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

Tumor necrosis factor (TNF)-a regulates various biological processes, including immune reaction, inflammation and apoptosis. Of two TNF receptors (TNF-R1 and R2), TNF-R1 is primarily responsible for the actions by TNF-a (1). TNF receptor family consists of more than 20 proteins, including TNF-R1, Fas, DR4 and DR5 (1). While Fas, DR4 and DR5 have main functions in cell death induction, TNF-R1 is involved in both NF-kB activation and apoptosis induction (1). Activation of TNF receptor family members is a major pathway for the induction of apoptosis in cells and tissues (1). Failure of apoptosis allows survival of transformed cells that are prone to undergo genetic damages and plays an important role in development of cancers. Somatic mutations of genes encoding TNF receptor family members, including Fas and Death Receptor 5 (DR5), have been reported in many human cancers, and many of the mutations were proven to inactivate cell death (2–6). Of note, most of the mutations were discovered in death domains (DD) of Fas andDR5. The DD of TNF receptor family members is an evolutionally conserved intracellular domain that transmits death signals from the ligands (1). Although the DD is crucial in apoptosis signaling and somatic mutations in the DD of Fas and DR5 are well known, currently the data on somatic mutations of TNF-R1 DD is lacking. In order to determine whether the DD of TNF-R1 is mutated in human cancers as well, we analyzed the exon 10 of TNF-R1gene by polymerase chain reaction (PCR) with three primer pairs and subsequent single-strand conformation polymorphism (SSCP) assay. The exon 10 contains DNA sequences that encode the DD of TNF-R1. The cancers analyzed consisted of methacarn-fixed tissues of 47 gastric adenocarcinomas, 47 colorectal adenocarcinomas, 47 breast invasive ductal carcinomas, 47 hepatocellular carcinomas and 47 non-small cell lung cancers, and non-fixed fresh tissues of 47 acute myelogenous leukemias (subgroup details available on request). Malignant cells and normal cells of the solid cancers were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection (2). Genomic DNA was extracted by a modified single-step method using proteinase K. Genomic DNA each from tumor and normal cells were amplified by PCR, and the PCR products were subsequently displayed on SSCP gels. 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 (2). On the SSCP, all of the PCR products from the cancers were clearly seen, but they did not reveal any aberrantly migrating band compared with wild-type bands from the normal tissues, indicating there was no evidence of somatic mutations in the DD of TNF-R1 in the cancers. We directly sequenced the PCR products of normal and cancers in five cases, but there was no sequence alteration. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure the specificity of the results, and found that the data were consistent. As a possible mechanism of apoptosis inactivation in common cancers, we analyzed TNF-R1 DD mutation in gastric, colorectal, breast, hepatocellular and lung carcinomas, and acute myelogenous leukemias. However, we detected no TNF-R1 mutation in the cancer tissues. This is in contrast to the high incidences of Fas DD mutations in lung cancers (7.7%), bladder cancers (23.3%) and multiple myelomas (10.4%), and DR5 DD mutations in lung cancers (8.7%) and breast cancers (10.5%) (2–6). Our data indicate that apoptosis resistance frequently observed in gastric, colorectal, lung, hepatocellular and breast cancers, and acute myelogenous leukemias may not be a result of TNF-R1 DD mutation in the cancer cells. The reason why we could not find any TNF-R1 DD mutation might be that the primary effect of the TNF-R1 receptor is activation of survival pathways through NF-kB activation (1), and not activation of apoptosis pathways. Apoptosis of cancer cells can be delayed or blocked by several APMIS 117: 547–548

Somatic mutation of the death domain of TNF-R1 is rare in common cancers.

