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.

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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.

Teratoma is a type of germ cell tumor that originates from totipotential germ cells that are present in gonads, which can differentiate into any of the cell types found in adult tissues. Ovarian teratomas are usually mature cystic teratomas (OMCTs, also known as dermoid cysts). Chromosome studies in OMCTs show that the chromosomes are uniformly homozygous with karyotype of 46, XX, indicating that they may be parthenogenic tumors that arise from a single ovum after thefirst meiotic division. However, the tissues in OMCTs have been known to be morphologically and immunophenotypically identical to the orthotopic tissues. Currently, expression profiles of tissue components in OMCTs are not known. To identify whether OMCT tissues are expressionally similar to or different from the orthotopic tissues, we adopted single-cell RNA-sequencing (scRNA-seq), and analyzed transcriptomes of individual cells in heterogenous tissues of two OMCTs. We found that transcriptome profiles of the OMCTs at single cell level were not significantly different from those of normal cells in orthotopic locations. The present data suggest that parthenogeneticlly altered OMCTs may not alter expression profiles of inrivirual tissue components in OMCTs.

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Single Cell Gene Transcriptome Analysis of Ovarian Mature Teratomas

Dear Editor, Mutations in CSF3R are a cause of Kostmann syndrome, also known as severe congenital neutropenia [1]. A recent study discovered that CSF3R somatic mutations were common in chronic neutrophilic leukemia (CNL) [2]. Of note, the most common recurrent mutation (13 out of 21 mutations) was a somatic mutation altering an amino acid residue at 618 (p.T618I). This mutation led to activation of JAK kinases and enhanced transformation of the mutant cells [3]. They also analyzed CSF3R mutation in other hematologic neoplasia (acute myelogenous leukemia (AML) 3/292 (1 %), T cell acute lymphoblastic leukemia (T-ALL) 0/8, early T cell precursor T cell ALL 1/3, and B cell ALL 0/41) [3]. Investigation of the CSF3R p.T618I mutation in additional AML cohort identified five cases (5/1446 cases 0.35 %) [2]. One of the aims of this study was to see whether the CSF3R p.T618I mutation was present in AML and solid tumors of Korean patients. For these, a total of 1807 tumor tissues from 702 hematologic (105 AML, 68 myelodysplastic syndromes, 11 T-ALL, 458 precursor B-ALL, and 60 multiple myelomas) and 1105 solid tumors (369 colon, 274 prostate, 147 lung, 180 gastric, 91 breast, and 44 hepatocellular carcinomas) were used in this study (Table 1). We analyzed somatic mutations of CSF3R p.T618I mutation using genomic DNA by single-strand conformation polymorphism (SSCP) and direct DNA sequencing as described previously [4, 5]. All institutional and national guidelines for the care were followed. Informed consent was obtained from all patients for being included in the study except for cases whose informed consents were waived for this study. PCR and subsequent SSCP analysis detected aberrant migrating SSCP bands in two AML, but neither in the other hematologic nor solid tumors. In the 105 adult AML, we were able to find the p.T618I mutation in two cases (1.9 %). They were not detected in matched normal tissues of the patients, indicating that they were somatic mutations. The mutations were identified in an AML with myelodysplasia-related changes (AML-MRC) and an AML with multilineage dysplasia (this case could also be classified by FAB as a refractory anemia with excess blasts in transformation (RAEB-T)). One of the main concerns in cancer genetics is to identify whether any mutation found in a cancer is common in other cancer types. Because CSF3R is involved not only in blood cell proliferation but also in non-hematopoietic cell proliferation, we expected some types of tumors would harbor the mutation as well. The present study, however, detected somatic mutation of CSF3R only in AML. Although our study observed a slightly higher prevalence (1.9 %) of the mutation in AML compared to the earlier data (1.0 and 0.35 %), there was no statistical difference (Fisher’s exact test, p>0.05). Our data revealed a novel finding that the CSF3R p.T618I mutation occurred in an AML-MRC and a RAEB-T. The discovery of the recurrent CSF3R mutation in cancers offered an opportunity for developing therapeutic and diagnostic tools targeting this mutation in human cancers. Our data, however, suggest that S. Y. Hwang :N. J. Yoo : S. H. Lee (*) Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, South Korea e-mail: suhulee@catholic.ac.kr

Mutational analysis of oncogenic CSF3R p.T618I in acute leukemias and common solid cancers.

Mutational analysis of pro-apoptotic ENDOG gene in common solid cancers and acute leukaemias.

