<div>Abstract<p><b>Purpose:</b> There is compelling evidence of a relationship between poly(ADP-ribosyl)ation and tumorigenesis; however, much less is known about the role of specific targets of poly(ADP-ribosyl)ation in tumor development. Two forms of the multifunctional transcription factor, CTCF, were previously identified: a 130-kDa protein (CTCF-130), characteristic for cell lines, and a 180-kDa protein (CTCF-180), modified by poly(ADP-ribosyl)ation. This study was aimed to investigate differential poly(ADP-ribosyl)ation of CTCF in normal and tumor breast tissues.</p><p><b>Experimental Design:</b> Western blot analysis, mass spectrometry, and immunohistochemical and immunofluorescent stainings were used to characterize CTCF-130 and CTCF-180 in breast cell lines, primary cultures, and normal and tumor breast tissues. The immunoreactivity score was used for CTCF-130 quantification in tissues.</p><p><b>Results:</b> We discovered that only CTCF-180 is detected in the normal breast tissues, whereas both CTCF-130 and CTCF-180 are present in breast tumors. Using an antibody specific for CTCF-130, we observed that 87.7% of breast tumors were positive for CTCF-130. A negative correlation existed between the levels of CTCF-130, tumor stage, and tumor size. Significantly, a transition from CTCF-180 to CTCF-130 was discovered in primary cultures generated from normal breast tissues, indicating a link between CTCF-130 and proliferation. Conversely, the appearance of CTCF-180 was observed following growth arrest in breast cell lines.</p><p><b>Conclusions:</b> Collectively, our data suggest that the loss of CTCF poly(ADP-ribosyl)ation is associated with cell proliferation and breast tumor development. We propose the use of CTCF-130 as a marker for tumor breast cells and lower levels of CTCF-130 as an indicator of unfavorable prognosis. (Clin Cancer Res 2009;15(18):5762–71)</p></div>
Supplementary Data from Decreased Poly(ADP-Ribosyl)ation of CTCF, a Transcription Factor, Is Associated with Breast Cancer Phenotype and Cell Proliferation
CTCF is a candidate tumor suppressor gene encoding a multifunctional transcription factor. Surprisingly for a tumor suppressor, the levels of CTCF in breast cancer cell lines and tumors were found elevated compared with breast cell lines with finite life span and normal breast tissues. In this study, we aimed to investigate the possible cause for this increase in CTCF content and in particular to test the hypothesis that up-regulation of CTCF may be linked to resistance of breast cancer cells to apoptosis. For this purpose, apoptotic cell death was monitored following alterations of CTCF levels induced by transient transfection and conditional knockdown of CTCF in various cell lines. We observed apoptotic cell death in all breast cancer cell lines examined following CTCF down-regulation. In addition, overexpression of CTCF partially protected cells from apoptosis induced by overexpression of Bax or treatment with sodium butyrate. To elucidate possible mechanisms of this phenomenon, we used a proteomics approach and observed that levels of the proapoptotic protein, Bax, were increased following CTCF down-regulation in MCF7 cells. Taken together, these results suggest that in some cellular contexts CTCF shows antiapoptotic characteristics, most likely exerting its functions through regulation of apoptotic genes. We hypothesize that CTCF overexpression may have evolved as a compensatory mechanism to protect breast cancer cells from apoptosis, thus providing selective survival advantages to these cells. The observations reported in this study may lead to development of therapies based on selective reduction of CTCF in breast cancer cells.
Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. It is the most aggressive form of breast cancer and clinical management is problematic due to lack of available targeted therapies. We have shown that approximately 30% of all TNBCs express estrogen receptor beta (ERβ), a ligand binding transcription factor, and a potential drug target for patients with this form of the disease. Methods: Using novel ERβ-expressing TN cell lines developed in our laboratory, we assessed the impacts of ERβ on proliferation, invasion, migration, and alterations in cell cycle progression following estrogen and ERβ-specific agonist treatment. We also characterized the ERβ transcriptome and cistrome in these models through microarray and ChIP-Seq, respectively. Finally, we determined the tumoral response of cell line xenografts and PDXs treated with 17β-estradiol. Results: We found that both estrogen and multiple ERβ-specific agonists elicit significant anti-tumor effects in ERβ+ TNBC cell lines and tumor xenografts. Activation of ERβ with estrogen and ERβ-specific agonists resulted in inhibition of cell proliferation primarily through a G1/S phase cell cycle arrest. Substantial reductions in cell migration and invasion were also observed following treatment. Microarray studies revealed that ERβ differentially regulated the expression of approximately 1000 genes following estrogen treatment. Of these genes, the most striking effects were observed in a family of small secreted cysteine protease inhibitors known as cystatins, which were highly induced following ERβ activation. ChIP-Seq and ChIP-PCR identified ERβ binding sites in the promoter region of each cystatin and demonstrated ERβ-mediated alterations in chromatin marks and recruitment of PolII around these promoters. We found that cystatins directly interact with TGFβ receptor 2 (TGFβR2) and block downstream TGFβ ligand-mediated activation of the canonical signaling pathway. Depletion of cystatins from conditioned media or through siRNA-mediated silencing reduced the ability of ERβ to elicit these anti-tumor effects. In vivo, estrogen treatment of mice harboring ERβ+ TNBC cell line xenografts or PDXs resulted in increased tumoral expression and serum levels of cystatins, and suppressed tumor growth. Conclusions: Our data demonstrated that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC, both in vitro and in vivo. These effects are partially mediated by cystatins which can interact with, and inhibit, canonical TGFβ signaling, a pathway known to drive TNBC progression. Given the lack of targeted therapies for TNBC patients, the present data suggests that estrogen or ERβ-specific agonists offer a novel approach to manage this subset of patients. Citation Format: Reese JM, Bruinsma ES, Suman VJ, Nelson AW, Chernukhin I, Carroll JS, Ingle JN, Goetz MP, Hawse JR. Biological functions of ERβ in triple negative breast cancer and its utility as a novel therapeutic drug target [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-20.
