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.

Accumulated evidences have established that aberrant regulation of histone deacetylases (HDACs) is one of major causes for development of human malignancies. Mammalian HDACs can be divided into three subclasses consisting of 11 homologous of HDACs and 7 of sirtuins, but little is known about HDAC2 causes for carcinogenesis in solid tumors. Here, in order to investigate the roles of HDAC2 in carcinogenesis of liver cancer progression, we analyzed the expression of HDAC2 in 62 human hepatocellular carcinomas by utilizing Immunohistochemistry. Moderate to strong expression of HDAC2 was found in 54 (87%) out of total 62 tumors. The majority of positive tumors were detected in nucleous, but normal hepatocytes did not express of HDAC2 or showed weak positive staining. Interestingly, we were also noted that HDAC2 expression was appeared to be associated with aggressiveness of the tumors by the fact that HDAC2 expression was observed with significances in high grade tumors (Edmonson grade II, III). Taken together, we found the aberrant expression of HDAC2 in hepatocellular carcinomas, and this suggests that HDAC2 may play an important role in the development of liver cancer.

Increased Expression of Histone Deacetylase 2 is Found in Human Hepatocellular Carcinoma

Frameshift mutations of TAF1C gene, a core component for transcription by RNA polymerase I, and its regional heterogeneity in gastric and colorectal cancers.

To the Editor Recent genome-wide somatic mutation analyses identified that SPOP gene encoding the speckle-type POZ protein was frequently mutated in some human cancers, including prostate and endometrial carcinomas (1–4). Also, functional analyses on SPOP gene indicated that the SPOP might be a tumor suppressor gene (TSG) (5). The SPOP protein is a BTB domain-containing protein that serves as an adaptor for Cul3-based ubiquitination (6). SPOP somatic mutations have been detected in 4–29% of prostate cancers (1–3), 8% of serous endometrial cancers (4), and 2% of lung cancers (1). Importantly, most of the SPOP somatic mutations have been found in exons 4–5 encoding a specific region in the N-terminal MATH domain (amino acid residues 68– 160) (1–4). Despite the evidence that SPOP gene is a TSG, it is still uncertain that SPOP acts as TSG in other cancers besides prostate and endometrial cancers. In this study, we analyzed the exons 4–5 of SPOP gene for the detection of somatic mutations in solid tumors (breast, lung, and liver cancer tissues) and acute leukemias. For this analysis, randomly collected methacarn-fixed solid tumor tissues [94 breast carcinomas, 160 non-small cell lung cancers (NSCLC), 42 hepatocellular carcinomas (HCC)] and bone marrow aspirates (488 acute leukemias) from Korean patients were used in this study. The hepatocellular carcinomas consisted of 3 grade I, 18 grade II, and 21 grade III cancers by Edmondson’s classification. All of the breast carcinomas were invasive ductal carcinomas. The lung carcinomas consisted of 84 adenocarcinomas and 76 squamous cell carcinomas. The acute leukemias consisted of 105 adult acute myelogenous leukemias (AML), 200 adult acute lymphoblastic leukemias (ALL), 41 multiple myelomas, 122 childhood ALL, and 20 childhood AML. Approval for this study was obtained from the Catholic University of Korea, College of Medicine’s institutional review board. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection as described previously (7). The normal tissues in solid tumor cases were from tissues (normal epithelial cells or lymphoid cells) away from tumors, while those in leukemias were from peripheral blood cells or bone marrow cells after complete remission. Up to now, most of the SPOP somatic mutations in tumors have been detected in exons 4 and 5 (1–4). Thus, we focused our analysis on these exons by polymerase chain reaction (PCR)-based singlestrand conformation polymorphism (SSCP) analysis. Genomic DNA each from tumor cells and normal cells of the same patients were amplified by PCR with a primer pair for exon 4 (5′-TTTTTCTATCTGTTTTGGACA-3′ and 5′-AGAGGAGAACATTTACCCAT-3′; product size: 190 bp) and a primer pair for exon 5 (5′-TTGATCTGGTTTTTGCGTAAC-3′ and 5′-CTCAGCAGAATACAAGGACTCAC-3′; product size: 204 bp). Radioisotope [(P) dCTP] was incorporated into the PCR products for detection by autoradiogram. After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and re-amplified for 35 cycles using the same primer set. Other procedures of the PCR-SSCP were described in our previous studies (7, 8). In the PCR-SSCP, we were able to find a SPOP mutation in a childhood precursor B-ALL (Fig. 1), but there was no evidence of the mutation in the other hematologic malignancies. In solid tumors (breast, lung, and liver cancers), there was no SPOP mutation, either. The mutation found in the ALL was a missense mutation that substituted amino acid residue Gly132 (p.Gly132Glu) (Fig. 1). This mutation is a novel mutation and close to the recurrent mutation site (p.Phe133Leu) (1). DNA from normal tissue from the same patient showed no evidence of the mutation in SSCP, indicating that the mutation had risen somatically. Ubiquitin modifications regulate a wide variety of cellular processes and more or less are involved in cancer pathogenesis (9). Also, earlier observations that SPOP, an adaptor for