Angela Stassinopoulos, Junji Sagara and John C. ReedChristian Stehlik, Sug Hyung Lee, Andrea Dorfleutner,http://www.jimmunol.org/content/171/11/6154J Immunol€2003; 171:6154-6163; ;Referenceshttp://www.jimmunol.org/content/171/11/6154.full#ref-list-1This article cites 41 articles, 20 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

a Regulator of Procaspase-1 Activation Is Containing a Caspase Recruitment Domain Apoptosis-Associated Speck-Like Protein

To the Editor: HACD4 (also known as PTPLAD2; protein tyrosine kinase-like A domain-containing protein 2) belongs to a family of enzymes that catalyze the dehydration step of very long chain fatty acid synthesis [1]. Esophageal squamous cell carcinomas show downregulation of HACD4 expression that is correlated with poor patient survival [2]. Knockdown of HACD4 increases STAT3 phosphorylation and cell proliferation [2]. In glioblastomas, the locus for HACD4 is frequently deleted [3]. TCPL10 encoding T-complex protein 10-like, a nuclear protein with transcription regulation functions, suppresses colony formation, cell cycle progression through G0/G1 phase and cell growth in vivo in liver [4]. Together, these data suggest that both HACD4 and TCP10L possess tumor suppressor gene (TSG) activities. However, the roles of HACD4 and TCP10L in colorectal (CRC) and gastric (GC) cancers are not known. About one third of CRC and GC have defects in mismatch repair that can cause microsatellite instability (MSI). TSGs are often observed to have mutations at monocleotide repeats in high MSI (MSI-H) CRC and GC [5]. There are mononucleotide repeats inHACD4 (A8) and TCP10L (G7) of their coding sequences that could be mutation targets in cancers with MSIH. In addition, it is well known that intratumoral heterogeneity (ITH) plays an important role in cancer development and progression and impedes proper diagnosis and treatment of cancer patients [6, 7]. The present study aimed to find whether HACD4 and TCP10L genes harbored frameshift mutation within the repeats and ITH. For this, we studied the mononucleotide repeats in HACD4 (A8) and TCP10L (G7) in 79 high MSI (MSI-H) CRCs, 45microsatellite stable (MSS) CRCs, 34 MSI-H GCs and 45 MSS GCs by single-strand conformation polymorphism (SSCP) assay. After SSCP, Sanger DNA sequencings were performed in cancers with mobility shifts in the SSCP to confirm the mutations [8]. SSCP and Sanger sequencing identified frameshift mutations of HACD4 in 3 cases of CRC and 1 case of GC, and TCP10L in 3 cases of GC. All of them were detected in CRC or GC with MSI-H, but neither in CRC nor GC with MSS. These mutations were not detected in their matched normal t issues. The HACD4 mutat ions (c.689delA (p.Lys230Argfsx42)) and the TCP10L mutations (c.641delG (p.Gly214Valfsx26)) were recurrent in all mutated cases (Table 1). For ITH of the mutations, we studied 16 cases of CRCs with 4 to 7 regional fragments per CRC. One of the 16 CRCs (6.3%) showed the deletion mutation of HACD4 mutations (c.689delA) in different tissue regions. No ITH of the TCP10L frameshift mutation was observed in the 16 CRCs. Clinical and histopathological parameters, however, could distinguish neither HACD4 frameshift mutation (+) and (−) cancers nor TCP10L frameshift mutation (+) and (−) cancers. The frameshift mutations of HACD4 and TCP10L identified in this study would result in truncation of HACD4 and TCP10L proteins respectively, suggesting that inactivation of their TSG functions would contribute to tumorigenesis in MSI-H GCs and CRCs. Also, one CRC (6.3%) exhibited ITH for the HACD4 frameshift mutation. ITH of the frameshift mutation in the CRC might suggest a possibility that there could be a mixed or ameliorated effect of the HACD4 mutation in the affected CRC. However, we were not able to find any distinguished clinicopathologic features of HACD4 mutation ITH-positive cancer. It * Sug Hyung Lee suhulee@catholic.ac.kr

Sug Hyung Lee

Papers

Single Cell Gene Transcriptome Analysis of Ovarian Mature Teratomas

Mutational analysis of oncogenic CSF3R p.T618I in acute leukemias and common solid cancers.

Mutational analysis of pro-apoptotic ENDOG gene in common solid cancers and acute leukaemias.

a Regulator of Procaspase-1 Activation Is Containing a Caspase Recruitment Domain Apoptosis-Associated Speck-Like Protein

Inactivating Frameshift Mutations of HACD4 and TCP10L Tumor Suppressor Genes in Colorectal and Gastric Cancers.