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.

/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

Recurrent missense somatic mutations in DICER1 gene affecting exons 25 and 26 have recently been reported in non-epithelial tumors of ovary, especially in Sertoli-Leydig cell tumors (SLCT). Such a recurrent nature of the mutations strongly suggests that DICER1 mutation may play an important role in the development of ovarian non-epithelial tumors. The aim of this study was to further characterize the DICER1 mutations in human tumor tissues. We analyzed the DICER1 mutations in 2,819 tumor tissues from ovary and other organs by single-strand conformation polymorphism analysis. We found the DICER1 exon 25 and 26 hot-spot mutations in 2 of 14 SLCT (14.3%) and one of 132 non-Hodgkin lymphomas (0.8%), but none in other tumors. In SLCT, DICER1 expression was observed in Sertoli cells, but neither in Leydig nor spindle-shaped cells. We also analyzed DICER1 expression in lung, stomach, colon and prostate cancer tissues. DICER1 expression was lost in 37% of prostate cancers, but not in lung, stomach and colon cancers. Our data indicate that DICER1 recurrent mutations commonly occur in SLCT, but the incidence was significantly lowerthan that in the earlier study. Our data suggest that both DICER1 mutation and its expression loss may be important in tumor pathogenesis and that they are tumor type-specific. Citation Format: Min Sung Kim, Eun Mi Je, Youn Jin Choi, Nam Jin Yoo, Sug Hyung Lee. Mutational and expressional analyses of DICER1 gene in ovarian stromal tumors and other common tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2013-1921

Abstract 1921: Mutational and expressional analyses ofDICER1gene in ovarian stromal tumors and other common tumors.

Inactivating mutations of tumor suppressor genes ABCA1 and CAPN13 in colorectal cancers

Mutational and expressional alterations of a candidate tumor suppressor HECA gene in gastric and colorectal cancers.

Sequestosome 1 (p62/SQSTM1) contains multiple domains that interact with key components in cellular processes and signaling, including autophagy, nuclear factor-κB (NF-κB) activation, and oxidative stress signaling.1 p62 behaves as an adaptor between autophagic machinery and ubiquitinated proteins and promotes autophagic degradation of ubiquitinated targets. Altered autophagy is accompanied by p62 accumulation and ubiquitinated proteins.1 p62 activates NF-κB signaling through binding with TRAF6 and RIP1. Additionally, by interacting with the Nrf2 binding site in Keap1, p62 competitively inhibits the Nrf2-Keap1 interaction, thereby activating the transcription of Nrf2 target genes that induces antioxidant responses.1 With respect to disease involvement, the p62-encoding gene is frequently mutated in Paget's disease of bone.1,2 With respect to cancer, p62 is involved in many signaling pathways whose functions are important in cancer development. p62 acts either as a tumor suppressor (via the Wnt pathway) or a pro-oncogenic protein (via the NF-κB and mammalian target of rapamycin pathways).1,2 p62 is highly expressed in breast, lung, gastric, colon, esophageal, and hepatocellular carcinomas and is associated with poor cancer patient prognosis.3-6 In contrast, a recent study reported that p62 was downregulated in colon carcinomas.7 The aim of our study was to confirm whether p62 expression is altered in gastric and colorectal cancers and to address whether it is altered in other cancers.

To determine whether alterations of p62 expression are present in other human cancers, we analyzed the expression of p62 in gastric cancer (GC), colorectal cancer (CRC), and prostate cancers (PCa) by immunohistochemistry using 11 tissue microarray (TMA) blocks. Cases of CRC (n=103) originated from the cecum (n=2), ascending colon (n=19), transverse colon (n=6), descending colon (n=4), sigmoid colon (n=28), and rectum (n=44). Cases of GC (n=100) consisted of 50 diffuse, 36 intestinal, and 14 mixed-type GC by Lauren classification, as well as four early GC and 96 advanced GC cases according to the depth of invasion. The PCa cases (n=107) consisted of one case with a Gleason score of 5, 10 with a score of 6, 47 with a score of 7, 10 with a score of 8, and 39 with a score of 9. For immunohistochemistry, we used the ImmPRESS System (Vector Laboratories, Burlingame, CA, USA) with a rabbit polyclonal antibody against human sequestosome 1 (Thermo Scientific, Rockford, IL, USA; dilution 1/2,000). By visual inspection under a microscope, we graded the immunoreactivity as -, +, or ++. Tumor and normal tissues were interpreted as positive by immunohistochemistry when they scored either + or ++. Other procedures for mutation and immunohistochemistry have been described in our previous reports.8,9 In the immunohistochemistry analysis, normal gastric mucosal epithelial, colonic mucosal epithelial, and prostate glandular cells exhibited positive p62 immunostaining in all cases (Fig. 1). All of the cases exhibited ++ immunopositivity. In the tumors, immunopositivity for p62 was observed in 97 cases (+, three; ++, 94 cases) of GC, 97 cases (+, two; ++, 95 cases) of CRC, and 100 cases (+, two; ++, 98 cases) of PCa. There was no significant difference in the immunopositivity between normal and GC, CRC, and PCa tumor tissues (Fisher exact test, p>0.05). There was no significant difference in the immunopositivity between the GC, CRC, and PCa tissues (Fisher exact test, p>0.05). There was no association between p62 expression and clinicopathological parameters, including Gleason score, invasion/metastasis, and TNM stage (chi-square test, p>0.05). Because p62 is involved in diverse cancer-related signaling, p62 expression has been studied in many cancers. However, our results demonstrated that p62 was expressed well in both normal and tumor cells of GC and CRC tissues and that there was no difference in the expression with respect to the clinicopathological data. Our data from Korean patients are in agreement with earlier data that demonstrated that both normal and cancer cells in gastric and colon cancers from Chinese patients expressed p62.6 However, these data are different from other data obtained in a Canadian population that demonstrated decreased expression of p62 in colon cancers compared with the high expression of p62 in normal colonic epithelial cells.7 One possibility for the disagreement may be ethnic differences (Asian vs Caucasian). Another explanation may be the method we used. Many researchers are concerned about the potential bias in TMAs, mostly due to the heterogeneity of expression patterns in TMA. One way to reduce bias in TMAs is to confirm the data in conventional large tissue sections. To validate the TMA data, we further analyzed the expression in another series of 20 additional colorectal adenocarcinomas and their normal mucosa tissue sections that were more than 1 cm2 in size and found that p62 was similarly expressed in the tumors (95%) and in the normal mucosa tissues (positive immunostaining in all cases). We also found that p62 expression was not altered in PCa. Our data indicate that alteration of p62 expression is not a common feature in all cancers and that p62 alterations may be tissue-specific.



