Unknown,Transcriptomics,Genomics,Proteomics

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Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer

ABSTRACT: We report the chromatin binding sites of HOXB7 transcription factor in BT-474 breast cancer cell line using ChIP-sequencing. We validated the chromatin binding sites in BT-474, MDA-MB-361, MCF7 and T-47D breast cancer cell lines using ChIP-qPCR. The ChIP experiments have been performed using HOXB7 antibody and IgG non-specific antibody as a negative control. The direct downstream target genes of HOXB7 were identified by analyzing the expression of genes located nearby HOXB7 binding sites in HOXB7 knockdown versus control cells using qRT-PCR. Examination of chromatin binding sites of HOXB7 in BT-474 breast cancer cell line using ChIP-seq. Four parallel IgG samples were sequenced, merged together and used as a control data set. Two parallel HOXB7 ChIP samples were sequenced and merged for each replicate, AF1 and AF2. Both HOXB7 ChIP replicates (AF1 and AF2) contained approximately the same amount of reads as the merged IgG control data set.

ORGANISM(S): Homo sapiens

SUBMITTER: Henna Heinonen

PROVIDER: E-GEOD-47164 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Accumulating evidence suggests that the androgen receptor (AR) and its endogenous ligands influence disease progression in breast cancer (BCa). However, AR-mediated changes in BCa differ among the various BCa subtypes according to their hormone receptor profile (i.e presence/absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, (HER2)). Thus, we explored the androgen-regulated transcriptomic changes in the ER+PR+HER2+ BCa cell line, BT-474, and compared them with PR-mediated changes. Methods: We performed RNA sequencing analysis, in treated BT-474 cells with dihydrotestosterone (DHT) and progesterone (PROG). Validation of the top ten differentially androgen-regulated genes and a number of other genes found in enriched signaling pathways was performed by qRT-PCR in BT-474 and other BCa cell lines. A parallel reaction monitoring targeted proteomic approach was developed for the verification of selected transcripts at the protein level. Results: We detected 19,450 transcripts, of which 224 were differentially regulated after DHT treatment. The increased expression of two well-known androgen-regulated genes, KLK2 (p<0.05) and KLK3 (p<0.001), confirmed the successful androgen stimulation in BT-474 cells. The transcription factor, ZBTB16, was the most highly upregulated gene, with ~1000-fold change (p<0.001). Pathway enrichment analysis revealed downregulation of the DNA replication processes (p<0.05), and upregulation of the androgen signaling and fatty acid metabolism pathways (p<0.05). Changes related to PROG-treatment showed opposite effects in gene expression than DHT treatment. Similar expression profiles were observed among other BCa cell lines expressing high levels of AR (ZR75.1 and MBA-MB-453). The parallel reaction monitoring targeted proteomic analysis further confirmed that altered protein expression (e.g. KLK3, and ALOX15B) in the supernatant and cell lysate of DHT-treated BT-474 cells, compared to control cells. Discussion: Collectively, our findings suggest that AR modulates the metabolism of BT-474 cells by affecting expression of a large number of genes and proteins. Based on further pathway analysis, we suggest that AR acts as a tumor suppressor in BT-474 cells.

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Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer

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