Genomic features of renal cell carcinoma developed during end-stage renal disease and dialysis.

Patients with end-stage renal disease (ESRD) or receiving dialysis have a much higher risk for renal cell carcinoma (RCC), but carcinogenic mechanisms and genomic features remain little explored and undefined. This study's goal was to identify the genomic features of ESRD RCC and characterize them for associations with tumor histology and dialysis exposure. In this study, we obtained 33 RCCs, with various histological subtypes, that developed in ESRD patients receiving dialysis and performed whole-genome sequencing and transcriptome analyses. Driver events, copy-number alteration (CNA) analysis and mutational signature profiling were performed using an analysis pipeline that integrated data from germline and somatic SNVs, Indels and structural variants as well as CNAs, while transcriptome data were analyzed for differentially expressed genes and through gene set enrichment analysis. ESRD related clear cell RCCs' driver genes and mutations mirrored those in sporadic ccRCCs. Longer dialysis periods significantly correlated with a rare mutational signature SBS23, whose etiology is unknown, and increased mitochondrial copy number. All acquired cystic disease (ACD)-RCCs, which developed specifically in ESRD patients, showed chromosome 16q amplification. Gene expression analysis suggests similarity between certain ACD-RCCs and papillary RCCs and in TCGA papillary RCCs with chromosome 16 gain identified enrichment for genes related to DNA repair, as well as pathways related to reactive oxygen species, oxidative phosphorylation and targets of Myc. This analysis suggests that ESRD or dialysis could induce types of cellular stress that impact some specific types of genomic damage leading to oncogenesis.

[Long-term survival of a breast cancer patient with carcinomatous pleuritis and carcinomatous cardiac tamponade successfully treated by multimodality therapy].

A 69-year old woman was admitted to our hospital because of dyspnea and pain in her left breast. Computed tomography revealed a massive quantity of left pleural effusion, a tumor in the left breast(5 cm in diameter), left cervical and supraclavicular lymph node metastasis, and a large left axillary metastatic mass. Based on a core needle biopsy, her breast tumor was diagnosed pathologically as scirrhous carcinoma, which was positive for estrogen receptor/progesterone receptor and negative for HER2 using the FISH assay, and left pleural metastasis was diagnosed cytologically. The carcinomatous pleural effusion was successfully controlled using pleural instillations of pirarubicin HCl and OK-432 after pleural drainage. A near clinical complete response was achieved by EC systemic chemotherapy(6 months)followed by endocrine therapy(letrozole), but 3 months later she was diagnosed cytologically with carcinomatous cardiac tamponade. After operative pericardial drainage, intrapericardial instillations of cisplatin and OK-432 successfully prevented re-accumulation of pericardial effusion. Systemic chemotherapy(weekly paclitaxel)for 11 months and endocrine therapy(letrozole)resulted in a clinical complete response. One year and 10 months after pericardial drainage, she underwent surgery(mastectomy and axillary lymph node dissection level II)because of two small tumors in the left breast which were found to be malignant using PET-CT. One tumor(diameter 1.6 cm)was found pathologically to consist of degenerated cancer cells, and another tumor(diameter 2 cm)was diagnosed as recurrent cancer. There was no lymph node metastasis in the axilla except for a single mass(1.4×0.7×0.3 cm), which was composed of extremely degenerative and necrotic non-lymphoid cancerous tissue. Since having the surgery, she has not experienced recurrence on hormone therapy with fulvestrant, and to date she is still alive, 3 years and 5 months since the left pleural metastasis episode.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

Novel homeodomain-interacting protein kinase family member, HIPK4, phosphorylates human p53 at serine 9.

We describe here the cloning and characterization of a novel mouse homeodomain-interacting protein kinase (HIPK)-like gene, Hipk4. Hipk4 is expressed in lung and in white adipose tissue and encodes a 616 amino acid protein that includes a serine/threonine kinase domain. We demonstrate that HIPK4 could phosphorylate human p53 protein at serine 9, both in vitro and in vivo. Among known p53-responsive promoters, activity of the human survivin promoter, which is repressed by p53, was decreased by HIPK4 in p53 functional A549 cells. Human BCL2-associated X protein-promoter activity was not affected. These findings suggest that phosphorylation of p53 at serine 9 is important for p53 mediated transcriptional repression.

Estrogen-related receptor alpha modulates the expression of adipogenesis-related genes during adipocyte differentiation.

