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.

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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.

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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.

Mounting evidence suggests that the alterations of Akt/protein kinase B (PKB) play an important role in tumorigenesis. Phosphorylated Akt regulates many of the key effector molecules involved in apoptosis, angiogenesis, and cell cycle progression during tumorigenesis. The expression of phosphorylated Akt has been described in some human malignancies, but not in primary human lung cancer. In this study, to understand the role of Akt in lung tumorigenesis we analyzed the expression of phosphorylated Akt in 43 non-small cell lung cancers (NSCLCs) by immunohistochemistry. Phosphorylated Akt was detected either in the cytoplasm (23 cases) or nucleus (6 cases) in 29 of 43 NSCLCs (67.4%). Squamous cell carcinomas, adenocarcinomas, and bronchioloalveolar carcinomas expressed phosphorylated Akt in 68.2%, 61.5% and 75%, respectively. We also analyzed the phosphorylated Akt expression between primary NSCLCs and their corresponding nodal metastasis; the expression was not, however, different between the primary and metastatic lesions. Taken together, these results indicate that Akt 1 is frequently activated in NSCLCs, irrespective of the histological subtypes, and suggest that phosphorylated Akt may play a role in the development of NSCLC rather than in the progression of NSCLC.

Non-small cell lung cancers frequently express phosphorylated Akt; an immunohistochemical study.

Increased oxygen free radicals produced in gastric mucosa by H. pylori induce DNA damage and lead to dyspalsia and gastric cancers. However, only a small percentage of individuals that carry H. pylori develop gastric cancer, indicating that other factors are involved. We have screened a set of 95 sporadic gastric cancers for mutations and allele loss of the DNA glycosylase MYH gene, which excises adenine misincorporated opposite unrepaired 8-oxoG. Two of 95 cancers had bi-allelic mutations of the MYH gene with somatic missense mutation of one allele and loss of the remaining allele. The mutations were missense mutations, P391S and Q400R, in exon 13 encoding NUDIX hydrolase domain of the gene. The patients were H. pylori-positive and the tumors were of advanced intestinal-type gastric cancer with lymph node metastasis. In addition, 4 (17.4%) of 23 informative cases showed allele loss at MYH locus. Therefore, our data suggest that somatic mutations of base excision repair gene MYH contribute to the development of a sub-set of sporadic gastric cancers.

Genetic alterations of the KLF6 gene in gastric cancer.

somatic mutations in the adenocarcinomas, bronchioloalveolarcarcinomas and large cell carcinomas of lung that all correlatedwith a clinical response to geﬁtinib. The mutations were de-tected in the exon 18, 19 and 20 that encode the intracellularkinase domain. The mutations detected in the exon 18 wouldsubstitute the amino acid G719 in the P-loop, while thosedetected in the exon 21 would substitute amino acids in theactivation domain. The mutations in the exon 19 were in-framedeletions that may alter the structure of C helix. All of the

Absence of EGFR mutation in the kinase domain in common human cancers besides non-small cell lung cancer.

Caspases play an essential role during apoptotic cell death, and alterations of caspases are known to contribute to human cancer development. In the current study, we analyzed the expression of caspase-2, -6 and -7 in 120 gastric carcinomas by immunohistochemistry using a tissue microarray approach. Caspase-2, -6 and -7 were expressed in 42 (35%), 63 (53%) and 39 (33%) of the gastric cancers, respectively. By contrast, the surface mucous cells and mucosal glandular cells in the normal gastric mucosa showed strong immunoreactivity for caspase-2, -6 and -7. Taken together, these results indicate that caspase-2, -6 and -7 expression in gastric cancer cells is decreased compared to in normal gastric mucosal cells, and suggest that loss of caspase-2, -6 and -7 expression might be involved in the mechanisms of gastric cancer development.

Loss of caspase-2, -6 and -7 expression in gastric cancers.

NOTCH proteins (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) play crucial roles in embryonic development. Also, mounting evidence indicates that NOTCH contributes to the pathogenesis of hematopoietic and solid malignancies. Recent studies reported a high incidence of gain-of-function mutations of the NOTCH1 gene in T-cell acute lymphoblastic leukemias (ALL). To see whether NOTCH1 mutation occurs in other malignancies, we analyzed NOTCH1 for the detection of somatic mutations in 334 malignancies, including 48 lung, 48 breast, 48 colorectal and 48 gastric carcinomas, and 142 acute leukemias (105 acute myelogenous leukemias, 32 B-ALLs and 4 T-ALLs) by single-strand conformation polymorphism assay. Also, to see whether other NOTCH genes harbor somatic mutations, we analyzed NOTCH2, NOTCH3 and NOTCH4 genes in the same tissue samples. Overall, we detected three NOTCH mutations in the cancers, which consisted of one NOTCH1 mutation in the T-ALLs (25.0%), one NOTCH2 mutation in the breast carcinomas (2.1%), and one NOTCH3 mutation in the colorectal carcinomas (2.0%). There was no NOTCH mutation in other malignancies analyzed. Our data indicate that NOTCH1 is mutated in T-ALL, but not in other common human cancers, and that NOTCH2, NOTCH3 and NOTH4 genes are rarely mutated in common human cancers. Despite the importance of NOTCH activation in many types of human cancers, mutation of NOTCH genes, except for NOTCH1 mutation in T-ALL, may not play an important role in the tumorigenesis of common cancers.

Sug Hyung Lee

Papers

Non-small cell lung cancers frequently express phosphorylated Akt; an immunohistochemical study.

Genetic alterations of the KLF6 gene in gastric cancer.

Absence of EGFR mutation in the kinase domain in common human cancers besides non-small cell lung cancer.

Loss of caspase-2, -6 and -7 expression in gastric cancers.

Mutational analysis of NOTCH1, 2, 3 and 4 genes in common solid cancers and acute leukemias.