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 serine protease Omi/HtrA2 is released from mitochondria into the cytosol after apoptosis stimuli, inducing apoptosis in a caspase-independent manner through its protease activity and in a caspase-dependent manner by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases. Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and chemotherapy is largely dependent upon apoptosis. Thus, analysis of the expression status of Omi/ HtrA2, a regulator of apoptosis, in cancer tissues is needed for an understanding of cancer development. In the current study, we analyzed the expression of Omi/HtrA2 in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity (defined as ≥30%) was observed for Omi/HtrA2 in 43 (72%) of the 60 cancers. By contrast, the surface mucous cells and mucous neck cells in the normal gastric mucosa showed no or weak expression of Omi/ HtrA2. Taken together, these results suggest that stomach cancer cells in vivo may need Omi/HtrA2 expression for apoptosis, and that Omi/HtrA2 expression might be involved in stomach cancer development.

Immunohistochemical analysis of Omi/HtrA2 expression in stomach cancer.

Cadherins (CDHs) are important in maintenance of cell adhesion and polarity, alterations of which contribute to tumorigenesis. Alterations of E-cadherin, a prototype CDH, have been reported in many cancers. However, alterations of unconventional CDHs, including CDH10, CDH24 and DCHS2 are largely unknown in cancers. Aim of this study was to explore whether CDH10, CDH24 and DCHS2 genes are mutated in gastric (GC) and colorectal cancers (CRC). In a public database, we found that CDH10, CDH24 and DCHS2 genes had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the mutations in 89 GC and 131 CRC (high MSI (MSI-H) or stable MSI/low MSI (MSS/MSI-L)) by single-strand conformation polymorphism analysis and DNA sequencing. We found six DCHS2, one CDH10 and one CDH24 frameshift mutations in them. All of the mutations were detected in cancers with MSI-H and there was a statistical difference in the frameshift mutation frequencies between the cancers with MSI-H (8/105) and MSS/MSI-L (0/115). The DCHS2 frameshift mutations were found in 8.8 % and 4.2 % of GC and CRC with MSI-H respectively. Our results show that unconventional CDH10, CDH24 and DCHS2 genes harbored frameshift mutations. These mutations might inactivate the cell adhesion-related functions and could be a feature of GC and CRC with MSI-H.

Frameshift mutations of cadherin genes DCHS2, CDH10 and CDH24 genes in gastric and colorectal cancers with high microsatellite instability.

Recent studies have revealed several recurrent mutations in oncogenes that could not only be underlying mechanisms of tumorigenesis, but also be potential targets for cancer therapies. Compared to carcinomas, genetic alterations of sarcomas are relatively unknown. To see whether recurrent oncogenes discovered in non-sarcomatous malignancies are present in sarcomas as well, we analyzed oncogenes with known mutations in various types of sarcomas. We performed mutational analysis of recurrent mutation sites of PIK3CA (exons 9 and 20), JAK2 (exon 14), BRAF (exon 15), FOXL2 (exon 1), IDH1 (exon 4), AKT1 (exon 3), and EZH2 (exon 16) genes in 108 sarcomas by single- strand conformation polymorphism and DNA sequencing. The sarcomas consisted of malignant fibrous histiocytomas, rhabdomyosarcomas, osteosarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas, synovial sarcomas, liposarcomas, angiosarcomas, chondrosarcomas, and Ewing sarcomas. Overall, we detected the two PIK3CA mutations and one JAK2 mutation (total: 3/108: 2.8%). Two rhabdomyosarcomas (16.7%) and one angiosarcoma (16.7%) harbored the mutations, whereas other sarcomas harbored none. The PIK3CA mutations were novel missense mutations that had not been detected in other cancers. The JAK2 mutation was an intron mutation. This study demonstrated that the somatic mutations of PIK3CA and JAK2 occurred in a small fraction of the sarcomas and that these mutations may not play a principal role in the development of sarcomas.

Mutational analysis of PIK3CA, JAK2, BRAF, FOXL2, IDH1, AKT1 and EZH2 oncogenes in sarcomas.

Application of amplified RNA and evaluation of cRNA targets for spotted-oligonucleotide microarray.

Cancer patients have serum antibodies that are reactive to their cancer cells, but not to normal cells. However, the nature of these cancer-specific epitopes is largely unknown. Many cancerspecific proteins resulting from somatic mutation reside in the intracellular space and cannot be accessible to antibodies. There are a few cancer-specific antigens on the cancer cell surface created by somatic mutations that could be potential targets for cancer immunotherapy (1). Tn antigen (also known as GalNAca1-Ser/ Thr) is a precursor of longer O-glycans such as core 1 (2). For the maturation of Tn antigen to core 1, C1b3Gal-T activity is required. Core 1b3Gal-T-specific molecular chaperone (COSMC; also known as C1GALT1C1) is a molecular chaperone that is required for C1b3Gal-T activity (3). Without COSMC, the C1b3Gal-T is targeted to the proteosome and degraded (3). The Tn antigen is overexpressed in many cancers, including colorectal and breast cancers (4, 5). Recent studies revealed that cancer-specific mutations in the COSMC gene remove C1b3Gal-T activity, disrupt O-glycan core 1 synthesis, lead to Tn antigen overexpression, and create a cancerspecific epitope on the cell surface (2, 3, 6). Somatic mutations of the COSMC gene have been found in both mouse (Ag104A fibrosarcoma and Neuro2A neuroblastoma) and human (Jurkat Tcell leukemia and LSC colorectal cancer) cancer cell lines (2, 3, 6). The COSMC mutations consisted of insertion of a T nucleotide (LSC), deletion of a T nucleotide (Jurkat), deletion of 78 nucleotides (Ag104A) and non-sense substitution of a nucleotide (Neuro2A). These mutations would result in premature stops of amino acid synthesis or deletion of a large portion of the

Sug Hyung Lee

Papers

Immunohistochemical analysis of Omi/HtrA2 expression in stomach cancer.

Frameshift mutations of cadherin genes DCHS2, CDH10 and CDH24 genes in gastric and colorectal cancers with high microsatellite instability.

Mutational analysis of PIK3CA, JAK2, BRAF, FOXL2, IDH1, AKT1 and EZH2 oncogenes in sarcomas.

Application of amplified RNA and evaluation of cRNA targets for spotted-oligonucleotide microarray.

Absence of COSMC gene mutations in breast and colorectal carcinomas