Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Evaluation of PSF1 as a prognostic biomarker for prostate cancer.

BACKGROUND: Partner of SLD5 1 (PSF1) is an evolutionarily conserved DNA replication factor. Previous studies have suggested that transcriptional activity of the PSF1 gene correlated with malignancy of cancer cells. The objective of the current study was to evaluate the relationship between PSF1 expression and the clinical features of prostate cancer. METHODS: We determined the expression of PSF1 in 120 needle biopsy samples of prostate cancer by immunohistochemistry. We divided patients into PSF1-positive or -negative groups and analyzed the relationships between the expression of PSF1, the Gleason score, PSA level, TNM classification and prognosis. RESULTS: Our results showed that the PSF1 expression correlated significantly with PSA values at diagnosis (P=0.0028), with tumor grade (P<0.0001), and with clinical stage (P=0.0005). Moreover, the PSF1 expression correlated significantly with overall survival (hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.17-15.8; P=0.003) and progression-free survival in 99 consecutive patients with prostate cancer. Noteworthy, the prognosis of PSF1-positive cases was also worse in patients with a Gleason score of 8-10 (HR 3.7; 95% CI 1.28-13.43; P=0.0143). Limitations include that this study had a retrospective design, that patients in the study were heterogeneous and included those with early and advanced cancer, and that small tumor fragments may not be representative of the entire carcinoma. CONCLUSIONS: PSF1 is expressed in high-grade prostate cancer and may be a useful biomarker to identify patients with a poor prognosis at the time of diagnosis.

Arl4c expression in colorectal and lung cancers promotes tumorigenesis and may represent a novel therapeutic target.

We recently demonstrated that expression of ADP-ribosylation factor (ARF)-like 4c (Arl4c) induced by a combination of Wnt/ß-catenin and epidermal growth factor/Ras signaling in normal epithelial cells grown in three-dimensional culture promotes cellular migration and proliferation, resulting in formation of tube-like structures, suggesting the involvement of Arl4c in epithelial morphogenesis. It is conceivable that there could be a common mechanism between epithelial morphogenesis and carcinogenesis. Therefore the current study was conducted to investigate whether Arl4c might be involved in tumorigenesis. Immunohistochemical analyses of tissue specimens obtained from colorectal and lung cancer patients revealed that Arl4c was not observed in non-tumor regions but was strongly expressed at high frequencies in tumor lesions. Inhibition of Wnt/ß-catenin or Ras/mitogen-activated protein kinase signaling reduced Arl4c mRNA levels in HCT116 colorectal cancer cells and A549 lung cancer cells. Knockdown of Arl4c inhibited Rac activity and also prevented nuclear localization of yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) in these cancer cells. Arl4c-depleted cancer cells consistently showed decreased migration, invasion and proliferation capabilities both in vitro and in vivo. Furthermore, direct injection of Arl4c small interfering RNA (siRNA) into HCT116 cell-derived tumors (in vivo treatment with siRNA) inhibited tumor growth in immunodeficient mice. These results suggest that Arl4c is involved in tumorigenesis and might represent a novel therapeutic target for suppressing proliferation and invasion of colorectal and lung cancer cells.

Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

OBJECTIVE: In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. METHODS: Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. RESULTS: High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. CONCLUSIONS: ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma.

MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression.

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin-focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3ß pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src-miR-542-3p-ILK-FAK circuit plays a crucial role in controlling tumor progression.

MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor growth remain unclear. Here we examined the involvement of microRNAs in the c-Src-mediated tumor growth. Microarray profiling revealed that c-Src activation downregulates a limited set of microRNAs, including miR-99a, which targets oncogenic mammalian target of rapamycin (mTOR) and fibroblast growth factor receptor 3 (FGFR3). Re-expression of miR-99a suppressed tumor growth of c-Src-transformed cells, and this effect was restored by the overexpression of mTOR. The downregulation of miR-99a was also observed in epidermal growth factor- and Ras-transformed cells, and it was suppressed by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Furthermore, miR-99a downregulation is associated with mTOR/FGFR3 upregulation in various human lung cancer cells/tissues. The tumorigenicity of these cells was suppressed by the introduction of miR-99a. These findings suggest that the miR-99a-mTOR/FGFR3 pathway is crucial for controlling tumor growth in a wide range of human cancers that harbor upregulation of the Src-related oncogenic pathways.

MITF-CM, a newly identified isoform of microphthalmia-associated transcription factor, is expressed in cultured mast cells.

The microphthalmia-associated transcription factor (MITF) gene encodes a basic helix-loop-helix and leucin zipper protein. In this study, we identified a novel MITF isoform, MITF-CM, which possesses a unique amino terminus. Exon 1CM is located 84 kb upstream of the exon encoding the B1b domain. MITF-CM was expressed in the human mast cell line HMC-1, the human basophilic cell line KU812, and CB-derived mast cells cultured for 10 weeks as well as bone marrow mononuclear cells. Transient transfection of MITF-CM cDNA in COS-7 cells resulted in the expression of a 64-kDa protein, detected by Western blotting, and nuclear localization of the protein, detected by immunostaining. The transient cotransfection of a luciferase construct under the control of the tyrosinase promoter and MITF-CM cDNA increased luciferase activity threefold. In contrast, none of the MITF isoforms transactivated both the tryptase and chymase gene promoters, indicating differences in the gene transactivation system between humans and mice.

Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

Epigenetic regulation of the expression of the novel stem cell marker CDCP1 in cancer cells.

CDCP1 is a novel stem cell marker that is expressed in several types of cancer. The mechanisms by which CDCP1 expression is regulated, and the clinical implications of this marker, have not been clarified. In this report, we examine the epigenetic regulation of CDCP1 expression in cell lines and clinical samples from patients with breast cancer. Many CpG sequences were localized around the transcription initiation site of CDCP1. These CpG motifs were found to be poorly methylated in cell lines with high levels of CDCP1 expression and heavily methylated in cell lines with low levels of CDCP1 expression. The in vitro methylation of CpG sites decreased CDCP1 promoter activity, and the addition of a demethylating reagent restored activity. In 25 breast cancer samples, an inverse correlation was noted between the CDCP1 expression level and the proportion of methylated to non-methylated CpG sites. Tumours with high-level CDCP1 expression showed higher levels of proliferation, as revealed by immunohistochemical detection of the MIB-1 antigen, than tumours with low-level CDCP1 expression. These findings indicate that the expression of CDCP1 is regulated by methylation of its promoter region in tumours. CDCP1 expression may prove to be useful in the further characterization of cancers.

Effect of MITF on transcription of transmembrane tryptase gene in cultured mast cells of mice.

Mouse mast cell protease (mMCP)-6, mMCP-7 and transmembrane tryptase (TMT) are all tryptases. The normal mi transcription factor (+-MITF) transactivated mMCP-6 gene by binding three consensus motifs in the promoter region, but no MITF-binding motifs were found in the mMCP-7 promoter. Instead, c-Jun transactivated mMCP-7 gene, and +-MITF cooperated with it. The mi-MITF encoded by mutant mi allele inhibited the transactivation by c-Jun and reduced the mMCP-7 promoter activity. Here, the effect of MITF on the TMT gene expression was examined. The +-MITF enhanced the TMT promoter activity by binding two consensus motifs. The mi-MITF showed the inhibitory effect on TMT gene expression. The effect of +-MITF on TMT gene was similar to the effect on mMCP-6 gene, and that of mi-MITF was similar to the effect on mMCP-7 gene. The effects of MITF on TMT gene appeared distinct from its effects on either mMCP-6 or mMCP-7 gene.

Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper transcription factor that is important for the development of mast cells. Cultured mast cells (CMCs) of mi/mi genotype express abnormal MITF (mi-MITF), but CMCs of tg/tg genotype do not express any MITFs. It was previously reported that mi/mi CMCs showed more severe abnormalities than tg/tg CMCs, indicating that mi-MITF had inhibitory function. Whereas mi-MITF contains a single amino acid deletion in the basic domain, MITF encoded by mi(ew) allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain. Here the effect of a large deletion of the basic domain was examined. In mi(ew)/mi(ew) CMCs, the expression pattern of genes whose transcription was affected by MITF was comparable to that of tg/tg CMCs rather than to that of mi/mi CMCs. This suggested that ew-MITF lacked any functions. The part of the basic domain deleted in ew-MITF appeared necessary for either transactivation or inhibition of transactivation.

Inhibitory effect of the mi transcription factor encoded by the mutant mi allele on GA binding protein-mediated transcript expression in mouse mast cells.

The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amounts of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. The synthesis of heparin is abnormal in the skin mast cells of mi/mi mice. Because N-deacetylase/N-sulfotransferase 2 (NDST-2) is essential for the synthesis of heparin, the amount of NDST-2 messenger RNA (mRNA) was compared among cultured mast cells (CMCs) of +/+, mi/mi, and tg/tg genotypes. The NDST-2 mRNA was detected by in situ hybridization in the skin mast cells of +/+ and tg/tg mice, but not in the skin mast cells of mi/mi mice. The amount of NDST-2 mRNA decreased significantly in CMCs derived from mi/mi mice when compared to the values of +/+ and tg/tg mice, suggesting that the defective form of MITF inhibited the expression of the NDST-2 transcript. The expression of NDST-2 transcript was mediated by the GGAA motif located in the 5'-untranslated region. GA binding protein (GABP) bound the GGAA motif and increased the amount of NDST-2 transcript. The mi-MITF appeared to inhibit the ability of GABP to express NDST-2 transcript by disturbing its nuclear localization. This is the first study to show that expression of an abnormal form of a bHLH-Zip transcription factor can dramatically alter the intracellular location of another DNA/RNA binding factor, which in turn brings about profound and unexpected consequences on transcript expression.

Mutant mice: a useful tool for studying the development of mast cells.

We have used various mouse mutants for studying the development of mast cells. The bone marrow origin of mast cells was shown by using giant granules of beige mice as a marker. Mast cell-deficient W/W(v) and Sl/Sl(d) mice are useful for investigation of the developmental processes. The mi locus encodes a member of the basic helix-loop-helix-leucine zipper protein family of transcription factors (MITF), and mast cells of mi/mi mice showed phenotypic abnormalities. Mast cells of mi/mi mice synthesized the mutant mi-MITF in normal amounts, and mi-MITF showed an inhibitory effect on the transcription of various mast cell-specific genes. On the other hand, mice of tg/tg possess the transgene insertional mutation in the 5' flanking region of the mi gene and do not express any MITFs. Genes whose transcription was suppressed were more numerous in mast cells of mi/mi mice than in those of tg/tg mice. The comparison between phenotypes of mi/mi mast cells and those of tg/tg mast cells gave some insights into the regulation of mast cell phenotypes by transcription factors.

Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mi(ce)/mi(ce) mutant mice of which MITF lacks the zipper domain.

The mi transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amount of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. Mast cells of mi/mi mice show more severe abnormalities than those of tg/tg mice, indicating that the mi-MITF possesses the inhibitory function. The MITF encoded by the mi(ce) mutant allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain using mi(ce)/mi(ce) mice. The amounts of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) messenger RNAs decreased in mast cells of mi(ce)/mi(ce) mice to levels comparable to those of tg/tg mice, and the amounts were intermediate between those of +/+ mice and those of mi/mi mice. Gr B mediates the cytotoxic activity of mast cells, and TPH is a rate-limiting enzyme for the synthesis of serotonin. The cytotoxic activity and serotonin content of mi(ce)/mi(ce) mast cells were comparable to those of tg/tg mast cells and were significantly higher than those of mi/mi mast cells. The phenotype of mi(ce)/mi(ce) mast cells was similar to that of tg/tg mast cells rather than to that of mi/mi mast cells, suggesting that the ce-MITF had no functions. The Zip domain of MITF appeared to be important for the development of mast cells. (Blood. 2001;97:2038-2044)

