Project description:Overexpression of c-Myc is one of the most common alterations in human cancers, yet it is not clear how this transcription factor acts to promote malignant transformation. To understand the molecular targets of c-Myc function, we have used an unbiased genome-wide location-analysis approach to examine the genomic binding sites of c-Myc in Burkitt's lymphoma cells. We find that c-Myc together with its heterodimeric partner, Max, occupy >15% of gene promoters tested in these cancer cells. The DNA binding of c-Myc and Max correlates extensively with gene expression throughout the genome, a hallmark attribute of general transcription factors. The c-Myc/Max heterodimer complexes also colocalize with transcription factor IID in these cells, further supporting a general role for overexpressed c-Myc in global gene regulation. In addition, transcription of a majority of c-Myc target genes exhibits changes correlated with levels of c-myc mRNA in a diverse set of tissues and cell lines, supporting the conclusion that c-Myc regulates them. Taken together, these results suggest a general role for overexpressed c-Myc in global transcriptional regulation in some cancer cells and point toward molecular mechanisms for c-Myc function in malignant transformation.
Project description:HER2 is a transmembrane receptor tyrosine kinase, which plays a key role in breast cancer due to a common genomic amplification. It is used as a marker to stratify patients in the clinic and is targeted by a number of drugs including Trastuzumab and Lapatinib. HER2 has previously been shown to translocate to the nucleus. In this study, we have explored the properties of nuclear HER2 by analysing the binding of this protein to the chromatin in two breast cancer cell lines. We find genome-wide re-programming of HER2 binding after treatment with the growth factor EGF and have identified a de novo motif at HER2 binding sites. Over 2,000 HER2 binding sites are found in both breast cancer cell lines after EGF treatment, and according to pathway analysis, these binding sites were enriched near genes involved in protein kinase activity and signal transduction. HER2 was shown to co-localise at a small subset of regions demarcated by H3K4me1, a hallmark of functional enhancer elements and HER2/H3K4me1 co-bound regions were enriched near EGF regulated genes providing evidence for their functional role as regulatory elements. A chromatin bound role for HER2 was verified by independent methods, including Proximity Ligation Assay (PLA), which confirmed a close association between HER2 and H3K4me1. Mass spectrometry analysis of the chromatin bound HER2 complex identified EGFR and STAT3 as interacting partners in the nucleus. These findings reveal a global role for HER2 as a chromatin-associated factor that binds to enhancer elements to elicit direct gene expression events in breast cancer cells.
Project description:Antiestrogens (AEs) are widely used for treatment of estrogen receptor alpha (ERα)-positive breast cancer, but display variable degrees of partial agonism in estrogen target tissues and breast cancer (BC) cells. The fact that BC cells resistant to selective ER modulators (SERMs) like tamoxifen (Tam) can still be sensitive to pure AEs, also called selective ER downregulators, suggests different mechanisms of action, some of which may contribute to the more complete suppression of estrogen target genes by pure AEs. We report herein that pure AEs such as fulvestrant induce transient binding of ERα to DNA, followed by rapid release after 30-40 min without loss of nuclear localization. Loss of DNA binding preceded receptor degradation and was not prevented by proteasome inhibition. Chromatin was less accessible in the presence of fulvestrant than with estradiol or Tam as early as 20 min following treatment, suggesting that chromatin remodeling by pure AEs at ERα target regions prevents transcription in spite of receptor binding. SUMO2/3 marks were detected on chromatin at the peak of ERα binding in cells treated with pure AEs, but not SERMs. Furthermore, decreasing SUMOylation by overexpressing the deSUMOylase SENP1 significantly delayed receptor release from DNA and de-repressed expression of estrogen target genes in the presence of fulvestrant, both in ERα-expressing MCF-7 cells and in transiently transfected ER-negative SK-BR-3 cells. Finally, mutation V534E, identified in a breast metastasis resistant to hormonal therapies, prevented ERα modification and resulted in increased transcriptional activity of estrogen target genes in the presence of fulvestrant in SK-BR-3 cells. Together, our results establish a role for SUMOylation in achieving a more complete transcriptional shut-off of estrogen target genes by pure AEs vs. SERMs in BC cells.
Project description:Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes.
Project description:To support cellular homeostasis and mitigate chemotherapeutic stress, cancer cells must gain a series of adaptive intracellular processes. Here we identify that NUPR1, a tamoxifen (Tam)-induced transcriptional coregulator, is necessary for the maintenance of Tam resistance through physical interaction with ESR1 in breast cancers. Mechanistically, NUPR1 binds to the promoter regions of several genes involved in autophagy process and drug resistance such as BECN1, GREB1, RAB31, PGR, CYP1B1, and regulates their transcription. In Tam-resistant ESR1 breast cancer cells, NUPR1 depletion results in premature senescence in vitro and tumor suppression in vivo. Moreover, enforced-autophagic flux augments cytoplasmic vacuolization in NUPR1-depleted Tam resistant cells, which facilitates the transition from autophagic survival to premature senescence. Collectively, these findings suggest a critical role for NUPR1 as a transcriptional coregulator in enabling endocrine persistence of breast cancers, thus providing a vulnerable diagnostic and/or therapeutic target for endocrine resistance.
Project description:Recent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells.Promoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays.We confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas.Our study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.
Project description:Cofactor of BRCA1 (COBRA1) was first identified as a protein that binds to the breast cancer susceptibility gene product BRCA1. COBRA1 modulates estrogen-dependent and independent transcription and suppresses the growth of breast cancer cells. Its expression is significantly reduced in metastatic and recurrent breast cancer, pointing to a tumor suppressor function in breast cancer development. In light of these initial implications of COBRA1 in human breast cancer, the current investigation sought to obtain more direct functional evidence that links COBRA1 with BRCA1 in transcriptional regulation in breast cancer cells. Small hairpin RNA (shRNA)-mediated gene knockdown and gene expression microarray were used to study the impact of COBRA1 and BRCA1 on global transcription in the same breast cancer cell background. The gene expression profiling study in tissue culture cells uncovers a significant overlap of COBRA1- and BRCA1-regulated genes, many of which have been previously implicated in breast cancer progression. The data shown herein support the notion that COBRA1 and BRCA1 may engage in common gene regulatory pathways to suppress breast cancer progression.