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Whole genome sequence analysis of BT-474 using Complete Genomics’ standard and Long Read Fragment technologies

ABSTRACT: Whole genome sequence analysis of BT-474 using Complete Genomics’ standard and Long Read Fragment technologies

PROVIDER: PRJEB10587 | ENA |

REPOSITORIES: ENA

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Project description:Background: Neuropilin-1 (NRP-1) is a non-tyrosine kinase glycoprotein receptor that elicits multiple functions depending on the ligand bound. It is known to promote cancer progression however; transcriptome-wide changes triggered by NRP-1 are not defined. In this study we aimed to identify novel molecules associated with NRP-1 expression using a global transcriptomic approach in a recombinant breast cancer cell model. Methods: NRP-1 was stably overexpressed in the BT-474 breast cancer cell line (BT-474 NRP-1) and subjected to next generation RNA sequencing using the Illumina HiSeq 4000 sequencing system. Two replicates each of the control BT-474 and BT474-NRP1 were utilized for sequencing. The count per million (CPM) method was utilized for filtering low counts/noise by NOISeq. The clean reads were mapped to reference genome using HISAT/ Bowtie2 tool. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method was utilized to calculate the expression levels. False Detection Rate (FDR) ≤ 0.001, absolute value of Log2 ratio ≥ 2 and consistency between the two replicates were used as the default threshold to identify significant DEGs. Results: A total of 22,655,647 clean reads from 22,914,684 raw sequencing reads were generated upon sequencing. 22 upregulated and 61 downregulated genes were identified. Among the DEGs, 11 upregulated and 11 downregulated genes were shortlisted based on their known roles in cancer and/or EMT for confirmation with real-time qPCR. 2 upregulated genes TNC and TARP and 5 downregulated genes ACE, APOD, DDIT3, P2RX6 and ATF3 indicated consistently differential expression with qPCR. Conclusion: We report for the first time the global transcriptome-wide changes elicited by NRP-1 overexpression in a breast cancer cell model. Our study identified the novel association between several candidate molecules such as TNC, a known oncogene, and NRP-1 expression that may controbute to the tumourigenic role of NRP-1 in breast cancer.

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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Whole genome sequence analysis of BT-474 using Complete Genomics’ standard and Long Read Fragment technologies

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