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

The regulation of cell growth and survival can be subverted by a variety of genetic defects that alter transcriptional programs normally responsible for controlling cell number. High throughput analysis of these gene expression patterns should ultimately lead to the identification of minimal expression profiles that will serve as common denominators in assigning a cancer to a given category. In the course of defining the common denominators, though, we should not be too surprised to find that cancers within a single category may nevertheless exhibit seemingly disparate genetic defects. The wnt pathway has already provided an outstanding example of this. We now know of three regulatory genes in this pathway that are mutated in primary human cancers and several others that promote experimental cancers in rodents (Fig. 1). In all of these cases the common denominator is the activation of gene transcription by -catenin. The resulting gene expression profile should provide us with a signature common to those cancers carrying defects in the wnt pathway. In this review, the wnt pathway will be covered from the perspective of cancer, with emphasis placed on molecular defects known to promote neoplastic transformation in humans and in animal models.

/pdf/wnt-signaling-and-cancer-2fdsemer2h.pdf

Wnt signaling and cancer

Beneath the complexity and idiopathy of every cancer lies a limited number of 'mission critical' events that have propelled the tumour cell and its progeny into uncontrolled expansion and invasion One of these is deregulated cell proliferation, which, together with the obligate compensatory suppression of apoptosis needed to support it, provides a minimal 'platform' necessary to support further neoplastic progression Adroit targeting of these critical events should have potent and specific therapeutic consequences

Proliferation, cell cycle and apoptosis in cancer

To the Editor Hippo signaling pathway (also known as the Salvador/Warts pathway) controls organ size in animals through the regulation of cell proliferation and apoptosis that are central to tumorigenesis as well (1). Perturbation of the Hippo pathway can trigger tumorigenesis in mice and flies, and altered Hippo pathway activity has been observed in human cancers (2). However, most Hippo pathway genes are not commonly mutated in human cancers except for neurofibromin 2 (NF2) that is mutationally inactivated in cancer and is considered a tumor suppressor gene (TSG) (3). In Hippo pathway, TEA domain family member 2 (TEAD-2) is a major transcription factor that interacts with YAP to form heterodimeric transcription factors that activate proliferative and anti-apoptotic gene expressions (1). In a public genome database (http://genome.cse. ucsc.edu/), we found that human TEAD2 gene had a mononucleotide repeat 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 colorectal cancers (CRC) with MSI (4). To date, however, it is not known whether TEAD2 gene is mutationally altered in GC and CRC with MSI. To see whether the mononucleotide repeat in TEAD2 gene is mutated in GC and CRC, we analyzed a C8 repeat in the exon 9 by polymerase chain reaction (PCR)-based single strand conformation 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). Radioisotope [(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 cancer cells with mobility shifts in the SSCP as described previously (5, 6). On the SSCP, we observed aberrant bands of the TEAD2 gene in nine cancers. 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 TEAD2 somatic mutations. All of the mutations were a type of heterozygous frameshift mutation (deletion of one base) in the C8 repeat (c.883delC) that would result in a frame shifting change with histidine 295 as the first affected amino acid, changing to a methionine and cresting a new reading frame ending in a stop at

TEAD2, a Hippo pathway gene, is somatically mutated in gastric and colorectal cancers with high microsatellite instability.

Many molecules are involved in DNA fragmentation during apoptosis, but there are two major apoptotic nucleases, termed caspase-activated DNAse (CAD) and endonuclease G (EndoG) (1). EndoG is a nuclease located in mitochondria yet it translocates into the nucleus of apoptotic cells (1). EndoG in the nucleus cleaves chromatin DNA into nucleosomal fragments and participates in the apoptotic process (1–5). Experimentally, blockade of EndoG expression by SiRNA protects cells from apoptosis conditions (6). Substantial evidence indicates that alterations in the control of apoptosis contribute to the pathogenesis of many human diseases, including cancer (7). Failure of apoptosis could allow survival of transformed cells that are prone to suffer further genetic damage and play an important role in the pathogenesis of cancers. Hepatocellular carcinoma (HCC) cells are frequently resistant to various apoptosis stimuli (8). However, the exact mechanisms of apoptosis resistance in HCC cells are largely unknown. EndoG is expressed in mouse and rat hepatocytes where apoptosis stimuli translocate EndoG from mitochondria to nuclei and induce apoptosis (3, 9). Also, Hep3B, a HCC cell line, expresses EndoG, which has a cell death activity in the cells (10). Meanwhile, at the human tissue level, EndoG expression is unknown in both HCC and normal hepatocytes. Because EndoG plays important roles in the apoptosis of cells, it can be hypothesized that EndoG function might be altered by aberrant expression in human HCCs. In the present study, we analyzed EndoG expression in HCC, cirrhosis and normal liver tissues by immunohistochemistry, and found EndoG expression is frequently lost in HCCs.

Decreased expression of endonuclease G (EndoG), a pro-apoptotic protein, in hepatocellular carcinomas

To the Editor,Both endogenous (e.g. metabolism) and exogenous (e.g. ultraviolet light) factors continuously cause DNA damage in cells. To maintain genomic stability, DNA damage is repaired by a ser...