Somatic mutation of SPOP tumor suppressor gene is rare in breast, lung, liver cancers, and acute leukemias.

Mutational analysis of caspase-14 gene in common carcinomas.

To the Editor: Chromatin remodeling is a dynamic modification process of chromatin architecture to allow access of genomic DNA to transcription machinery proteins, and control gene expression. ATP-dependent chromatin remodeling is a main mechanism for eukaryote chromatin remodeling [1]. HELLS (also known as SMARC6 and LSH) gene encodes a protein related to the SNF2 family of chromatin-remodeling ATPase. This protein is essential for correct establishment of DNA methylation level and for efficient repair of DNA double-strand breaks [2]. Somatic mutations in chromatin remodeling genes have been detected in many cancers, including gastric, ovary and renal cancers [3]. For example, ARID1A in the SWI-SNF remodeling complex is mutated in many cancers and is considered a tumor suppressor gene [3]. Also, HELLS is known to be involved in maintenance of genome stability [2], loss of which is considered a hallmark of tumor cells. However, alterations of HELLS in human cancers have not been reported , yet. In a public genome database (http://genome.cse.ucsc. edu/), we found that human HELLS gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with microsatellite instability (MSI). Frameshift mutation of genes containing mononucleotide repeats is a feature of gastric (GC) and colo-rectal cancers (CRC) with MSI [4]. To see whether the mononucleotide repeat in human HELLS gene is mutated in GC and CRC, we analyzed an A8 repeat in exon 7 and another A8 repeat in exon 16 by polymerase chain reaction (PCR)-based single strand confor-mation polymorphism (SSCP) assay. For this, we used methacarn-fixed tissues of 34 GC with high MSI (MSI-H), 45 GC with stable MSI (MSS), 99 CRC with MSI-H and 45 CRC with MSS. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [5,6]. Radioisotope ([ 32 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 [6]. On the SSCP, we observed aberrant bands of the HELLS gene in eight cancers (seven CRC and one GC). DNA from normal tissue showed no shifts in SSCP, indicating the aberrant bands had risen somatically (Fig. 1). DNA sequencing analysis confirmed that aberrant bands represented HELLS somatic mutations. All of the mutations were frameshift mutations (deletion of one base) in …

Sug Hyung Lee

Papers

Increased Expression of Histone Deacetylase 2 is Found in Human Hepatocellular Carcinoma

Frameshift mutations of TAF1C gene, a core component for transcription by RNA polymerase I, and its regional heterogeneity in gastric and colorectal cancers.

Somatic mutation of SPOP tumor suppressor gene is rare in breast, lung, liver cancers, and acute leukemias.

Mutational analysis of caspase-14 gene in common carcinomas.

Mutation of HELLS, a chromatin remodeling gene, gastric and colorectal cancers.