Abstract 2800: Target RNA sequencing analysis of hypoxia induced expression of stem cell genes in primary cultures of prostate cancer cells

Abstract

Abstract Hypoxia plays important regulatory roles in expanding the tumor stem cell population. In prostate cancer, hypoxia is a feature of poor prognosis. The association of prostate cancer stem cells with hypoxia has been investigated. The altered stem cell gene expression in prostate cancer cells under the hypoxic condition has been documented. The majority of the investigations were performed with established prostate cancer cell lines. However, little is known about the expression of stem cell genes in primary cultures of prostate cancer. In this study, we examined the hypoxia-regulated expression of stem cell genes with primary cultures of human prostate cancer. Cells were isolated from three patient's prostate cancer samples. Isolated cells were cultured in prostate epithelium culture medium CnT-52. After 48 hours of exposure to 1% oxygen, RNA was extracted and quantified. The library was prepared with Illumina's TruSeq Targeted RNA Expression Stem Cell Panel. The sequencing was performed on Miseq platform with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter and analyzed further by Truseg Target RNA v1.0(Illumina), Cufflinks Assembly &DE(Illumina), and Biomedical Genomics Workstation(Qiagen).The results showed that of the 100 tested stem cell genes, seven genes were significantly upregulated(FDR p-value<0.05). They are BMP1, BMP2, FZD7, JUND, NOTCH1, NOTCH3, and PPARD. Three genes, PSEN2, SDAD1, and SOX2, were significantly down-regulated. To validate sequencing results, we examined NOTCH1 and NOTCH3 expression with RT-PCR. Compared to cells under normoxic condition, hypoxia induced expression of NOTCH1 increased by 3.7, 3.9 and 3.1 fold, respectively among three hypoxic cultures, while the expression of NOTCH3 increased by 8.1, 5.9, and 7.5 fold, respectively. However, the investigation of hypoxia affected expression of stem cell genes in LNCaP, an established prostate cell line, showed that the significant decreased expression of BMP1, FZD7, PPARD, and NOTCH3 was seen under hypoxic condition(FDR p-value <0.05). RT-PCR analysis showed that expression of NOTCH3 in hypoxic LNCaP, PC3, and DU145 cells was decreased by 7.7, 6.1, and 1.2 fold, respectively. The expression profile of stem cell genes in response to hypoxia was different between primary cultures of prostate cancer cells and established prostate cancer cell lines. The activation of BMP-NOTCH signaling could be one character of stemness of prostate cancer in response to hypoxia. Citation Format: Hong Yin, Adam H. Greer, Glenn Mills. Target RNA sequencing analysis of hypoxia induced expression of stem cell genes in primary cultures of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2800.