Yuka Yanagisawa

NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Constitutive activation of nuclear factor‐κB is preferentially involved in the proliferation of basal‐like subtype breast cancer cell lines

Constitutive nuclear factor (NF)‐κB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF‐κB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF‐κB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF‐κB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal‐like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF‐κB activation by expression of IκBα super‐repressor reduced proliferation of the basal‐like subtype cell lines. Expression levels of mRNA encoding NF‐κB‐inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference‐mediated knockdown of NIK reduced NF‐κB activation in a subset of the basal‐like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF‐κB activation, partially dependent on NIK, is preferentially involved in proliferation of basal‐like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. (Cancer Sci 2009; 100: 1668–1674)

Age‐related hypermethylation of the hMLH1 promoter in gastric cancers

To determine whether methylation of the hMLH1 promoter is related to increasing age and gastric carcinogenesis, we examined hMLH1 methylation and expression in 100 gastric cancers. hMLH1 methylation and aberrant protein expression were observed in 9 and 13 cancers, respectively. Normal and intestinal metaplastic tissues adjacent to cancers with hypermethylation did not exhibit any hMLH1 methylation, indicating that it may be specific to gastric cancers. The frequency of hMLH1 methylation significantly increased with age. These results suggest that hMLH1 methylation plays an important role in gastric carcinogenesis in old people.

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

The human DNA methyltransferases DNMT3A and DNMT3B have two types of promoters with different CpG contents

DNA modification that is established by de novo methylation is involved in the epigenetic control of genome functions. The DNMT3A and DNMT3B genes encode putative de novo methyltransferases. In this paper, we investigated the transcriptional regulatory regions of the human DNMT3A and DNMT3B genes. We found that the DNMT3A and DNMT3B genes have multiple transcriptional start points (TSPs) that are separated on the chromosome and the expressions of these genes are controlled by multiple promoters. The DNMT3A gene has at least four TSPs and the expression is controlled by three different promoters. All three promoters lack typical TATA sequences adjacent to the TSPs. Two of them bear CpG-rich promoters and the other a CpG-poor promoter. The DNMT3B gene has at least two TSPs which exist in different exons and the expression is controlled by different promoters. Both promoter regions of the DNMT3B gene lack typical TATA sequences, where one promoter contains a CpG-rich area near the TSP, the other promoter is CpG-poor. Together with the data that the human DNMT1 gene has both CpG-rich and CpG-poor promoters, it is suggested that transcriptional regulation by two types of promoters, CpG-rich and CpG-poor, might be common characteristics in the DNMT gene family.

Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability

Microsatellite instability (MSI), which is recognized as an important mechanism in tumorigenesis, has been reported in familial gastric cancers (FGC). However, genetic defects responsible for this phenotype, that is, mutations in mismatch‐repair genes such as hMLH1 and hMSH2, have not been detected in most FGC cases. Earlier studies have shown that the promoter region of the hMLH1 gene was methylated in some sporadic colorectal and endometrial cancers. To determine how FGC acquire MSI, we examined the MSI status, hMLH1‐protein expression and methylation status of the hMLH1‐promoter region in FGC cases. Out of 9 cancers, 6 from 8 FGC kindreds showed MSI at one or more loci; no germline mutations in the hMLH1 or hMSH2 genes were detected; 4 cancers exhibiting MSI displayed aberrant hMLH1 expression: complete loss in one, decreased level in another, and partially staining pattern in the remaining 2. Methylation in the hMLH1‐promoter region was found in these 4 cases. In contrast, the cancers displaying hMLH1‐protein expression were not methylated in the hMLH1‐promoter region. Our data show a significant association between the absence of hMLH1 expression and methylation of its promoter in FGC cases with MSI. This suggests that the mechanism of inactivation of hMLH1 is epigenetic and that there are other genes responsible for FGC. Int. J. Cancer 85:50–53, 2000.

