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.

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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.

To the Editor: Receptor tyrosine kinases (RTK) that regulate biological processes in cell growth and survival are a major oncogene family. The ERBB family of RTK consists of ERBB1 (EGFR/HER1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) (1). When compared to the other three ERBBs, ERBB3 has a weaker kinase activity (1). Upon binding with ligand, ERBB3 heteromerizes with other RTK, especially ERBB2, and transmits downstream signals (1). Mounting evidence indicates that ERBB genes are altered in human cancers. For example, lung adenocarcinomas harbor EGFR mutations and breast cancers harbor ERBB2 amplification (2, 3). These genetic alterations are therapeutically targeted and currently used in clinic (2, 3), indicating that oncogenic mutations of ERBB genes are common targets of genetic alterations in human cancers. A recent study discovered that ERBB3 somatic mutations were common in colorectal and gastric cancers (~11%), and less common in other solid cancers (4). Of note, most of the ERBB3 mutations were discovered in the extracellular ligand-binding domain (ECD) and were recurrent at specific amino acids. Of these, somatic mutations altering an amino acid residue at 104 (p.Val104Met and p.Val104Leu) were the most common recurrent mutations. These mutations conferred transformation activities in cells in a ligand-independent fashion. Furthermore, anti-ERBB antibodies and small molecule inhibitors blocked the mutant ERBB3-related oncogenic activities. Although ERBB3 signaling and alterations are well known in solid tumors, but they are not known well in hematologic neoplasia (5). Thus, it is interesting to know whether recurrent ERBB3 mutation occurs in neoplasia from hematopoietic tissues as well. For this, we analyzed somatic mutations of ERBB3 gene using genomic DNA from in fresh bone marrow aspirates of 811 hematologic tumors (acute myelogenous leukemias (AML), acute lymphoblastic leukemias (ALL), multiple myelomas, and myelodysplastic syndromes) (Table 1) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed the gene in paraffin-embedded tissues of 63 non-Hodgkin lymphomas (NHL). Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Genomic DNA each from tumor cells and normal cells (remission bone marrow cells in the cases of leukemias) was used in this study. Genomic DNA each from tumor cells and corresponding normal cells was amplified with a primer pair covering the exon 3 of ERBB3 gene that contained coding sequences of amino acid residue Val104. Radioisotope was incorporated into the PCR products for detection by autoradiogram. After the SSCP, DNAs showing mobility shifts were cut out from the dried gel, and re-amplified for 30 cycles using the same primer sets. In colon carcinomas, we were able to find the recurrent mutations p.Val104Met and p.Val104Leu, which were subsequently used as positive controls in SSCP. However, none of the SSCP from the hematologic neoplasia revealed aberrantly migrating bands compared with wild-type bands from the normal tissues (Fig. 1), indicating there was no evidence of ERBB3 mutation altering the Val104 in the neoplasia analyzed. To confirm the SSCP results, we repeated the experiments twice and found that the data were consistent.

Oncogenic ERBB3 mutations altering p.Val104 is rare in acute leukemias and non‐Hodgkin lymphomas

Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers.

