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.

Dear Editor, Rho GTPases are involved in major cellular functions including cell motility, growth and differentiation. TRIO, one of the Rho guanine nucleotide exchange factors (GEFs), contains three functional domains: a serine/ threonine kinase domain and two GEF domains [1], suggesting that TRIO may play a crucial role in signaling pathways that control cell proliferation. In several cancers, TRIO expression is significantly increased and is correlated with poor prognosis [2]. In addition, many cancers are reported to harbor somatic mutations of TRIO gene that include truncating mutations as well as missense mutations [3, 4]. However, the functional consequences of the TRIO mutations are largely unknown. About 10% of gastric cancer (GC) and colorectal cancer (CRC) show microsatellite instability (MSI) phenotype that has defects in mismatch repair [5]. TRIO gene has mononucleotide repeats (A7, T8 and C7) in their coding sequences that could be targets for frameshift mutation in cancers with MSI. Intratumoral heterogeneity (ITH) plays an important role in cancer development and progression and impedes proper diagnosis and treatment of cancers [6]. In this study, we analyzed somatic frameshift mutation of TRIO gene and its mutational ITH in GC and CRC. We analyzed the G7 repeat in 34 GCs with MSI-H, 45 GCs with MSS, 79 CRCs with MSI-H and 45 CRCs with MSS by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. After SSCP, Sanger DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP [7]. We found TRIO somatic frameshift mutations in GCs (5/34, 14.7%) and CRCs (11/79, 13.9%) with MSI-H, but not in CRCs (0/45) and GCs (0/45) with MSS (p < 0.001). All of the mutations were deletion or duplication mutations in the repeats that would cause premature stops, which would lead to termination of amino acid translation. For ITH of the mutation, we studied 16 cases of CRCs with 4 to 7 regional fragments per CRC. One CRC showed a frameshift mutation in the T8 (T8 to T7), which exhibited ITH of the mutation (mutation in four of seven regional biopsies and wild type in the other three) (Tables 1 and 2). Another CRC showed a frameshift mutation in the C7 (C7 to C6), but the mutation was identified in all regional biopsies, indicating no ITH of the mutation in this CRC. The frameshift mutations identified in this study would result in truncation of TRIO protein, suggesting that TRIO may be inactivated in MSI-H GCs and CRCs by this frameshift mutation. Recent studies suggested evidence that TRIO might have oncogenic activity [1, 2]. Also, our data on inactivating TRIO mutations in MSI-H cancers may suggest that TRIO could have tumor suppressor activities. However, because to our knowledge there is no definite experimental data on TRIO functions in MSI-H tumor development, it is not possible to guess consequences of the frameshift mutations of TRIO. On the other hand, provided that TRIO activation promotes oncogenic activities, the Eun Ji Choi and Min Sung Kim contributed equally to this work.

TRIO Gene Encoding Trio Rho Guanine Nucleotide Exchange Factor Harbors Frameshift Mutations of in Gastric and Colorectal Cancers.

Intratumoral heterogeneity of CSNK1G3 mutations, a casein kinase 1, in colon cancers.

O-137 Clinical results of stereotactic body frame based fractionated radiosurgery for stage I non-small cell lung cancer

One of the central aims of cancer research is to identify functional cancer-specific mutations that are causally implicated in tumorigenesis. Recently, Sjöblom et al. analyzed 13,023 genes (approximately one half of all human genes), a set of protein-coding genes termed the consensus coding sequences (1). In 35 breast and 35 colorectal cancer tissues, they identified 189 genes that were mutated at significant frequencies. In addition to genes that had been reported to be mutated in colorectal or breast cancers, including TP53, APC and KRAS, many other genes were newly found to be mutated in the cancers (1). Of the genes with mutations, excision-repair cross-complementing, group 6 (ERCC6) was found to be frequently mutated in breast carcinomas (3/35; 8.6%) and colorectal carcinomas (2/35; 5.7%) (1). The ERCC6 gene is involved in the nucleotide excision repair pathway that eliminates a broad spectrum of structural DNA lesions, including ultraviolet (UV)-induced cyclobutane pyrimidine dimers, bulky chemical adducts, and DNA cross-links (2). ERCC6 consisted of 21 exons that encode a 1493-amino acid protein. Germline mutations of ERCC6 have been reported in Cockayne’s syndrome, which is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation (3). Defects of nucleotide repair pathways are a feature of cancer cells. However, mutations of ERCC6 have not been reported in human cancers, except for in the study of Sjöblom et al. (1). The aim of this study was to confirm the presence of ERCC6 gene mutations in colorectal and breast carcinomas, and to see whether ERCC6 mutations also occur in other types of cancers.

Absence of ERCC6 gene mutation in common cancers of Korean patients

Twelve concrete prisms pretensioned with 2,400-MPa strands were fabricated to investigate the effects of initial prestress (fpi), concrete compressive strength at prestress release (fci′), 

Sug Hyung Lee

Papers

TRIO Gene Encoding Trio Rho Guanine Nucleotide Exchange Factor Harbors Frameshift Mutations of in Gastric and Colorectal Cancers.

Intratumoral heterogeneity of CSNK1G3 mutations, a casein kinase 1, in colon cancers.

O-137 Clinical results of stereotactic body frame based fractionated radiosurgery for stage I non-small cell lung cancer

Absence of ERCC6 gene mutation in common cancers of Korean patients

Prediction of Transfer Lengths Inclusive of Conventional and High-Strength Strands