Frequent somatic mutations of the transcription factor ATBF1 in human prostate cancer.

Cancer often results from the accumulation of multiple genetic alterations. Although most malignancies are sporadic, only a small number of genes have been shown to undergo frequent mutations in sporadic cancers. The long arm of chromosome 16 is frequently deleted in human cancers, but the target gene for this deletion has not been identified. Here we report that ATBF1, which encodes a transcription factor that negatively regulates AFP and MYB but transactivates CDKN1A, is a good candidate for the 16q22 tumor-suppressor gene. We narrowed the region of deletion at 16q22 to 861 kb containing ATBF1. ATBF1 mRNA was abundant in normal prostates but more scarce in approximately half of prostate cancers tested. In 24 of 66 (36%) cancers examined, we identified 22 unique somatic mutations, many of which impair ATBF1 function. Furthermore, ATBF1 inhibited cell proliferation. Hence, loss of ATBF1 is one mechanism that defines the absence of growth control in prostate cancer.

Infrequent mutation of ATBF1 in human breast cancer.

Deletion at chromosome 16q is frequent in prostate and breast cancers, suggesting the existence of one or more tumor suppressor genes in 16q. Recently, the transcription factor ATBF1 at 16q22 was identified as a strong candidate tumor suppressor gene in prostate cancer, and loss of ATBF1 expression was associated with poorer prognosis in breast cancer. In the present study, we examined mutation, expression, and promoter methylation of ATBF1 in 32 breast cancer cell lines. Only 2 of the 32 cancer cell lines had mutations, although 18 nucleotide polymorphisms were detected. In addition, 24 of 32 (75%) cancer cell lines had reduced ATBF1 mRNA levels, yet promoter methylation was not involved in gene silencing. These findings suggest that ATBF1 plays a role in breast cancer through transcriptional downregulation rather than mutations.

Regulation of tissue factor in microvascular dermal endothelial cells.

Inflammation is accompanied by activation of the coagulation cascade, manifested by thrombosis and fibrin generation. Whereas endothelial cells normally provide a nonthrombogenic surface, inflammatory mediators may induce the expression of tissue factor, rendering their surface thrombogenic. In order to define the mechanisms regulating the expression of tissue factor in the skin microvasculature, we examined tissue factor expression in human dermal microvascular endothelial cells. Quiescent human dermal microvascular endothelial cells did not constitutively express tissue factor protein, but were induced to express tissue factor by treatment with tumor necrosis factor-alpha in a time- and concentration-dependent fashion. Increased expression of tissue factor protein was accompanied by increases in steady-state mRNA levels. Tumor necrosis factor-alpha treatment resulted in increased expression of tissue factor heterogeneous nuclear RNA without changes in mRNA stability, suggesting that increased mRNA was mediated primarily via increased tissue factor gene transcription. In order to define the pathways regulating tissue factor induction, we examined the effects of MG-132, an inhibitor of nuclear factor-kappaB activation, PD98059, an inhibitor of MEK1 action, and SB203580, an inhibitor of activated p38 activity. MG132 only partially blocked tumor necrosis factor-alpha-induced tissue factor protein expression, despite an almost complete inhibition of tumor necrosis factor-alpha-induced E-selectin expression. In contrast, SB203580, almost completely inhibited tumor necrosis factor-alpha-induced tissue factor expression but inhibition of MEK1 by PD98059 had a minimal effect on tumor necrosis factor-alpha-mediated tissue factor induction in human dermal microvascular endothelial cells. Both SB203580 and MG132 treatment inhibited tumor necrosis factor-alpha-mediated increases in tissue factor mRNA and tissue factor gene transcription as measured by expression of tissue factor heterogeneous nuclear RNA. These data support a transcriptional role for both nuclear factor-kappaB and p38 mitogen-activated protein kinase, but not MEK1 in tissue factor gene expression in human dermal microvascular endothelial cells.

Iron chelators inhibit VCAM-1 expression in human dermal microvascular endothelial cells.