To the Editor Werner syndrome, an autosomal recessive disorder causing premature aging, is caused by truncating mutations in Werner syndrome gene (WRN) that encodes a DNA helicase with exonuclease activity [1]. Patients with Werner syndrome have an increased cancer incidence as well, suggesting that the lack normal WRN function affects tumorigenesis [2]. Both helicase and exonuclease activities ofWRN protein contribute to DNA repair in cells [3]. Also, cells with defective WRN show genomic instability [4]. These features are frequently observed in cancers, suggesting a possibility of WRN gene alterations in cancers. However, it remains unknown whether inactivating mutation of WRN is common in gastric cancer (GC) and colorectal cancer (CRC). About one third of GC and CRC are classified as high microsatellite instability (MSI-H) cancers [5]. Many tumor suppressor genes such as BAX and TGFBR2 harbor frameshift mutations at monocleotide repeats in MSI-H cancers [5]. In the human genome database, we observed that WRN gene possesses nucleotide repeats in coding sequences that might be mutated in MSI-H cancers. In this study, we analyzed an A8 repeat in exon 2 and an A7 repeat in exon 28 of WRN by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. In this study, we used 79 GCs and 124 CRCs. The GCs were 34 GCs with MSI-H, 45 GCs with microsatellite stable/low MSI (MSS/ MSI-L), 79 CRCs with MSI-H and 45 CRCs with MSS/ MSI-L. In cancer tissues, malignant cells and normal cells were selectively procured by microdissection. Radioisotope ([P]dCTP) was incorporated into the PCR products, which were subsequently displayed in SSCP gels and analyzed with direct DNA sequencing [6]. Additionally, to see whether the WRN mutations possess intra-tumor heterogeneity (ITH) that contributes to tumor aggressiveness [7], we studied 16 CRCs with four to seven regional biopsies per CRC. In the SSCP, we found aberrantly migrating bands in three GCs and three CRCs, but not in their matched normal samples. DNA sequencing analysis confirmed that the aberrant bands represented WRN somatic mutations, which consisted of frameshift mutations by a deletion (c.15delA (p. Lys5AsnfsX15)) in exon 2 and another deletion (c.3382delA (p. Ser1128ValfsX34)) in exon 28 within the repeat (Table 1). The mutations were found in GCs (3/34, 8.8%) and CRCs (3/79, 3.8%) with MSI-H (3/113, 5.3%), but not in GCs (0/45) and CRCs (0/45) with MSS/MSI-L (Fisher’s exact test, p = 0.028). The frameshift mutation in exon 2 showed ITH in one of 16 CRCs (6.3%). A CRC (# 41) showed the c.15delA mutation in three regional biopsies (#41–1, 41–3 and 41–4), but there was no such mutation in the two regional biopsies (#41–6 and 41–7) (Fig. 1). We could not find any significant histological difference among the ITH regions in this case. Cancer-related functions (DNA repair and maintenance of genomic stability) and increased cancer incidence in Werner syndrome [1] led to us to analyze inactivating mutations of WRN gene in GC and CRC. In the present study, we found that six cases (5.3%) of GCs and CRCs with MSI-H harbored WRN frameshift mutations, indicating that WRN is mutated Ju Hwa Lee and Sung Soo Kim contributed equally to this work.

WRN, the Werner Syndrome Gene, Exhibits Frameshift Mutations in Gastric and Colorectal Cancers

Apoptosis is a biological process that plays a critical role in the development and homeostasis of multicellular organisms [1]. The extrinsic apoptosis pathway initiates at the cell surface through...

Mutational analysis of proapoptotic death associated protein 3 (DAP3) P-loop domain in common human carcinomas

TRAF2 and TRAF3 genes of tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family are involved in diverse cell signaling, and function as both tumor suppressor gene and oncogene. Alterations of TRAF2 and TRAF3 in colon cancer (CC) along with their regional difference and microsatellite instability (MSI) are largely unknown. In the present study, we analyzed TRAF2 and TRAF3 frameshift mutations in 168 sporadic CCs (100 high MSI (MSI-H) and 68 microsatellite-stable (MSS) CCs). We identified TRAF2 and TRAF3 frameshift mutations in 4 (4%) and 3 CCs (3%) with MSI-H, respectively, but none in 68 cases of MSS CCs. Of the 168 CCs, we analyzed the mutations in multi-regions for 39 CCs (16 MSI-H and 23 MSS CCs), and discovered that 12.5% (2/16) and 6.3% (1/16) of MSI-H CCs exhibited regional difference in TRAF2 and TRAF3 mutations, respectively. In the multi-region samples of 23 MSS CCs, neither TRAF2 nor TRAF3 frameshift mutation was found. In 40% of CCs, both TRAF2 and TRAF3 expressions were increased compared to normal colon cells. Our data indicate that TRAF2 and TRAF3 frameshift mutations and their regional difference as well as altered expressions are present in MSI-H CCs, which could contribute to MSI-H cancer development.

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Sug Hyung Lee

Papers

Oncogenic ERBB3 mutations altering p.Val104 is rare in acute leukemias and non‐Hodgkin lymphomas

Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers.

WRN, the Werner Syndrome Gene, Exhibits Frameshift Mutations in Gastric and Colorectal Cancers

Mutational analysis of proapoptotic death associated protein 3 (DAP3) P-loop domain in common human carcinomas

Brief Research Report Regional Difference in TRAF2 and TRAF3 Gene Mutations in Colon Cancers.