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

Apoptosis is a principal type of cell death, and it has a profound effect on the development of cancer. It is also well known that anti-cancer agents induce apoptosis, and defects in the apoptosis pathways reduce the treatment sensitivity. Of the many pathways that induce apoptosis, the mechanisms of the intrinsic and extrinsic apoptosis pathways are well established. Non-small cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, yet the exact molecular mechanisms of its development remain unclear. Apoptosis deregulations may underlie the development and pathogenesis of NSCLC. This review discusses the general mechanisms of apoptosis, the constituents of the apoptosis machinery and the alterations of the apoptosis-related genes in NSCLC. (J Lung Cancer 2008;7(2):59�� 64)

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Alterations of the Apoptosis Genes and Their Products in Non-small Cell Lung Cancer Tissues

Purpose: In a recent study of Wilms' tumors, a new X chromosome gene, Wilms' tumor gene on the X chromosome (WTX), was discovered that was found to harbor small deletions and point mutations. The WTX protein negatively regulates Wnt/ β-catenin signaling, and is considered to be a tumor suppressor gene. One of the questions about the WTX gene is whether the genetic alterations of the WTX gene are specific only to Wilms' tumors. The aim of this study was to explore whether the WTX gene mutation is a characteristic of human non-small cell lung cancer (NSCLC). Materials and Methods: In the current study, we analyzed the part of the WTX gene encoding the N-terminal of WTX, where most of the WTX point mutations have been detected in Wilms' tumors. Forty-eight NSCLC tissues were analyzed by a single-strand conformation polymorphism assay and DNA sequencing. Results: SSCP analysis revealed no evidence of somatic mutations in the DNA sequences encoding the N-terminal of the WTX gene in the 48 NSCLC tissues. Conclusion: The data presented here indicate that the WTX gene may not be somatically-mutated in human NSCLCs, and suggest that NSCLCs may not utilize mutational events of the WTX gene in the process of pathogenesis. (J Lung Cancer 2008;7(1):22�� 24)

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Mutational Analysis of the Tumor Suppressor WTX Gene in Non-small Cell Lung Cancer

To the Editor To identify new cancer genes implicated in breast cancer development, Stephens et al. (1) analyzed whole genomes of 100 breast cancers by next-generation sequencing. Somatic driver substitutions and small insertions/deletions (indels) were identified in cancer genes previously implicated in breast cancer development, including AKT1, BRCA1, CDH1, GATA3, PIK3CA, PTEN, RB1, and TP53 (1). In addition, they found new cancer genes, including TBX3 (1). TBX3 is a member of the T-box family of genes that encode developmentally important transcription factors (2). TBX3 plays important roles in development of several organs, including heart, limb, and mammary gland. In the report of Stephens et al. (1), somatic mutations of TBX3 were observed in 6% of the breast cancers (6/100 cases). Of note, all of the TBX3 mutations were recurrently found in exon 2, encoding the DNA-binding domain. The TBX3 mutations consisted of two c.635_637delACA, one c.487_488insT, one c.629_631delCCA, one c.489T > A, and one c.591G > A. In addition to the somatic mutations, increased levels of TBX3 have been identified in many cancers, including melanoma, breast, liver, bladder, and pancreatic cancers (3–5), suggesting a possibility that TBX is important in many cancers. To see whether TBX3 exon 2 mutation is widespread as well, we analyzed the mutation in a large number of human tumors from various origins by polymerase chain reaction (PCR)-based singlestrand conformation polymorphism (SSCP) assay in this study. For this, 2218 tumor tissues from Korean patients were randomly collected and used for this study. They consisted of breast carcinomas (N = 93), gastric carcinomas (N = 187), colorectal carcinomas (N = 401), prostate carcinomas (N = 275), non-small cell lung cancers (N = 149), esophageal squamous cell carcinomas (N = 67), laryngeal squamous cell carcinomas (N = 30), basal cell carcinomas of skin (N = 17), squamous cell carcinomas of skin (N = 8), hepatocellular carcinomas (N = 27), hepatoblastomas (N = 26), renal cell carcinomas (N = 30), ovarian epithelial tumors (N = 51), ovarian stromal tumors (N = 59), meningiomas (N = 30), uterine leiomyomas (N = 68), gastrointestinal stromal tumors (N = 20), sarcomas (N = 82), acute leukemias (N = 423), nonHodgkin lymphoma (N = 107), and myelodysplasias (N= 68). For the solid tumors, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection as described previously (6). Approval for this study was obtained from the Catholic University of Korea, College of Medicine’s institutional review board. Genomic DNA each from tumor cells and normal cells of the same patients were amplified by PCR with two primer pairs (5′-TTTTATAGGCGAATGTT TCC -3′ and 5′-C TGTCCGGGTGAATGTA3′; 5′CGAAATG CCAAAGAGGATG-3′ and 5′-TTGACTGA GTGAAGGAGGAGAA-3′) that encompassed the exon 2. Radioisotope [(P)dCTP] was incorporated into the PCR products for detection by autoradiogram. Other procedures of the PCR-SSCP were described in our previous studies (6). After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and sequenced by Sanger sequencing (ABI Prism Genetic Analyzer; Applied Biosystem, Foster City, CA, USA). For breast cancers that had been reported to harbor TBX3 mutations, we performed direct DNA sequencing as well for the detection of TBX3mutations. PCR and subsequent SSCP analyses identified three tumors with aberrantly migrating bands in SSCP (Fig. 1). None of the corresponding normal samples from the same patients showed evidence of mutations by SSCP, indicating that the mutations had risen somatically. DNA sequencing analysis of the aberrant bands led us to identify that these aberrant bands represented TBX3 mutations (Fig. 1). One of the TBX3 mutations (c.558delT (p.His187fsx21)) was detected in a prostate carcinoma. The other (c.604G > A (p.Val202Ile) was recurrently detected in two non-Hodgkin lymphomas (diffuse large B-cell lymphomas). Also, we repeated the mutational screening by a direct sequencing in the breast