Vascular cell adhesion molecule (VCAM)-1 expression may be coupled to redox-sensitive regulatory pathways, and iron may play a role in generation of reactive oxygen species that participate in these signaling pathways. To investigate the role of iron in TNF alpha-induced VCAM-1 gene expression, human dermal microvascular endothelial cells (HDMEC) were stimulated with TNF alpha in the presence of iron chelators and examined for expression of VCAM-1. The iron chelators dipyridyl (DP) and desferoxamine (DFO) inhibited VCAM-1 protein and mRNA induction in a concentration- and time-dependent manner. The induction of VCAM-1 was not inhibited by nonmetal binding reactive oxygen species (ROS) scavengers, implying a direct effect of iron in the expression of these adhesion molecules. The effect of iron was mediated at the level of gene transcription since pretreatment with DP abrogated the TNF alpha-mediated up-regulation of VCAM-1 heterogeneous nuclear RNA. Pretreatment of HDMEC with DP prior to TNFalpha treatment had no effect on p65 nuclear localization, DNA binding, or serine phosphorylation. DP pretreatment inhibited TNF alpha- and IFN gamma-mediated interferon regulatory factor 1 (IRF-1) protein expression, although restoration of IRF-1 expression failed to reconstitute VCAM-1 expression. DP treatment also blocked VCAM-1 induction in human umbilical vein endothelium and blocked induction of a host of NF-kB activated genes in HDMEC including ICAM-1, IL-8, and tissue factor. I kappa B alpha, an NF-kappa B inducible and constitutively accessible gene not requiring chromatin remodeling for transcription, was not affected by DP treatment. These data suggest that iron plays a critical role in TNF alpha mediated VCAM-1 induction in HDMEC, and the target for iron effects may be IRF-1, NF-kappa B, and potentially chromatin remodeling.

Induction of interferon regulatory factor 1 expression in human dermal endothelial cells by interferon-gamma and tumor necrosis factor-alpha is transcriptionally regulated and requires iron.

Interferon regulatory factor-1 is a transcription factor that is linked to the expression of genes important in the initiation of the inflammatory response and the control of cell cycle. In this study, we determined that the generation of interferon regulatory factor-1 expression in human dermal microvascular endothelial cells was transcriptionally mediated by tumor necrosis factor-alpha or interferon-gamma via iron-dependent pathways. The induction of interferon regulatory factor-1 protein and the up-regulation of interferon regulatory factor-1 mRNA levels was inhibited when cells were pretreated with the iron chelators 2-2-dipyridyl or deferoxamine. This inhibition of interferon regulatory factor-1 expression was associated with loss of interferon regulatory factor-1 binding to the interferon-stimulated response element as assessed by electrophoretic mobility shift assay. Addition of exogenous iron with the iron chelator resulted in reconstitution of cytokine responsiveness, thus demonstrating iron as the target for the chelator effect. Both tumor necrosis factor-alpha and interferon-gamma-induced interferon regulatory factor-1 gene transcription, as assessed by the measurement of unspliced, nascent, heterogeneous nuclear RNA, and treatment with iron chelators blocked tumor necrosis factor-alpha or interferon-gamma mediated interferon regulatory factor-1 gene transcription. Iron was not essential, however, for the association of interferon regulatory factor-1 mRNA with polyribosomes, suggesting iron was not essential for interferon regulatory factor-1 protein translation. Through such inhibitory regulation on pro-inflammatory transcription factors, iron chelators may serve as anti-inflammatory agents.

Regulation of KLF5 involves the Sp1 transcription factor in human epithelial cells.

Human Kruppel-like factor 5 (hKLF5) is a transcription factor with a potential tumor suppressor function in prostate and breast cancers. In the majority of cancer samples examined, a significant loss of expression for KLF5 has been detected. Whereas hemizygous deletion appears to be responsible for KLF5's reduced expression in about half of the cases, the mechanism for reduction is unknown in the remaining half; gene promoter methylation does not appear to be involved. In this report, we studied the regulation of KLF5 and cloned and functionally characterized a 1944-bp fragment of the 5'-flanking region of the hKLF5 gene. Several mitogens as well as global demethylation induced the expression of KLF5, implicating multiple factors in the regulation of KLF5. KLF5's promoter lacks a TATA box and has a GC-rich region. Deletion mapping in combination with promoter activity assay showed that multiple cis-elements are involved in the transcriptional regulation of KLF5, some of which may play a repressor role whereas some others play an enhancer role. The Sp1 site between position -239 and -219 is essential for a basal promoter activity. Deletion or mutations of this Sp1 site significantly reduced promoter activity in several epithelial cell lines. Electrophoretic mobility shift assays (EMSAs) revealed that the Sp1 site binds Sp1 protein in nucleic extracts of different cell lines. In addition, overexpression of Sp1 protein transactivates KLF5 promoter activity. These findings suggest that Sp1 is a key transcription factor in KLF5's dynamic transcriptional regulation.

