Makoto Tsuneoka

JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation

The rate‐limiting step in ribosome biogenesis is the transcription of ribosomal RNA, which is controlled by environmental conditions. The JmjC enzyme KDM2A/JHDM1A/FbxL11 demethylates mono‐ and dimethylated Lys 36 of histone H3, but its function is unclear. Here, we show that KDM2A represses the transcription of ribosomal RNA. KDM2A was localized in nucleoli and bound to the ribosomal RNA gene promoter. Overexpression of KDM2A repressed the transcription of ribosomal RNA in a demethylase activity‐dependent manner. When ribosomal RNA transcription was reduced under starvation, a cell‐permeable succinate that inhibited the demethylase activity of KDM2A prevented the reduction of ribosomal RNA transcription. Starvation reduced the levels of mono‐ and dimethylated Lys 36 of histone H3 marks on the rDNA promoter, and treatment with the cell‐permeable succinate suppressed the reduction of the marks during starvation. The knockdown of KDM2A increased mono‐ and dimethylated Lys 36 of histone H3 marks, and suppressed the reduction of ribosomal RNA transcription under starvation. These results show a novel mechanism by which KDM2A activity is stimulated by starvation to reduce ribosomal RNA transcription.

Mina53 as a Potential Prognostic Factor for Esophageal Squamous Cell Carcinoma

Purpose: We previously identified mina53, a novel Myc target gene. Here we investigated whether mina53 is related to esophageal squamous cell carcinoma (ESCC), a disease with poor prognosis. Experimental Design: Mina53 expression was suppressed in ESCC cell lines by a RNA interference method to investigate whether Mina53 is involved in cell proliferation. Expression of Mina53 was investigated by Western blotting in tissue sections from patients with ESCC. Immunohistochemical analysis of Mina53 was carried out and compared with that using anti–Ki-67 antibody. Finally, the level of Mina53 expression was compared with the length of survival of patients with ESCC. Results: Reduction of mina53 expression by RNA interference suppressed cell proliferation in ESCC cell lines. Western blot analysis of surgically resected ESCC specimens indicated that the expression of Mina53 in tumors was increased compared with that in adjacent nonneoplastic tissues in all four specimens examined. When formalin-fixed specimens from 52 patients with ESCC were stained immunohistochemically, it was found that Mina53 was highly expressed in 83% of specimens. Anti-Mina53 antibody stained tumors more efficiently than antibody against Ki-67, a cell proliferation biomarker, in some cancer specimens. Patients with high expression of Mina53 had shorter survival periods, whereas the expression level of Ki-67 in ESCC showed no relationship to patient outcome. Conclusions: Taken together, our results indicate that expression of Mina53 is a characteristic feature of ESCC and suggest that immunostaining by anti-Mina53 antibody may be useful as a potential prognostic indicator.

Ras/MEK signaling suppresses myc-dependent apoptosis in cells transformed by c-myc and activated ras

Cooperation of myc and activated ras has been suggested to cause malignant cell transformation but the mechanism is still unknown. Here we isolated a transformed cell line in which activation of c-Myc and Ras are independently controllable, and show that after establishment of the transformed state by c-myc and activated ras, removal of activated Ras initiates apoptosis that is dependent on c-Myc activity. Apoptosis is also initiated by an inhibitor of MEK (MAPK/ERK kinase), a kinase downstream of Ras, and apoptosis is blocked by activated Mek1. These results suggest that one of the conditions required for establishment of the transformed state is a block of apoptosis involving MEK activity. We tested the effect of MEK inhibition on cells transformed by various oncogenes. Suppression of apoptosis by MEK is not critical in general, but in cells transformed by c-myc plus a gene that activates the MAPK cascade it is necessary to avoid cell death. Activated Ras/MEK did not suppress c-myc-dependent apoptosis due to serum-limitation. Overexpression of chicken bcl-xL suppressed apoptosis under serum-limiting conditions, but not apoptosis initiated by Ras/MEK inhibition in cells transformed by myc and activated ras. Altogether, these results suggest the existence of a novel regulatory mechanism for myc-dependent apoptosis in certain transformed cells.

c-myc activates RCC1 gene expression through E-box elements.

