Kei Koizumi

Human Breast Cancer Tissues Contain Abundant Phosphatidylcholine(36∶1) with High Stearoyl-CoA Desaturase-1 Expression

Breast cancer is the leading cause of cancer and mortality in women worldwide. Recent studies have argued that there is a close relationship between lipid synthesis and cancer progression because some enzymes related to lipid synthesis are overexpressed in breast cancer tissues. However, lipid distribution in breast cancer tissues has not been investigated. We aimed to visualize phosphatidylcholines (PCs) and lysoPCs (LPCs) in human breast cancer tissues by performing matrix assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a novel technique that enables the visualization of molecules comprehensively. Twenty-nine breast tissue samples were obtained during surgery and subjected to MALDI-IMS analysis. We evaluated the heterogeneity of the distribution of PCs and LPCs on the tissues. Three species [PC(32∶1), PC(34∶1), and PC(36∶1)] of PCs with 1 mono-unsaturated fatty acid chain and 1 saturated fatty acid chain (MUFA-PCs) and one [PC(34∶0)] of PCs with 2 saturated fatty acid chains (SFA-PC) were relatively localized in cancerous areas rather than the rest of the sections (named reference area). In addition, the LPCs did not show any biased distribution. The relative amounts of PC(36∶1) compared to PC(36∶0) and that of PC(36∶1) to LPC(18∶0) were significantly higher in the cancerous areas. The protein expression of stearoyl-CoA desaturase-1 (SCD1), which is a synthetic enzyme of MUFA, showed accumulation in the cancerous areas as observed by the results of immunohistochemical staining. The ratios were further analyzed considering the differences in expressions of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki67. The ratios of the signal intensity of PC(36∶1) to that of PC(36∶0) was higher in the lesions with positive ER expression. The contribution of SCD1 and other enzymes to the formation of the observed phospholipid composition is discussed.

Increased Polyamine Intake Inhibits Age-Associated Alteration in Global DNA Methylation and 1,2-Dimethylhydrazine-Induced Tumorigenesis

Polyamines (spermine and spermidine) play many important roles in cellular function and are supplied from the intestinal lumen. We have shown that continuous high polyamine intake inhibits age-associated pathologies in mice. The mechanism by which polyamines elicit these effects was examined. Twenty-four week old Jc1:ICR male mice were fed one of three experimental chows containing different polyamine concentrations. Lifetime intake of high polyamine chow, which had a polyamine content approximately three times higher than regular chow, elevated polyamine concentrations in whole blood, suppressed age-associated increases in pro-inflammatory status, decreased age-associated pathological changes, inhibited age-associated global alteration in DNA methylation status and reduced the mortality in aged mice. Exogenous spermine augmented DNA methyltransferase activity in Jurkat and HT-29 cells and inhibited polyamine deficiency-induced global alteration in DNA methylation status in vitro. In addition, increased polyamine intake was associated with a decreased incidence of colon tumors in BALB/c mice after 1,2-demethylhydrazine administration; 12 mice (60%) in the low polyamine group developed tumors, compared with only 5 mice (25%) in the high polyamine group (Fishers exact probability = 0.027, p = 0.025). However, increased polyamine intake accelerated the growth of established tumors; maximal tumor diameter in the Low and High groups was 3.85±0.90 mm and 5.50±1.93 mm, respectively (Mann-Whitney test, p = 0.039). Spermine seems to play important roles in inhibiting age-associated and polyamine-deficient induced abnormal gene methylation as well as pathological changes including tumorigenesis.

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability

BackgroundRecent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.MethodsWith an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.ResultsUnsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3′ end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).ConclusionsSomatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Array-based identification of common DNA methylation alterations in ulcerative colitis.

Patients with long-standing ulcerative colitis (UC) have higher risk of developing colorectal cancer. Albeit the causes remain to be understood, epigenetic alterations have been suggested to play a role in the long-term cancer risk of these patients. In this work, we developed a novel microarray platform based on methylation-sensitive amplified fragment length polymorphism (MS-AFLP) DNA fingerprinting. The over 10,000 NotI sites of the human genome were used to generate synthetic primers covering these loci that are equally distributed into CpG rich regions (promoters and CpG islands) and outside the CpG islands, providing a panoramic view of the methylation alterations in the genome. The arrays were first tested using the colon cancer cell line CW-2 showing the reproducibility and sensitivity of the approach. We next investigated DNA methylation alterations in the colonic mucosa of 14 UC patients. We identified epigenetic alterations affecting genes putatively involved in UC disease, and in susceptibility to develop colorectal cancer. There was a strong concordance of methylation alterations (both hypermethylation and hypomethylation) shared by the cancer cells of the CW-2 cell line and the non-cancer UC samples. To the best of our knowledge, this work defines the first high-throughput aberrant DNA methylation profiles of the colonic mucosa of UC patients. These epigenetic profiles provide novel and relevant knowledge on the molecular alterations associated to the UC pathology. Some of the detected alterations could be exploited as cancer risk predictors underlying a field defect for cancerization in UC-associated carcinogenesis.