Mutations in exon 2 of TBX3 are rare in common human tumors.

To the Editor: Protein kinases play an important role in the regulation of normal cellular functions, and alterations of the kinases contribute to cancer development (1). Recently, Parsons et al. (2) analyzed 340 serine/threonine kinase genes in 204 colorectal cancer tissues for the detection of somatic mutations. They identified 23 mutations in eight genes. Of them, five genes (AKT2, MYLK2, MAP2K4, PDPK1, MARK3, and PAK4) were mutated in more than one tumor. AKT, known as protein kinase B, represents a subfamily of the serine/ threonine protein kinases, and three isoforms of them have been identified, AKT1, 2 and 3 (3). AKTs are major downstream targets of growth factor receptor tyrosine kinases (3). Myosin light chain kinase 2, skeletal muscle (MYLK2) is a serine/threonine kinase that is exclusively expressed in skeletal and cardiac muscles, but the function of this protein in the cell is largely unknown (4). The presence of somatic mutations of AKT2 and MYLK2 in colorectal cancers (2) led us to analyze as to whether acute leukemias also harbor the mutations of these kinase genes. Leukemia DNA samples were extracted from the bone marrow of 143 acute leukemia patients (age range 20– 80 years). The leukemias consisted of eight acute myelogenous leukemias (AMLs) minimally differentiated, 16 AMLs without maturation, 32 AMLs with maturation, 10 acute myelomonocytic leukemias, seven acute monoblastic and monocytic leukemias, one AML with t (8;21) (q22;q22), two acute erythroid leukemias, 14 acute promyelocytic leukemias (AML with t (15;17) (q22;q12), 12 AMLs with multilineage dysplasia, one acute myeloid leukemia and myelodysplastic syndrome, 14 biphenotypic acute leukemias, two T-cell acute lymphoblastic leukemias (ALL) and 24 B-cell ALLs according to the WHO classification. Both G-loop and activation segment in the kinase domains are frequentlymutated in protein kinases in human cancers (1, 2). Also, the previous study identifiedAKT2mutations in the exon 11 encoding a part of the kinase domain (2). Thus, we analyzed the exon 6 encoding the G-loop, the exon 10 encoding the activation segment and the exon 11 in the AKT2 gene. In the case of MYLK2 gene, we analyzed the exons 6 and 7, because the MYLK2 kinase domain mutations in the colorectal cancers were detected only within the exons 6 and 7 in the previous study (2). Genomic DNA each from tumor cells and normal cells were amplified by polymerase chain reaction (PCR). Radioisotopewas incorporated into the PCR products for detection by single-strand conformation polymorphism (SSCP) autoradiogram. Other procedures of PCR and SSCP analysis were performed as described previously (5). On the SSCP autoradiograms, all of the PCR products were clearly seen. However, the SSCP from the whole samples analyzed did not reveal any aberrantly migrating band compared with the wildtype bands from the normal tissues. To confirm the SSCP results, we repeated the experiments twice, including PCR, SSCP and direct DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent. Because genetic alterations of protein kinaseencoding genes profoundly contribute to the development of cancers (1), we tried to find MYLK2 and AKT2 mutations in acute leukemia in the present study. However, we detected no mutation of these genes in the samples, suggesting that the kinase domain mutation of AKT2 and MYLK2 is rare in acute leukemias. Protein kinase mutations could be the therapeutic targets in the treatment of cancer patients. However, the present data suggest a low possibility of targeting MYLK2 and AKT2 kinase domain mutations in the treatment of acute leukemias.