Inhibitory effect of the transcription factor encoded by the mutant mi microphthalmia allele on transactivation of mouse mast cell protease 7 gene.

The transcription factor encoded by the mi locus (MITF) is a transcription factor of the basic-helix-loop-helix zipper protein family. Mice of mi/mi genotype express a normal amount of abnormal MITF, whereas mice of tg/tg genotype do not express any MITFs due to the transgene insertional mutation. The effect of normal (+) and mutant (mi) MITFs on the expression of mouse mast cell protease (MMCP) 6 and 7 was examined. Both MMCP-6 and MMCP-7 are tryptases, and their coding regions with high homology are closely located on chromosome 17. Both MMCP-6 and MMCP-7 genes are expressed in normal cultured mast cells (+/+ CMCs). Although the transcription of MMCP-6 gene was severely suppressed in both mi/mi and tg/tg CMCs, that of MMCP-7 gene was severely suppressed only in mi/mi CMCs. The study identified the most significant segment for the transcription in the 5' flanking region of MMCP-7 gene. Unexpectedly, no CANNTG motifs were found that are recognized and bound by +-MITF in this segment. Instead, there was an AP-1 binding motif, and binding of c-Jun to the AP-1 motif significantly enhanced the transcription of MMCP-7 gene. The complex formation of c-Jun with either +-MITF or mi-MITF was demonstrated. The binding of +-MITF to c-Jun enhanced the transactivation of MMCP-7 gene, and that of mi-MITF suppressed the transactivation. Although the former complex was located only in the nucleus, the latter complex was predominantly found in the cytoplasm. The negative effect of mi-MITF on the transcription of MMCP-7 gene appeared to be executed through the interaction with c-Jun.

mi-transcription factor as a regulator of mast cell differentiation.

Masts cells are progeny of the hematopoietic stem cell. For the differentiation of mast cells, a transcription factor encoded by the mouse mi locus (MITF) plays an important role. The expression of many genes encoding proteins that are essential for the function of mast cells is regulated by MITF. Because various mutant mice are available at the mi locus and because cultured mast cells are easily obtained from the spleen of these mutant mice, this system may be a good model for studying the regulation of hematopoietic cell differentiation by a transcription factor.

Involvement of mi-transcription factor in expression of alpha-melanocyte-stimulating hormone receptor in cultured mast cells of mice.

The microphthalmia (mi) locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (MITF). We have reported that expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi mice due to a defective transactivation ability of mutant MITF (mi-MITF). We also found that mi/mi CMCs did not express a receptor (MC1R) for alpha-melanocyte-stimulating hormone. The overexpression of the wild-type (+/+) MITF but not mi-MITF normalized the expression of the MC1R in mi/mi CMCs, indicating the involvement of +-MITF in the MC1R gene expression. Next, we analyzed the promoter region of the MC1R gene by the transient cotransfection assay. The luciferase construct under the control of the MC1R promoter and the cDNA-encoding +-MITF or mi-MITF were cotransfected into NIH/3T3 fibroblasts. The cotransfection of +-MITF but not mi-MITF increased the luciferase activity. There were five CANNTG motifs recognized by bHLH-Zip-type transcription factors in the cloned promoter region. We found +-MITF bound two of five CANNTG motifs, and both motifs were essential for the transactivation of the MC1R gene by +-MITF. These results indicated that +-MITF directly transactivated the MC1R gene through these two motifs.