The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals

Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers.

Aberrant methylation of PSD disturbs Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis in ulcerative colitis-associated carcinogenesis

We previously reported that the Pleckstrin and Sec7 domain-containing (PSD) gene is preferentially methylated in patients with ulcerative colitis (UC) who developed colorectal cancer (CRC), and is implicated in UC-associated carcinogenesis through its inhibition of apoptosis. This study aimed to determine the potential effect of PSD methylation on its downstream molecule, Ras-related C3 botulinum toxin substrate 1 (Rac1), which governs neutrophil chemotaxis and apoptosis signaling. PSD was knocked down in a normal human fibroblast cell line (HNDF) and a neutrophil-like cell line (HL-60). Both NHDF and HL-60 cells exhibited numerous filamentous-actin (F-actin) rich membrane extensions, resulting in the activation of Rac1; this activation was hampered by PSD silencing. Lipopolysaccharide, a reactive oxygen species (ROS) inducer, stimulated NHDF cells to release ROS and activated caspase-3/7 in the presence of neutrophils, which was inhibited by PSD knockdown. Migration assays demonstrated that chemotaxis of HL-60 cells was affected by PSD silencing in NHDF cells. Tissue sections from 6 UC patients with CRC and 15 UC patients without CRC were examined. To verify Rac1-mediated chemotaxis in tissue sections, we evaluated the grade of neutrophil infiltration by histological assessment and assessed F-actin and PSD expression by immunohistochemistry. Neutrophil infiltration, F-actin and PSD expression were significantly decreased in specimens from UC patients with PSD methylation compared with those without. Decreased levels of F-actin expression were observed in colorectal mucosa, as well as in infiltrating cells with PSD methylation. PSD expression was preferentially inhibited in colorectal mucosa by PSD methylation, whereas PSD expression was rarely observed in infiltrating cells, regardless of PSD methylation status. These data indicate that aberrant methylation of PSD occurs in UC-associated colorectal mucosa, enabling circumvention of Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis, and thus plays a pivotal role in the mechanisms underlying UC-associated carcinogenesis.

Aberrant methylation of the Pleckstrin and Sec7 domain-containing gene is implicated in ulcerative colitis-associated carcinogenesis through its inhibitory effect on apoptosis.

The Pleckstrin and Sec7 domain-containing (PSD) gene, which regulates skeletal rearrangements, has been found to be more frequently methylated both in ulcerative colitis (UC)-associated colorectal cancer tissues (5 of 7; 71.4%) and matched normal epithelia (4 of 7; 57.1%) compared to non-neoplastic UC epithelia (6 of 22; 27.3%) and sporadic colorectal cancer tissues (6 of 32; 18.8%). The levels of PSD mRNA were positively correlated with the methylation status of PSD, as shown by both MSP and bisulfite sequencing. To determine the potential role of PSD silencing in the mechanisms underlying UC-associated carcinogenesis, the levels of senescence, proliferation and apoptosis were evaluated in a normal human fibroblast cell line (NHDF) in which 93% of PSD expression was knocked down by a small-interfering RNA (si-RNA). Although there were no significant differences in the levels of senescence and proliferation caused by PSD knockdown, the level of apoptosis was significantly decreased by PSD knockdown (5.3% in siControl-treated cells vs. 0.67% in siPSD-treated cells, p=0.0001). In addition, reactive oxygen species inducers accelerated apoptosis in NHDF and a neutrophil-like cell line, which was significantly reduced by PSD knockdown. To verify the effect of PSD methylation in tissue sections including 21 samples from UC patients with or without tumors, we elucidated PSD promoting accumulation of filamentous-actin (F-actin) and apoptosis by immunohistochemistry and TUNEL assay, respectively. Both levels of accumulation of F-actin and apoptosis were significantly decreased in specimens from UC patients with PSD methylation compared to those without PSD methylation (F-actin: 0.69±0.86 with vs. 1.57±0.51 without, p=0.0031, apoptotic index: 0.31±0.63 with vs. 1.0±0.88 without, p=0.0277). In conclusion, our results indicate that PSD methylation plays a significant role in the mechanisms underlying UC-associated carcinogenesis through its inhibitory effect on apoptosis in the interaction between colorectal mucosa and neutrophils.