Absence of the mutation of serine/threonine kinase genes AKT2 and MYLK2 in acute leukemias.

To the Editor Protein tyrosine phosphorylation is crucial for many cellular processes, including cell growth, differentiation, and survival. Protein phosphorylation is activated by protein tyrosine kinases but switched off by protein tyrosine phosphatases (PTPs) [1]. Although many of the PTPs are considered to have tumor suppressor functions, PTPN11 (also known as SHP2) shows oncogenic activities [1]. Germline mutations of PTPN11 are associated with LEOPARD syndrome and Noonan syndrome, which are characterized by multiple congenital anomalies [1]. Somatic mutations of PTPN11 are observed in many hematologic neoplasia, including juvenile myelomonocytic leukemia (~35%), B-cell acute lymphoblastic leukemia (~7%), acute myelogenous leukemia (~5%), myelodysplastic syndromes (MDS) (1–5%), and chronic myelomonocytic leukemia (CMML) (~2%) [1]. Somatic mutations of PTPN11 in these tumors are localized in N-SH2 or PTP domain, structural alterations of which disrupt autoinhibitory interface and generate activated PTPN11 [1]. However, some PTPN11 mutations identified in Noonan syndrome and leukemias have not been detected in MDS [1]. In Korean population, subtype prevalence of MDS is different from those in Western populations [MDS without ring sideroblast (RS) is far less common than that of MDS with RS] [2]. These data suggest a possibility that somatic PTPN11 mutations in MDS might harbor different spectrum in Korean patients. In this study, we analyzed PTPN11 exon 3 encoding the N-SH2 and exon 13 encoding the PTP for the detection of somatic mutations using genomic DNA from in fresh bone marrow (BM) aspirates of 61 MDS, 3 CMML, 1 MDS/ myeloproliferative disorder, and 1 MDS-related acute myelogenous leukemia by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. DNAs were extracted from the whole BM aspirates of the patients. The DNA samples from the same patients were extracted from the BMs after complete remission if available and used as possible normal genomic DNA samples of the same patients. Ages of the MDS patients ranged from 16 to 69 years with an average of 50.7 years. The MDS patients included 2 refractory anemias (RA), 1 RA with RS (RARS), 29 RA with multilineage dysplasia (RCMD), 12 RA with excess blasts-1 (RAEB-1), 11 RAEB-2, and 6MDS unclassified. Of these, six (one RARS and five RCMD-RS with >15% RSs) were considered MDS with RS, whereas the other 55 were considered

Nam Jin Yoo

Papers

Alterations of the Apoptosis Genes and Their Products in Non-small Cell Lung Cancer Tissues

Mutational Analysis of the Tumor Suppressor WTX Gene in Non-small Cell Lung Cancer

Mutations in exon 2 of TBX3 are rare in common human tumors.

Absence of the mutation of serine/threonine kinase genes AKT2 and MYLK2 in acute leukemias.

Novel oncogenic PTPN11 mutations in myelodysplastic syndrome in Korean patients