ABSTRACT: Purpose: To examine the effects of proL1 upregulation on global gene expression in PC3 cells using RNA sequencing (RNA-Seq) Methods: PC3 cells were transformed to overexpress proL1 using lentiviral vectors. The proL1 overexpressing variant (PC3 proL1+) and their parental cell line were plated in 6-well plates in triplicate (3 wells for each cell line). After 3 days, their RNA was isolated using the Qiagen RNeasy Mini kit and their global gene expression patterns were analyzed using RNA-Seq Results: We identified 1698 differentially expressed genes between PC3 proL1+ and its parental cell line. Of these differentially expressed genes (DEGs), 209 were in common with LNCaP when proL1 was overexpressed. To identify biological functions that may be regulated through overexpression of proL1 in LNCaP cells, the list of DEGs was submitted to the DAVID, GOC, and KEGG databases, which identified 1250, 1366, and 480 unique genes within each database, respectively, which were then used for ontological analysis. According to these databases, proL1 overexpression regulated gene expression in ontological groups involved "anatomical structure development/morphogenesis" (more specifically "prostate gland morphogenesis"), extracellular matrix organization," "signaling," and "signal transduction. DEGs were also overrepresented in ontological groups potentially involved in vascularization and blood flow regulation. Analysis of the list of DEGs using the KEGG database identified the ontologic group "pathways in cancer" as having the greatest number of DEGs. DEGs were also identified with significant overrepresentation in specific biochemical pathways, such as the P13K-Akt, VEGF, and MAPK signaling pathways as well as steroid metabolic pathways. However, there were differences in the regulation of specific genes related to the androgen response between LNCaP and PC3. Unlike PC3, where overexpression of ProL1 caused a significant increase in expression of the androgen receptor gene (AR) and a decrease in expression of the estrogen receptor (ESR1), in LNCaP neither of these genes were changed in expression. In contrast, proL1 overexpression in LNCaP reduced expression of the progesterone receptor gene (PGR) but not in PC3 cells. Conclusion: Global analysis of the changes in gene expression caused by proL1 overexpression in PC3 and LNCaP cells supports that it has a role both in the modulation of genetic pathways involved in both overcoming hypoxia and the development of androgen-insensitivity. Opiorphins have previously been shown to be directly involved in regulation of blood-flow to tissues through their modulation of smooth muscle tone, and therefore their upregulation in tumors may directly contribute to overcoming the hypoxic barrier that develops in the growing tumor. In addition, overexpression of ProL1 in both PC3 and LNCaP cells modulated expression of genes involved in angiogenesis and morphogensis; the activation of these pathways would likely be involved in vascularization of the tumor and thereby also contribute to overcoming hypoxia. Overexpression of proL1 also affected genes involved in steroid metabolism and response pathways in both LNCaP and PC3, which could potentially contribute to the modulation of androgen sensitivity. In PC3 proL1+ cells we observed a significant upregulation of the Androgen Receptor (AR). Increased activity of the AR could subsequently lead to the activation of secondary messengers involved in modulating the activity of other signaling pathways such as the PI3K-Akt signaling and MAPK pathways, which had significant over-representation of DEGs in PC3 proL1+ cells. The PI3K-Akt signaling pathway is considered one of the most commonly dysregulated pathways in prostate cancer with approximately 40% of early stage, and 70-100% of advanced staged cancer exhibiting dysregulation of this pathway.