Fig. 1

Visualization of p62 expression in gastric, colorectal, and prostate cancer tissues by immunohistochemistry. (A) Normal gastric epithelial cells exhibit positive immunostaining. (B) In a case of gastric cancer, the cancer cells exhibit p62 immunostaining. ...

Alteration of p62/SQSTM1 Expression Is Uncommon in Gastrointestinal and Prostate Cancer Tissues

To the editor,

Human MITOSTATIN gene encodes a mitochondrial protein, which promotes apoptosis and inhibits cell growth.1 The MITOSTATIN gene is somatically mutated in cancer cell lines (gastric, prostate and pancreas cell lines) and is down-regulated in bladder and breast cancer tissues.1 These data suggest that MITOSTATIN gene may be a tumor suppressor gene, inactivation of which plays an important role in cancer development. Although somatic mutations of MITOSTATIN gene have been identified in cancer cell lines, its mutation status has not yet been studied in cancer tissues. Frameshift mutations of genes containing mononucleotide repeats are features of gastric (GC) and colorectal carcinomas (CRC) with microsatellite instability (MSI).2 Many cancer-associated genes, such as TGFBR2, BAX, IGFR2 and TCF4, have been found to harbor mutations at mono- or dinucleotide repeats in the coding sequences in these cancers with MSI.2 There is a polyadenine tract (A8) in the exon 5 of the MITOSTATINgene, but the frameshift mutation at this site is not known.

To see whether the mononucleotide repeat A8 in the exon 5 of MITOSTATIN gene is mutated in GC and CRC, we analyzed the exon 5 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. For the mutation analysis, methacarn-fixed tissues of 42 GC and 51 CRC with MSI were used in this study. These cancers consisted of 31 GC with high MSI (MSI-H), 11 GC with low MSI (MSI-L), 37 CRC with MSI-H and 14 CRC with MSI-L. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator.3 Genomic DNA each from tumor cells and corresponding normal cells were amplified with a primer pair by PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. After SSCP, migration of the PCR products on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. Other procedures of the PCR and SSCP were described in our previous studies.3,4

On the SSCP, all of the PCR products from the cancers were clearly seen. The PCR-SSCP analysis identified aberrant bands in one (3.2%) of the 31 GC with MSI-H, but not in the other cancers. DNA from normal tissues from the same patients showed no evidence of the mutation in SSCP, indicating the mutations had risen somatically. A direct DNA sequence analysis of the GC with the aberrant SSCP bands led to identification of a MITOSTATIN deletion mutation within the A8 repeat sequences (Fig. 1). The mutation was c.522delA, which would result in premature stops of the amino acid synthesis (p.Lys174AsnfsX26). We repeated the experiments twice, including PCR, SSCP and DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent.



Fig. 1

Frameshift mutation of MITOSTATIN in a gastric carcinoma with microsatellite instability. Direct DNA sequence analysis of MITOSTATIN exon 5 from a gastric adenocarcinoma shows a heterozygous A deletion in tumor tissue relative to normal tissue.



Failure of apoptosis allows survival of cells and plays an important role in development of cancers.3 As a mechanism of apoptosis resistance in cancer cells, somatic mutations of pro-apoptosis genes have been reported in many human cancers, and many of the mutations were proven to inactivate cell death.3,5 Because the main tumor suppressor function of MITOSTATIN appears to depend on apoptosis activity, we analyzed frameshift mutation of MITOSTATIN gene at a mononucleotide repeat that may inactivate the apoptosis. However, we detected only one MITOSTATIN mutation in the cancers with MSI, indicating that the mutation at the repeat is rare in GC and CRC with MSI. In the earlier study1 that first disclosed tumor suppressor functions of MITOSTATIN, the authors found that loss of expression and mutation of the gene in solid tumors and cell lines, respectively. Our and the earlier data1 suggest that MITOSTATIN may be inactivated by various mechanisms. To see whether loss of function of MITOSTATIN is a feature of human cancers, the status of MITOSTATIN gene should be further analyzed at various levels in many cancer types.

Nam Jin Yoo

Papers

Abstract 1921: Mutational and expressional analyses ofDICER1gene in ovarian stromal tumors and other common tumors.

Inactivating mutations of tumor suppressor genes ABCA1 and CAPN13 in colorectal cancers

Mutational and expressional alterations of a candidate tumor suppressor HECA gene in gastric and colorectal cancers.

Alteration of p62/SQSTM1 Expression Is Uncommon in Gastrointestinal and Prostate Cancer Tissues

Mutational Analysis of MITOSTATIN, a Candidate Tumor-Suppressor Gene, at a Mononucleotide Repeat in Gastric and Colorectal Carcinoma.