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.

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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.

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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

Somatic mutation of CYLD gene is rare in hematologic and solid malignancies.

A scaffold protein DAB2 and its interaction partner DAB2IP have putative tumor suppressor gene (TSG) functions. Previous studies identified that both DAB2 and DAB2IP genes were inactivated by promoter hypermethylation in human cancers, but their mutational alterations in cancers remain largely unknown. The aim of our study was to find whether DAB2 and DAB2IP were mutated in gastric (GCs) and colorectal cancers (CRCs) by DNA sequencing. Both DAB2 and DAB2IP have mononucleotide repeats in their coding sequence that could be mutation targets in high microsatellite instability (MSI-H) cancers. We analyzed GC and CRC tissues and found that 8 of 34 GCs (23.5%) and 15 of 79 CRCs (20.0%) with MSI-H harbored DAB2IP frameshift mutations. DAB2 frameshift mutations were found in 2 of 79 CRCs (2.5%) with MSI-H. These mutations were not detected in microsatellite stable (MSS) cancers. We also found intratumoral heterogeneity (ITH) of DAB2IP frameshift mutations in 7 of 16 CRCs (43.8%). Loss of DAB2IP protein expression was found in approximately 20% of GCs and CRCs irrespective of MSI and DAB2IP frameshift mutation status. Our study shows that the TSG DAB2IP harbored frameshift mutations and ITH as well as expression loss. Together these tumor alterations might play a role in tumorigenesis of GC and CRC with MSI-H by down-regulating the tumor-inhibiting activities of DAB2IP.

DAB2IP with tumor-inhibiting activities exhibits frameshift mutations in gastrointestinal cancers.

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.

Nam Jin Yoo

Papers

Somatic mutation of CYLD gene is rare in hematologic and solid malignancies.

DAB2IP with tumor-inhibiting activities exhibits frameshift mutations in gastrointestinal cancers.

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