The role of BRCA1 in transcriptional regulation and cell cycle control

The exact functions of BRCA1 have not been fully described but it now seems apparent that it has roles in DNA damage repair, transcriptional regulation, cell cycle control and most recently in ubiquitylation. These functions of BRCA1 are most likely interdependent but this review will focus on the role of BRCA1 in relation to transcriptional regulation and in particular how this impacts upon cell cycle control. We will (i) describe the structure of BRCA1 and how it may contribute to its transcription function; (ii) describe the interaction of BRCA1 with the core transcriptional machinery (RNA polII); (iii) describe how BRCA1 may regulate transcription at an epigenetic level through chromatin modification; (iv) discuss the role of BRCA1 in modulating transcription through its association with sequence-specific transcription factors. Finally, we will discuss the possible effects of BRCA1 transcriptional regulation on downstream targets with known roles in cell cycle control.

Interleukin-8 signaling attenuates TRAIL- and chemotherapy-induced apoptosis through transcriptional regulation of c-FLIP in prostate cancer cells

Chemotherapy-induced interleukin-8 (IL-8) signaling reduces the sensitivity of prostate cancer cells to undergo apoptosis. In this study, we investigated how endogenous and drug-induced IL-8 signaling altered the extrinsic apoptosis pathway by determining the sensitivity of LNCaP and PC3 cells to administration of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL induced concentration-dependent decreases in LNCaP and PC3 cell viability, coincident with increased levels of apoptosis and the potentiation of IL-8 secretion. Administration of recombinant human IL-8 was shown to increase the mRNA transcript levels and expression of c+FLIPL and c-FLIPS, two isoforms of the endogenous caspase-8 inhibitor. Pretreatment with the CXCR2 antagonist AZ10397767 significantly attenuated IL-8-induced c-FLIP mRNA up-regulation whereas inhibition of androgen receptor- and/or nuclear factor-kappa B-mediated transcription attenuated IL-8-induced c-FLIP expression in LNCaP and PC3 cells, respectively. Inhibition of c-FLIP expression was shown to induce spontaneous apoptosis in both cell lines and to sensitize these prostate cancer cells to treatment with TRAIL, oxaliplatin, and docetaxel. Coadministration of AZ10397767 also increased the sensitivity of PC3 cells to the apoptosis-inducing effects of recombinant TRAIL, most likely due to the ability of this antagonist to block TRAIL- and IL-8-induced up-regulation of c-FLIP in these cells. We conclude that endogenous and TRAIL-induced IL-8 signaling can modulate the extrinsic apoptosis pathway in prostate cancer cells through direct transcriptional regulation of c-FLIP. Therefore, targeted inhibition of IL-8 signaling or c-FLIP expression in prostate cancer may be an attractive therapeutic strategy to sensitize this stage of disease to chemotherapy.

Progressive lung cancer determined by expression profiling and transcriptional regulation

Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.

Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15

Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1.

Bioinformatic evaluation of transcriptional regulation of WNT pathway genes with reference to diabetic nephropathy

Objective: Diabetic nephropathy (DN) is a microvascular complication of diabetes. Members of the WNT/ β-catenin pathways have been implicated in interstitial fibrosis and glomerular sclerosis, characteristic hallmarks of DN. These processes are controlled, in part, by transcription factors (TFs), proteins which bind to gene promoter regions attenuating their regulation. We sought to identify predicted cis-acting transcription factor binding sites (TFBS) over-represented within the promoter regions of WNT pathway members compared to genes across the genome.Methods: We assessed the frequency of 62 TFBS motifs from the JASPAR databases on 65 WNT pathway genes. P-values were estimated on the hypergeometric distribution for each TF. Gene expression profiles of enriched motifs were examined from DN-related datasets to assess clinical significance.Results: TFBS motifs transcription factor AP-2 alpha (TFAP2A), myeloid zinc finger 1 (MZF1), and specificity protein 1 (SP1) were significantly enriched within WNT pathway genes (P-values<6.83x10-29, 1.34x10-11 and 3.01x10-6 respectively). MZF1 gene expression was significantly increased in DN in a whole kidney dataset (fold change = 1.16; 16% increase; P = 0.03). TFAP2A gene expression was decreased in an independent dataset (fold change = -1.02; P = 0.03). SP1 was not differentially expressed in any datasets examined.Conclusions: Three TFBS profiles are significantly enriched within the WNT pathway genes examined highlighting the use of in silico analyses for identifying key regulators of this pathway. Modification of TF binding to gene promoter regions involved in DN pathology may limit progression, making refinement of targeted therapeutic strategies possible through clearer delineation of their role.

Role played by BRCA1 in transcriptional regulation in response to therapy

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor, implicated in the hereditary predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of cellular processes including DNA repair and recombination, cell cycle checkpoint control, chromatin remodelling and ubiquitination. In addition, substantial data now exist to suggest a role for BRCA1 in transcriptional regulation; BRCA1 has been shown to interact with the Pol II holoenzyme complex and to interact with multiple transcription factors, such as p53 and c-Myc. We have previously identified a range of BRCA1 transcriptional targets and have linked these to specific cellular pathways, including cell cycle checkpoint activation and apoptosis. Current research is focused on the transcriptional mechanisms that underpin the association of BRCA1 deficiency with increased sensitivity to DNA damage-based chemotherapy and resistance to spindle poisons.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

The regulation and role of osteopontin in malignant transformation and cancer.

Osteopontin (OPN) is a predominantly secreted extracellular matrix glycophosphoprotein which binds to alpha v-containing integrins and has an important role in malignant cell attachment and invasion. High OPN expression in the primary tumor is associated with early metastasis and poor outcome in human breast and other cancers. Forced OPN overexpression in benign cells may induce neoplastic-like cell behaviour including increased attachment and invasion in vitro as well as the ability to metastasize in vivo. Conversely, OPN inhibition by antisense cDNA impedes cell growth and tumor forming capacity. OPN is not mutationally activated in cancer but its expression is regulated by Wnt/Tcf signaling, steroid receptors, growth factors, ras, Ets and AP-1 transcription factors. Presumably these factors are implicated in induction of OPN overexpression in cancer. Greater understanding of the role of OPN in neoplastic change and its transcriptional regulation may enable development of novel cancer treatment strategies

Regulation of cyclin D1 RNA stability by SNIP1

Cyclin D1 expression represents one of the key mitogen-regulated events during the G1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP–associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1a (IRE1a)-dependent manner. Knockdown of XBP1 or IRE1a by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.