells and provides some explanation for clinical observations, which indicate the anticancer activity of statins and bisphosphonates.
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