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERRalpha in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERRalpha and ERRalpha-related transcriptional coactivators, peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) and PGC-1beta, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERRalpha-specific siRNA results in mRNA down-regulation of fatty acid binding protein 4, PPARgamma, and PGC-1alpha in 3T3-L1 cells in the adipogenesis medium. ERRalpha and PGC-1beta mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERRalpha in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERRalpha may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.

c-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements.

c-Ski inhibits transforming growth factor-beta (TGF-beta) signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-beta-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. In this study, we examined how c-Ski inhibits both TGF-beta-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-beta1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-beta signaling.

Molecular basis of constitutive production of basement membrane components. Gene expression profiles of Engelbreth-Holm-Swarm tumor and F9 embryonal carcinoma cells.

Engelbreth-Holm-Swarm (EHS) tumors produce large amounts of basement membrane (BM) components that are widely used as cell culture substrates mimicking BM functions. To delineate the tissue/organ origin of the tumor and the mechanisms operating in the BM overproduction, a genome-wide expression profile of EHS tumor was analyzed using RIKEN cDNA microarrays containing approximately 40,000 mouse cDNA clones. Expression profiles of F9 embryonal carcinoma cells that produce laminin-1 and other BM components upon differentiation into parietal endoderm-like cells (designated F9-PE) were also analyzed. Hierarchical clustering analysis showed that the gene expression profiles of EHS and F9-PE were the most similar among 49 mouse tissues/organs in the RIKEN Expression Array Database, suggesting that EHS tumor is parietal endoderm-derived. Quantitative PCR analysis confirmed that not only BM components but also the machineries required for efficient production of BM components, such as enzymes involved in post-translational modification and molecular chaperones, were highly expressed in both EHS and F9-PE. Pairs of similar transcription factor isoforms, such as Gata4/Gata6, Sox7/Sox17, and Cited1/Cited2, were also highly expressed in both EHS tumor and F9-PE. Time course analysis of F9 differentiation showed that up-regulation of the transcription factors was associated with those of BM components, suggesting their involvement in parietal endoderm specification and overproduction of the BM components.

Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage.

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Lymphoid enhancer factor 1 makes cells resistant to transforming growth factor beta-induced repression of c-myc.

The activation of lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated transcription by sustained expression of beta-catenin and the loss of transforming growth factor beta (TGF-beta) signaling are essential steps in carcinogenesis, particularly for cancers of the colon, breast, and liver. The oncogene c-myc is a common target of both of these signaling pathways and a key regulator of cell cycle progression. Here we have identified a novel LEF/TCF-responsive element in the promoter of the human c-myc gene. beta-Catenin activated the transcriptional activity of the c-myc promoter by binding to this element in various cell lines. When TCF-4 was bound to this element, TGF-beta dissociated beta-catenin and repressed the transcriptional activity of the c-myc promoter. However, TGF-beta could not dissociate beta-catenin and could not repress c-myc transcription when LEF-1 was bound to the element instead of TCF-4. These findings suggest that enhanced expression of LEF-1, which occurs frequently in colon cancer, may make cells refractory to the down-regulation of c-myc and the subsequent growth arrest induced by TGF-beta.

c-myc is a downstream target of the Smad pathway.

c-Myc is one of the most potent regulators of cell cycle progression in higher eukaryotes. Down-regulation of c-Myc is a critical event for growth inhibition induced by transforming growth factor-beta (TGF-beta) and is frequently impaired in cancer cells. We determined a Smad-responsive element in the c-myc promoter. This element is a complex of the TGF-beta1 inhibitory element (TIE) originally identified in the transin/stromelysin promoter and an E2F site responsible for transcriptional activation of the c-myc promoter. Smad3 and E2F-4 directly bound to the element (TIE/E2F), and substitution of two nucleotides in TIE/E2F impaired binding of both Smad3 and E2F-4 as well as serum-induced activation and TGF-beta-induced suppression of the c-myc promoter activity. Smads bound TIE/E2F within 1 h after stimulation with TGF-beta, before the suppression of c-myc transcription, whereas binding of p130 to TIE/E2F became augmented later than 12 h. TGF-beta signaling did not compete with E2F-4 for binding to TIE/E2F, but reduced p300 co-immunoprecipitating with E2F-4. Therefore, TGF-beta signaling may suppress c-myc promoter activity by dissociating p300 from E2F-4.