sisted of 105 cases of AML, 54 cases of ALL (43 B-ALL and 11 T-ALL) and 1 undifferentiated acute leukemia. The AML samples included 8 cases of AML minimally differentiated, 15 cases of AML without maturation, 21 cases of AML with maturation, 7 acute myelomonocytic leukemia samples, 7 acute monoblastic and monocytic leukemia samples, 2 acute erythroid leukemia samples, 15 cases of AML with t(8; 21)(q22;q22), 6 cases of AML with abnormal bone marrow eosinophils Inv(16) (p13; q22), 12 acute promyelocytic leukemia samples [AML with t(q22;q12)], 11 AML samples with multilineage dysplasia and 1 AML and myelodysplastic syndrome, therapy-related according to the WHO classification. The acute leukemia DNA samples were extracted from bone marrows of acute leukemia patients (age range 20–80 years). All of the patients were Asians (Korean). Approval for this study was obtained from the institutional review board, College of Medicine, Catholic University of Korea. Most of the FBXW7 mutations have been reported within the exon 8–9 [3] . Thus, we analyzed FBXW7 gene in these 2 exons. Genomic DNA was isolated and analyzed for potential mutations in the FBXW7 exon 8–9 by polymerase chain reaction (PCR) with primer pairs ( table 1 ). Radioisotope ( 32 P-dCTP) was incorporated into PCR products for detection by autoradiogram. The PCR products were subsequently analyzed by a single-strand conformation polymorphism (SSCP) analysis, as described previously [10, 11] . On SSCP autoradiogram, all of the PCR products were clearly seen. However, the SSCP from the acute leukemia Cell cycle deregulation is important in the pathogenesis of human cancers. Cyclin E couples with Cdk2 and is involved in the G1 progression of the cell cycle. Degradation of cyclin E depends on ubiquitin-mediated proteolysis by the SCF ubiquitin ligase complex. FBXW7 (also known as hCDC4) is a component of the SCF complex [1, 2] . Inactivation of FBXW7 causes an increased expression of cyclin E and chromosomal instability of the affected cells [2, 3] . In a mouse model, the loss of 1 FBXW7 allele promoted tumor transformation, suggesting that FBXW7 gene may be a haploinsufficient tumor suppressor gene [4] . FBXW7 gene was found to be somatically mutated in several human tumors, including endometrial, colorectal and ovarian tumors (2.9–16% of the endometrial carcinomas, 13% of the colorectal tumors and 2% of the ovarian carcinomas) [1, 3, 5, 6] . Mutational analyses of FBXW7 have been performed in acute leukemia samples [7–9] . FBXW7 mutation was detected in both pediatric and adult T cell acute lymphoblastic leukemia (T-ALL), but neither in B-ALL nor in acute myelogenous leukemia (AML) [7–9] . Additionally, CCRF-CEM, a cell line derived from T-ALL, contains a FBXW7 mutation (R465C) [1] , suggesting that FBXW7 mutation may play a role in the pathogenesis of T-ALL. However, because all of the FBXW7 mutational analyses were performed in Western countries, we would like to know whether the pattern of FBXW7 mutation in acute leukemia in an Asian country is similar to that in Western countries. In this study, we selected 160 acute leukemia samples and analyzed the FBXW7 mutation. The samples conReceived: July 17, 2007 Accepted after revision: August 30, 2007 Published online: November 9, 2007

FBXW7 gene mutation is rare in acute leukemia samples of Korean patients.

dulthood AML 206 206 0 (0) dulthood ALL 156 156 0 (0) hildhood AML 21 21 0 (0) hildhood ALL 201 200 1 (0.5) ultiple myeloma 75 75 0 (0) yelodysplasia 68 68 0 (0) on-Hodgkin lymphoma 105 105 0 (0) on-small cell lung cancer 208 208 0 (0) astric carcinoma 201 201 0 (0) olorectal carcinoma 417 414 3 (0.7) reast carcinoma 81 81 0 (0) rostate carcinoma 263 263 0 (0) varian epithelial tumours 15 15 0 (0) varian granulosa cell tumours 69 68 1 (1.5) epatocellular carcinomas 36 36 0 (0) epatoblastomas 20 20 0 (0) sophageal squamous cell carcinomas 61 61 0 (0) aryngeal squamous cell carcinomas 44 45 0 (0) eiomyoma 68 68 0 (0) astrointestinal stromal tumours 20 20 0 (0) alignant peripheral nerve sheath mour 22 21 1 (4.5)

Mutational analysis of H3F3B gene in acute leukaemias and solid tumours.

Promoter mutation in long non-coding RNA NEAT1 is not common in common solid cancers.

cell lymphomas with immunophenotype similar to mantle cell lymphoma but negative for Cyclin D1 and t(11;14) are best classified as CD5 B-cell chronic lymphoproliferative disorders (B-CLPD) or atypical chronic lymphocytic leukemia (aCLL). A French study [2] segregated CD5-positive, Cyclin D1-negative B-CLPD into 2 groups based on the intensity of CD20. Patients with dim CD20 profile represent a homogeneous subgroup very close to the B-CLL on morphologic, immunophenotypic and cytogenetic criteria. In contrast, they found the group expressing bright CD20 to be heterogeneous with a more aggressive course and non CLL like profile. Our patient’s lymphoma demonstrated bright CD20 and no typical cytogenetic abnormalities for CLL. The presence of bone marrow and peripheral blood involvement just a year after initial presentation (leukemic phase) demonstrates the aggressiveness of his disease. This aggressive nature may also be attributed to the atypical co-expression of CD10 by the lymphoma [3]. This case demonstrates that further studies are required to properly classify and determine prognosis of CD5 non mantle cell, non CLL B-CLPD’s and that provisions should be made in the WHO classification to accommodate these aberrant cases. Although a causal relationship between B-cell lymphoma and Hepatitis B virus has not been described to date, the possibility of chronic HBV infection as a trigger for monoclonal B-cell proliferation due to chronic antigenic stimulation may not be entirely speculative.

Nam Jin Yoo

Papers

Somatic mutation of the death domain of TNF-R1 is rare in common cancers.

FBXW7 gene mutation is rare in acute leukemia samples of Korean patients.

Mutational analysis of H3F3B gene in acute leukaemias and solid tumours.

Promoter mutation in long non-coding RNA NEAT1 is not common in common solid cancers.

Somatic mutation of TRAF3 gene is rare in common human cancers and acute leukemias