Cathepsin L is highly expressed in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract that are diagnosed by c-kit staining in most cases. A lysosomal cysteine proteinase termed cathepsin L has been commonly associated with malignancy in several cancer types, but this finding has not been reported for GISTs. We analyzed the cathepsin L mRNA and protein expression in GISTs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that cathepsin L levels were higher in GISTs than those in gastric or colorectal tumors; this finding was supported by results of the Western blot analysis. Immunohistochemistry revealed that cathepsin L was localized to the cytoplasm of GIST cells as an intense granular signal, which was not observed in the cells of leiomyoma, a mesenchymal tumor that was analyzed as a control specimen. Double immunofluorescence microscopy revealed that a portion of the granular signal colocalized with lysosome-associated membrane protein-1 (LAMP-1), which is a lysosomal marker. Moreover, immunohistochemical analysis of 43 tumor specimens revealed that 86.0% (n=37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity but not with other clinicopathological factors, including gender, age, region, size, mitosis and risk of recurrence. From these results, we conclude that cathepsin L is highly expressed in GISTs compared to its expression in other cancerous lesions; this identifies cathepsin-L as a new diagnostic marker for GISTs.

FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma

Personalized therapy for non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma, has recently been significantly improved by the discovery of various molecular targets. However, this has not been the case for lung squamous cell carcinoma (SCC). In the present study, we identified the family with sequence similarity 83, member B (FAM83B) as a candidate marker for SCC through a comprehensive gene expression analysis and examined its correlations with various clinicopathological factors. The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection from 2005 to 2011 at the Fukushima Medical University Hospital (Fukushima, Japan). They included 102 patients with adenocarcinoma and 113 with SCC. FAM83B expression was first examined in some of the samples by gene expression analysis and western blotting, and then all clinical specimens were evaluated by immunohistochemistry (IHC). The relationship between the quantitative values for IHC and clinicopathological factors was statistically analyzed. The results showed that FAM83B mRNA expression was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this trend. Specimens containing >10% positive area for FAM83B were judged as ‘positive’; 94.3% (107/113) of SCC and 14.7% (15/102) of adenocarcinoma were positive. Patients were divided into two subgroups according to expression (54 high-expression and 53 low-expression patients); the high-expression group was associated with a better disease-free survival (DFS) rate (P=0.042, log-rank test). In conclusion, FAM83B may be a reliable diagnostic and prognostic biomarker for SCC. Detailed analyses of FAM83B function in lung cancer are required to understand how its expression is associated with better prognosis in SCC.

Gene expression profiling of acute cellular rejection in rat liver transplantation using DNA microarrays

Acute cellular rejection (ACR) is still a major problem in organ transplantation, and its genetic and molecular mechanisms remain poorly understood. We used DNA microarrays to investigate the gene expression profiles in ACR. We hypothesized that changes of gene expression in grafts could also be detected in peripheral blood leukocytes. We first compared the gene expression profiles in liver isografts (Lewis to Lewis) and allografts (Dark Agouti to Lewis) harvested from rats at days 1, 3, 5, and 7 after transplantation. Hierarchical clustering analysis indicated that gene expression started to change on day 3, and 89 differentially expressed genes were extracted from allografts in comparison with isografts at day 3. Most of the up‐regulated genes were associated with graft‐infiltrating leukocytes. We then confirmed the similarity of gene expression changes in peripheral leukocytes by quantitative real‐time polymerase chain reaction. We also investigated the gene expression changes in other inflammatory and liver dysfunction models. Two interferon‐gamma inducible genes, interferon regulatory factor 1 and guanylate nucleotide binding protein 2, were overexpressed in both the peripheral leukocytes and liver graft during ACR. Although further studies are necessary, these 2 genes in peripheral leukocytes could be potentially useful markers for rejection or immunosuppression. Liver Transpl 15:509–521, 2009.

Novel clusters of highly expressed genes accompany genomic amplification in breast cancers

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.