CTCF is a transcriptional repressor of the c‐ myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down‐regulated during terminal differentiation in most cell lines tested; (ii) CTCF down‐regulation is retarded and less pronounced than that of c‐ myc ; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post‐transcriptional levels.
In Arabidopsis thaliana, HEAT SHOCK TRANSCRIPTION FACTORA1b (HSFA1b) controls resistance to environmental stress and is a determinant of reproductive fitness by influencing seed yield. To understand how HSFA1b achieves this, we surveyed its genome-wide targets (ChIP-seq) and its impact on the transcriptome (RNA-seq) under non-stress (NS), heat stress (HS) in the wild type, and in HSFA1b-overexpressing plants under NS. A total of 952 differentially expressed HSFA1b-targeted genes were identified, of which at least 85 are development associated and were bound predominantly under NS. A further 1780 genes were differentially expressed but not bound by HSFA1b, of which 281 were classified as having development-associated functions. These genes are indirectly regulated through a hierarchical network of 27 transcription factors (TFs). Furthermore, we identified 480 natural antisense non-coding RNA (cisNAT) genes bound by HSFA1b, defining a further mode of indirect regulation. Finally, HSFA1b-targeted genomic features not only harboured heat shock elements, but also MADS box, LEAFY, and G-Box promoter motifs. This revealed that HSFA1b is one of eight TFs that target a common group of stress defence and developmental genes. We propose that HSFA1b transduces environmental cues to many stress tolerance and developmental genes to allow plants to adjust their growth and development continually in a varying environment.
Megestrol-Acetate (MA) has activity after progressive disease (PD) on aromatase inhibitors, providing a disease control rate of 40% and a duration of clinical benefit of 10 months. Ligand-bound progesterone receptor modulates estrogen receptor (ER) activity and reprograms ER-regulated transcription in breast cancer (BC), leading to regression of tumour xenografts. MEGA trial [NCT03024580] aimed to evaluate modulation of steroid receptor activity in advanced BC. Here we present the clinical and preliminary translational data. Eligible patients (pts) had metastatic ER+ HER2- BC, and were randomized to receive MA or another HT agent (AHT), Tamoxifen or Exemestane, with 10 pts planned for each arm. The primary endpoint was PFS, secondary were clinical benefit rate and OS. Tumour biopsies and blood for translational analysis (TA) were collected before day 1 of treatment and at PD. Paired samples were analysed by RNA-seq and by chromatin immunoprecipitation and sequencing (ChIPseq) for histone acetylation (H3K27-Ac). From Aug/17 to Jan/20, a total of 20 pts were recruited and evaluable for efficacy. 16 pts evaluable for TA. No difference was observed between both arms in mPFS, MA = 121 vs AHT = 137 days (p=0.66), or mOS, MA=26 vs AHT=30 months, (p=0.35). The best clinical response at 12 weeks (w) was stable disease for MA was (50%) and AHT (58%) and the duration of response >24w for both was 25%. 10 pts in the MA arm had samples evaluable for TA. RNA-seq (n=7) analysis revealed enrichment for estrogen early and late response pathways in MA resistant samples. There was a global increase in H3K27 acetylation (n=10) at ESR1 motifs in MA resistant samples at PD, although a subgroup of pts (n=3 of 10) showed decreased H3K27Ac at these loci globally. Numerically higher median PFS and OS was observed among pts with decreased (N=3) vs increased (N=7) H3K27Ac at ESR1 motif at PD, respectively mPFS 223 (84-337) vs 137 days (46-291), (p=0.25) and mOS 30 (26-35) vs 14 (7-63) months (p=0.42). Translational analysis find evidence of increased transcriptional activity at ER regulated genes in most resistant samples. This could indicate that MA resistant tumours will retain sensitivity to further HT, requiring future research to proof this hypothesis.
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