Sox2 is an androgen receptor-repressed gene that promotes castration-resistant prostate cancer.

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways.

Stress-activated kinase pathway alteration is a frequent event in bladder cancer.

OBJECTIVES: The stress-activated MAP kinases (SAPK) signaling pathways play a critical role in the cellular response to toxins and physical stress, mediate inflammation, and modulate carcinogenesis and tumor metastasis. The stress-activated MAP kinases (MAPK) c-Jun N-terminal kinase (JNK) and p38 are activated upon phosphorylation by a widely expressed and conserved family of upstream MAP kinase kinases (MAP2K). Signaling mediated by p38 and JNK has well-established importance in cancer, yet the contribution of this pathway in urothelial bladder cancer is not understood. This study evaluated stress-activated MAP kinase pathway expression in cell lines derived from human urothelial carcinomas. MATERIALS AND METHODS: Total protein lysates from a panel of human urothelial bladder cancer cell lines (RT4, T24, UMUC-3, J82, 5637, 253J, and 253J-BV) were analyzed by immunoblotting for the JNK and p38 MAPKs, as well as MKK3, MKK4, MKK6, and MKK7. Quantitative real time PCR was utilized to determine mRNA expression levels of the MAP2Ks. Stress stimuli (sorbitol, hydrogen peroxide, and UV irradiation) were used to active p38, which was measured by phospho-antibody. RESULTS: Although protein levels were variable, all cell lines expressed p38 and JNK. On the other hand, with the exception of the well-differentiated cell line RT4, each cell line had a reduction or absence of expression of one or more MAP2K. 253J and 253J-BV exhibited no expression of MKK6, even when an excess of protein was queried. mRNA levels indicated that both transcriptional and post-transcriptional mechanisms are involved in the regulation of MAP2Ks. Decreased MAP2K expression correlated with decreased ability to activate p38 in response to stress stimuli. CONCLUSIONS: Aberrant MAP2K protein expression indicates that altered cellular signal transduction mediated via JNK and p38 may be common in bladder cancer. Down-regulation of MAP2Ks likely occurs at both the transcriptional and post-transcriptional levels. Consistent with the known function of p38 and JNK in apoptosis, defects in normal pathway function caused by decreased expression of upstream MAP2Ks may provide a survival advantage to bladder cancer cells. Further investigations should focus on identifying a functional role for these pathways in bladder cancer development.

KLF5 promotes cell proliferation and tumorigenesis through gene regulation and the TSU-Pr1 human bladder cancer cell line.

KLF5 is a transcription factor that plays important roles in multiple physical and pathological processes, including cell growth, cell cycle regulation, and angiogenesis. To better characterize KLF5 function in bladder carcinogenesis, we established stable TSU-Pr1 cell clones expressing different levels of KLF5. These clones were then characterized for cell growth, cell cycle progression, tumorigenesis, and alteration in gene expression. Overexpression of KLF5 promoted tumorigenesis of the TSU-Pr1 cancer cells in mice. Consistently, KLF5 increased G1 to S phase transition, which was accompanied by the upregulation of cyclin D1, phosphorylation of MAPK and Akt, and reduced protein levels for CDK inhibitors p27 and p15. Microarray analysis combined with expression verification in different cell systems identified a number of additional genes that are potentially regulated by KLF5, including HBP17, ITGA6, and RAIG1. These findings suggest that the KLF5 transcription factor plays an oncogenic role in the TSU-Pr1 bladder cancer cell line through the regulation of a subset of genes.