Proto-oncogene c-myc is implicated in proliferation of mammalian cells. Although c-Myc protein has been demonstrated to function as a transcription factor recognizing an E-box (CACGTG) element, few c-myc-regulated genes have been identified and the specific role of c-myc is still unclear. RCC1 is necessary for mammalian cells to proliferate. Four CACGTG elements exist within 1.3u2009kb downstream of the major transcription start site for the human RCC1 gene in HeLa cells. Stimulation of HeLa cells with serum increased c-myc expression and RCC1 expression. Therefore the relationship between the expression of RCC1 and c-myc was investigated. Rat 3Y1 cells overexpressing c-myc contained about twice as much RCC1 mRNA as control cells. When a chimeric protein comprised of c-myc and the estrogen binding domain of estrogen receptor was activated by addition of 4-hydroxytamoxifen (OHT), expression of RCC1 mRNA increased twofold. To examine whether c-Myc functions through the CACGTG elements, a DNA fragment of RCC1 intron 4, exon 5 and part of intron 5 was joined to firefly luciferase cDNA to construct a reporter plasmid. In transient expression experiments using HeLa cells, co-transfection with c-myc stimulated the luciferase activity up to 2.5-fold in a dose-dependent manner. When the CACGTG elements in the reporter plasmid were destroyed, stimulation by c-myc was not observed. The four CACGTG elements did not contribute equally to the stimulation by c-myc. Gel retardation experiments suggest that c-Myc with Max binds to the CACGTG elements in the context of the RCC1 gene sequence in vitro. These results indicate that c-Myc can regulate expression of RCC1 through the E-box elements.

Mina53, a novel c-Myc target gene, is frequently expressed in lung cancers and exerts oncogenic property in NIH/3T3 cells.

PurposeMina53, whose expression is directly induced by c-Myc, is overexpressed in various cancers and plays an important role in cell growth. To clarify the involvement of Mina53 in lung cancers, we investigated its expression in human lung cancer tissues as well as in various lung cancer cell lines.MethodsMina53 expression was determined by real-time RT-PCR, western blotting, and immunohistochemistry using lung cancer cell lines, normal human bronchial epithelial cells, and lung cancer tissues. Biological effects of Mina53 were evaluated by soft agar colony formation assay and tumorigenicity in nude mice using Mina53-transfected NIH/3T3 cells. cDNA microarray analysis was performed to determine the gene alteration by Mina53 and confirmation was made using real-time RT-PCR with mina53 expression plasmid or mina53 shRNA-transfected NIH/3T3 cells.ResultsWe observed that 62% of patients evidenced overexpression of Mina53 from the early clinical stages of lung cancer. Differences according to gender, smoking status, or histologic type were not statistically significant. Forced expression of Mina53 in NIH/3T3 cells induced cell transformation, and mina53-transfected NIH/3T3 clones produced tumors in nude mice, demonstrating that Mina53 has oncogenic potential. cDNA microarray revealed that 254 genes had altered expression in a mina53-transfected NIH/3T3 clone. Mina53 regulates several genes related to cell adhesion and metabolism, which have also been reported to be regulated by c-Myc. Genes regulated by Mina53, but not by c-Myc included cytokine/growth factor related genes such as EGFR, IL-6, and HGF.ConclusionOur results suggest that Mina53 plays an important role in carcinogenesis and may be a target for cancer prevention.

Accelerated expression of a Myc target gene Mina53 in aggressive hepatocellular carcinoma

Aim:u2002 Expressions of the myc target genes Mina53 and mimitin are high in esophageal squamous cell carcinoma and colon cancer, and their relationship to cell proliferation and patient prognosis has been reported. Because c‐myc gene expression is closely related to hepatocellular carcinoma (HCC) growth or formation and/or maintenance, we examined the Mina53 and mimitin expressions in HCC.

Expression of Mina53, a novel c-Myc target gene, is a favorable prognostic marker in early stage lung cancer.

Mina53, a novel target gene product of c-Myc, is overexpressed in various malignancies. We previously demonstrated that Mina53 is overexpressed in lung cancer patients from the early clinical stages. In this paper, the association between disease prognosis and Mina53 expression in lung cancer patients is analyzed; we found that overexpression of Mina53 in lung cancer patients is associated with favorable prognosis. Statistical analysis using the Kaplan-Meier method showed that patients with negative staining for Mina53 had significantly shorter survival than patients with positive staining for Mina53, especially in stage I or with squamous cell carcinoma. Because the major cause of death in lung cancer patients after surgery is distant metastasis, the effect on cancer cell invasiveness was analyzed for the mechanisms involved in the association with favorable outcome. Overexpression of Mina53 in H226B, a lung squamous cell carcinoma cell line, inhibited cancer cell invasion. Transfection with mina53 shRNA increased the number of invading cells. These results suggest that Mina53 immunostaining is a useful prognostic marker--especially in the early stage of lung cancer--and that Mina53 negative patients should be managed particularly carefully after surgery.