Two cases of diabetic mastopathy: MR imaging and pathological correlation

Diabetic mastopathy is a rare benign condition associated with long-standing diabetes mellitus and presents with breast lumps. This report describes two cases in which diffusion-weighted images (DWI) on magnetic resonance imaging were quite different from each other. In case 1, there were hyperintense lesions on DWI, and surgically removed specimens revealed ductitis with marked lymphocytic infiltration. In case 2, no abnormal intensity was depicted on DWI, and biopsy specimens showed dense stromal fibrosis with mild perivascular lymphocytic infiltration that corresponded to previous reports. Although it is reported that diabetic mastopathy is composed of dense fibrous tissue with low cellularity that results in no hyperintense lesion on DWI, in cases with marked lymphocytic infiltration, strong hyperintensity can be seen on DWI mimicking malignant breast tumors.

P4-05-05: Imaging Mass Spectrometry Based Lipid Metabolites Analysis for Breast Cancer.

Background: Activation of lipid metabolism is an early event of carcinogenesis and a central hallmark of many cancers including breast cancer. Recent findings argue that stearoyl CoA desaturase-1 (SCD1), a key regulator of the fatty acid (FA) composition and the endoplasmic reticulum resident enzyme that converts saturated FA (SFA) into monounsaturated FA (MUFA) is a novel regulator of carcinogenesis. The distinctive lipids composition of membrane in cancer cells and the biological functions of SCD1, however, still remain uncertain. Imaging mass spectrometry (IMS) is a mass spectrometry-based analyzing technique that enables visualization of the individual molecules without requiring antibodies. It allows comprehensive detection of a wide range of biomolecules, such as lipids. We attempted to visualize the localization of lipids in breast cancer by IMS for better understanding of cancer proliferation. Materials and methods: 13 specimens were obtained from the primary breast cancer patients. All were Japanese woman and aged 41–86 years (mean 61.5y.o.). Only one patient received preoperative systematic therapy. 6 were estrogen receptor (ER) and/or progesterone receptor (PgR) positive and human epidermal growth factor receptor 2 (HER2) negative, 2 were ER and/or PgR positive and HER2 positive, 2 were both ER and PgR negative and HER2 positive and 2 were triple negative. IMS: Samples were immediately chilled in liquid Hexan and stored at −80°. All specimens were sliced into 10 mm thin sections, mounted onto one indium-tin oxide-coated glass slides (Bruker Daltonics) and then sprayed by 2,5-Dihydroxybenzoic acid. Matrix assisted laser desorption ionization (MALDI) technique was used as a soft ionization method. We used time of flight (TOF)/TOF type instrument (Ultraflex, Bruker Daltonics) and all the spectrum were acquired automatically using Fleximaging software (Bruker Daltonics). Each spectral intensity at any mass-to-charge ratio (m/z) was measured at 16 regions of interest (ROI); 13 ROI were picked up from cancerous parts and 3 were from non-cancerous parts. Spectral intensities were compared and statistical analysis was performed by Mann Whitney test. The software was also used to create two-dimensional ion-density maps. Results: In the cancerous parts of all the 13 specimens, two distinct peaks of the molecular ions were detected at m/z 798.5 and 810.5, which were not found in the non-cancerous parts. Median intensity of the molecular ions at m/z 798.5 and 810.5 were 38.9 and 3.18 in the cancerous part, while they were 0.84 and 1.02 in the non-cancerous part (p=0.010 and 0.015, respectively). Tandem mass spectrometry analysis for these two molecules revealed that they were two kinds of phosphatidylcholine (PC), PC (16:0/18:1) and PC (18:0/18:1). Localization of the individual PC was visualized by means of IMS, which showed that in cancerous part accumulation of PCs containing MUFA was more pronounced than those containing SFA only. Conclusion: Two kinds of PC containing MUFA were found to highly accumulate in cancerous parts, which may suggest involvement of SCD1 in the membrane composition regulation and cancer proliferation. Further studies may thus be warranted to explore the relation between PC localization and the SCD1 expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-05.