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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Interest in the topic of tumour metabolism has waxed and waned over the past century of cancer research. The early observations of Warburg and his contemporaries established that there are fundamental differences in the central metabolic pathways operating in malignant tissue. However, the initial hypotheses that were based on these observations proved inadequate to explain tumorigenesis, and the oncogene revolution pushed tumour metabolism to the margins of cancer research. In recent years, interest has been renewed as it has become clear that many of the signalling pathways that are affected by genetic mutations and the tumour microenvironment have a profound effect on core metabolism, making this topic once again one of the most intense areas of research in cancer biology.

/pdf/regulation-of-cancer-cell-metabolism-1dhy51t2fl.pdf

Regulation of cancer cell metabolism

Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Global changes in the epigenetic landscape are a hallmark of cancer. The initiation and progression of cancer, traditionally seen as a genetic disease, is now realized to involve epigenetic abnormalities along with genetic alterations. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer including DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs, specifically microRNA expression. The reversible nature of epigenetic aberrations has led to the emergence of the promising field of epigenetic therapy, which is already making progress with the recent FDA approval of three epigenetic drugs for cancer treatment. In this review, we discuss the current understanding of alterations in the epigenetic landscape that occur in cancer compared with normal cells, the roles of these changes in cancer initiation and progression, including the cancer stem cell model, and the potential use of this knowledge in designing more effective treatment strategies.

/pdf/epigenetics-in-cancer-53kplki7aw.pdf

Epigenetics in Cancer

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

The PI3K signaling pathway regulates many normal cellular processes, and perturbation of the PI3K signaling results in deregulated kinase activity and malignant transformation [1]. Phosphatidylinos...

Somatic mutation of PIK3R1 gene is rare in common human cancers

Kinase domain mutation of ERBB family genes is uncommon in acute leukemias.

To the Editor: Recent advance in genome sequencing technologies such as whole-exome sequencing has provided huge data sets of somatic mutations in major cancer types [1]. The Pan-Cancer project integrated overall genome sequencing data, subsequently identified mutations across the cancers, found their organ specificity and identified molecular commonalities across tumor types [1, 2]. During the Pan-Cancer efforts, many of the mutations that occurred at a low frequency in a cancer type were shared by sets of cancer types and were predicted as significantly mutated genes [1–3]. One of such genes is ASPM that is the human ortholog of the Drosophila melanogaster abnormal spindle gene, which is essential for normal mitotic spindle function in the cells [4]. Germline mutations in ASPM are associated with microcephaly primary type 5 [4]. ASPM was initially identified as a protein that regulated neurogenesis but it was later known to be widely expressed in a variety of normal and malignant tissues [5]. ASPM is overexpressed in many cancers and considered a poor prognostic marker [6]. ASPM mutations have been identified in several cancers with a low prevalence, but it became a significantly mutated gene by the Pan-Cancer analysis [3]. Colorectal cancer (CRC) and gastric cancer (GC) harbored ASPM mutation in 4.35 % and 1.31 %, respectively [3], all of which were missense or splicing-site mutations (search at www.intogen.org). Through a search in the UCSC public genome database (http://genome.cse.ucsc.edu/), we found that the ASPM gene harbors a mononucleotide repeat in the coding sequence that might serve as a target for mutation in GC and CRCs exhibiting microsatellite instability (MSI) [6]. To see whether ASPM gene harbored frameshift mutations of the repeat in GC and CRC, we analyzed the exon 18 A7 repeats in 34 GC with high MSI (MSI-H), 45 GC with stable MSI/low MSI (MSS/MSI-L), 76 CRC with MSI-H and 45 CRC with MSS/MSI-L by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously [7]. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [7, 8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [7, 8]. In addition, to see whether frameshift mutations in the ASPM gene repeat possess intra-tumor heterogeneity (ITH) that contributes to tumor aggressiveness [9], we analyzed 16 CRCs with four to seven regional biopsies per CRC. On the SSCP, we observed aberrant SSCP bands of ASPM gene in three CRCs and two GCs. DNA from the patients’ normal tissues showed no shifts in SSCP, indicating the aberrant bands had risen somatically. DNA sequencing analysis confirmed that the aberrant bands Eun Ji Choi and Min Sung Kim contributed equally to this work.

Frameshift Mutation of ASPM Gene in Colorectal Cancers with Regional Heterogeneity

In humans, oxidative stress is involved in the development of many diseases, including cardiovascular diseases and cancers. Nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor, binds antioxidant response element DNA sequences and activates transcription of detoxifying genes such as NADPH quinine oxidoreductase and glutathione S-transferase (1). In normal cells, NRF2 plays a role in removing oxidative damages and may protect the cells from transforming to cancer cells. For example, NRF2-deficient mice had a significantly higher burden of gastric neoplasia (2). However, NRF2 protects not only normal cells but also cancer cells from cellular stress, and enhances survival of both normal and cancer cells (3), indicating roles of NRF2 in both cancer prevention and promotion. Increased expression of NRF2 was observed in head ⁄neck cancers (4). An enhanced activation of NRF2 is related to an increased proteasome activity in colon cancer (5). As NRF2 plays an important role in cancer cell survival, it might be possible that aberrant expression of NRF2 protein is prevalent in other cancers as well. In the present study, we analyzed the expression of NRF2 protein in gastric cancer (GC) tissues. For this purpose, six tissue microarray blocks were used in this study. The GC (n = 100) consisted of 50 diffuse, 36 intestinal and 14 mixed-type GC by Lauren’s classification. The tumor, lymph node and metastasis (TNM) stages of the GC were four IA, 15 IB, 79 II and two IIIA. From each donor GC block, two cylinders (one from normal and one from cancer tissues of each patient) of 2.0 mm diameter tissue were taken. Ages of the GC patients ranged from 29 to 89 years with an average of 60 years. For immunohistochemistry, we used the DAKO REAL EnVision System (DAKO, Glostrup, Denmark) with a polyclonal antibody against human NRF2 (Atlas Antibodies, Stockholm, Sweden). Sections were incubated

Expression of NRF2, a cytoprotective protein, in gastric carcinomas

Among the systems triggering apoptosis, the Fas-Fas ligand (FasL) system is recognized as a major pathway for the induction of apoptosis in cells and tissues Ligation of Fas by either an agonistic antibody or FasL transmits a 'death signal' to the target cell, potentially triggering apoptosis Alterations of genes along the Fas-mediated apoptosis pathway have been reported in many human cancers However, there have been no data regarding FasL gene mutations in human cancers We hypothesized that FasL gene mutation might be involved in the development of non-Hodgkin lymphoma (NHL) In this study, we analyzed the entire coding region of the FasL gene for the detection of somatic mutations in a series of 111 NHLs and found that one tumor had a FasL gene mutation in the cytoplasmic domain To evaluate the functional alterations of the mutant in apoptosis, we overexpressed the mutant in 293T cells, but couldn't find any significant loss of cell death compared to the wild-type FasL Together, these data suggest that FasL is occasionally mutated in human NHL and that FasL mutations appear to play no role in the pathogenesis of the vast majority of NHLs

Sug Hyung Lee

Papers

Somatic mutation of PIK3R1 gene is rare in common human cancers

Kinase domain mutation of ERBB family genes is uncommon in acute leukemias.

Frameshift Mutation of ASPM Gene in Colorectal Cancers with Regional Heterogeneity

Expression of NRF2, a cytoprotective protein, in gastric carcinomas

Mutational analysis of Fas ligand gene in human non-Hodgkin lymphoma