Haptoglobin Gene Promoter Polymorphism and Haplotypes Are Unique in Different Populations

ABSTRACT We investigated the distribution of haptoglobin (HP) alleles and haplotypes among Africans (Ghanaians), Europeans (from South Africa), and Chinese. HP*1F was present only in Africans and Europeans, whereas HP*del was unique to Chinese. Six base substitutions at the promoter region were population specific. Only 3 out of 18 haplotypes were shared among the populations. A probable application of HP in human population genetics appears legitimate.

Mild Glucose Starvation Induces KDM2A-Mediated H3K36me2 Demethylation through AMPK To Reduce rRNA Transcription and Cell Proliferation

ABSTRACT Environmental conditions control rRNA transcription. Previously, we found that serum and glucose deprivation induces KDM2A-mediated H3K36me2 demethylation in the rRNA gene (rDNA) promoter and reduces rRNA transcription in the human breast cancer cell line MCF-7. However, the molecular mechanism and biological significance are still unclear. In the present study, we found that glucose starvation alone induced the KDM2A-dependent reduction of rRNA transcription. The treatment of cells with 2-deoxy-d-glucose, an inhibitor of glycolysis, reduced rRNA transcription and H3K36me2 in the rDNA promoter, both of which were completely dependent on KDM2A in low concentrations of 2-deoxy-d-glucose, that is, mild starvation conditions. The mild starvation induced these KDM2A activities through AMP-activated kinase (AMPK) but did not affect another AMPK effector of rRNA transcription, TIF-IA. In the triple-negative breast cancer cell line MDA-MB-231, the mild starvation also reduced rRNA transcription in a KDM2A-dependent manner. We detected KDM2A in breast cancer tissues irrespective of their estrogen receptor, progesterone receptor, and HER2 status, including triple-negative cancer tissues. In both MCF-7 and MDA-MB-231 cells, mild starvation reduced cell proliferation, and KDM2A knockdown suppressed the reduction of cell proliferation. These results suggest that under mild glucose starvation AMPK induces KDM2A-dependent reduction of rRNA transcription to control cell proliferation.

SF-KDM2A binds to ribosomal RNA gene promoter, reduces H4K20me3 level, and elevates ribosomal RNA transcription in breast cancer cells

Regulation of rRNA transcription is an important factor for control of cell proliferation. We previously found that the JmjC domain-containing demethylase KDM2A reduces H3K36me2 in the rRNA gene promoter and rRNA transcription under starvation, which results in suppression of cell proliferation. The KDM2A gene also produces another protein product, SF-KDM2A, which lacks a JmjC domain and has no demethylase activity. As yet, the function of SF-KDM2A is not clear. Recently, it was reported that KDM2A was frequently amplified and that elevated expression of KDM2A was significantly associated with short survival of breast cancer patients. SF-KDM2A was more abundant than full-length KDM2A in a subset of breast cancers. In the present study, we report that SF-KDM2A localized in nucleoli and bound to the rRNA gene promoter in breast cancer cells. Overexpression of SF-KDM2A stimulated the transcription of rRNA. While the zf-CXXC domain was required for SF-KDM2A binding to the rRNA gene promoter, SF-KDM2A with mutations in the zf-CXXC domain lost the binding to the rRNA gene promoter and did not stimulate rRNA transcription. Knockdown of SF-KDM2A reduced rRNA transcription and cell proliferation. When SF-KDM2A was overexpressed, a transcriptionally repressive mark, H4K20me3, in the rRNA gene promoter was specifically reduced in a zf-CXXC domain-dependent manner, and knockdown of SF-KDM2A increased the H4K20me3 level. Taken together, these results demonstrate that SF-KDM2A binds to the rRNA gene promoter, reduces the H4K20me3 level, and activates rRNA transcription, suggesting that the stimulation of rRNA transcription by SF-KDM2A may contribute to tumorigenesis in breast cancer.