Somatic mutation of EXO1 gene in gastric and colorectal cancers with microsatellite instability

To the Editor: USP9X is a member of the peptidase C19 family that encodes a protein that is similar to ubiquitin-specific proteases. Two opposite activities (a tumor suppressor gene (TSG) and oncogenic) of TMEFF2 have been identified [1–4]. USP9X stabilizes MCL1 in human follicular lymphomas and diffuse large B-cell lymphomas and increases the survival of tumor cells [1]. USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer [2]. These data suggest oncogenic activity of USP9X in cancers. By contrast, USP9X was identified as a TSG in pancreatic ductal adenocarcinoma by ‘sleeping Beauty’ transposon-mediated insertional mutagenesis model [3]. Although previous work had attributed oncogenic roles to USP9X in human tumors [1, 2], this study found instead that loss of USP9x enhances transformation and protects pancreatic ductal adenocarcinoma cells from cell death [3]. In human cancers, low USP9X expression correlates with poor survival and treatment with 5-aza-2-primedeoxycytidine elevates USP9X expression in pancreatic ductal adenocarcinoma cell lines [3]. Also, USP9X downregulation renders breast cancer cells resistant to tamoxifen [4]. These reports [3, 4] suggest that USP9X is a TSG with prognostic and therapeutic relevance in cancers. Collectively, these studies suggest that alteration of USP9X may be tumor typedependent. However, somatic inactivating mutation status of USP9X remains undetermined in most carcinomas. There is a mononucleotide repeat (A7) in the coding sequence of USP9X that could be a target for frameshift mutation in cancers with microsatellite instability (MSI) such as colorectal cancers (CRC) [5]. To see whether USP9X gene harbored frameshift mutations within the repeat in CRC, we analyzed the A7 repeat in exon 43 in 79 CRCs with high MSI (MSI-H) and 45 microsatellite-stable/low MSI (MSS/MSI-L) CRCs by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay as described previously [6]. The MSI evaluation system used five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24 and MONO27), tumoral MSI status of which was characterized as: MSIH, if two or more of these markers show instability, MSI-L, if one of the markers shows instability and MSS, if none of the markers shows instability [7]. In cancer tissues, malignant cells and corresponding normal tissue were selectively procured by microdissection and their DNAs were used in the PCR [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, Sanger DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [6]. In the SSCP, we found aberrantly migrating bands in four CRCs with MSI-H (5.1 %) (Fig. 1), but not in their normal DNA. DNA sequencing analysis confirmed that the aberrant bands represented USP9X somatic mutations, which consisted of frameshift mutations by a deletion of one base (c.7440delA (p.Ala2481Profsx7)) and a duplication of one base (c.7440dupA (p.Ala2481Serfsx17)) within the repeat (Fig. 1). The mutations were detected in CRCs with MSIH, but not in those with MSS/MSI-L. Clinical and histopathological parameters could not distinguish USP9X mutation (+) and (−) cancers, possibly due to the small number of the mutated cases. * Sug Hyung Lee suhulee@catholic.ac.kr

USP9X, a Putative Tumor Suppressor Gene, Exhibits Frameshift Mutations in Colorectal Cancers

Many genes act as both tumor suppressor gene (TSG) and proto-oncogene depending on cellular context and cancer type. Nemo-like kinase (NLK) encoding a serine/threonine kinase, Yin Yang 1 (YY1) encoding a zinc-finger transcription factor and PA2G4 encoding an ErbB3 binding protein have both of these two opposing functions. In the present study, we analyzed NLK, YY1 and PA2G4 frameshift mutations in sporadic GC and CRC with high microsatellite instability (MSI-H). Also, regional intratumoral heterogeneity (ITH) of frameshift mutations of these genes was analyzed in CRCs. We found frameshift mutations of NLK, YY1 and PA2G4 in CRC and GC with MSI-H (17/132: 12.9%), but not in those with MSS (0/90). Two (12.5%), one (6.3%) and one (6.3%) CRC (s) of the 16 CRCs exhibited ITH of NLK, YY1 and PA2G4 mutations among the 4–7 regions, suggesting that ITH of the frameshift mutations might be frequent in the CRCs. These results suggest that frameshift mutations of NLK, YY1 and PA2G4 along with the ITH might contribute to MSI-H cancer pathogenesis.

Nam Jin Yoo

Papers

TEAD2, a Hippo pathway gene, is somatically mutated in gastric and colorectal cancers with high microsatellite instability.

Decreased expression of endonuclease G (EndoG), a pro-apoptotic protein, in hepatocellular carcinomas

Somatic mutation of EXO1 gene in gastric and colorectal cancers with microsatellite instability

USP9X, a Putative Tumor Suppressor Gene, Exhibits Frameshift Mutations in Colorectal Cancers

Somatic Mutations and Intratumoral Heterogeneity of Cancer-Related Genes NLK, YY1 and PA2G4 in Gastric and Colorectal Cancers.