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Analyzing The Effects of ProL1 Upregulation On LNCaP Global Gene Expression

ABSTRACT: Purpose: To examine the effects of proL1 upregulation on global gene expression in LNCaP cells using RNA sequencing (RNA-Seq) Methods: LNCaP cells were transformed to overexpress proL1 using lentiviral vectors. The proL1 overexpressing variant (LNCaP 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 1110 differentially expressed genes between LNCaP proL1+ and its parental cell line. Of these differentially expressed genes (DEGs), 209 were in common with PC3 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. According to these databases, proL1 overexpression regulated gene expression in ontological groups involved in morphogenesis and signal transduction. DEGs were also overrepresented in ontological groups potentially involved in vascularization and blood flow regulation. 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. For example, overexpression of ProL1 in LNCaP reduced expression of the progesterone receptor gene (PGR). Activation of PGR is a positive modulator of cell division and the reduced expression of PGR in LNCaP proL1 cells might therefore explain the negative effect on tumor growth in female mice. Overall, our data associates the development of PrCa with overexpression of opiorphin and suggests genetic pathways by which they contribute to tumor growth and development of androgen-insensitivity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156218 | GEO | 2021/08/12

REPOSITORIES: GEO

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Project description: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.

Project description:Following androgen ablation therapy (AAT), the vast majority of prostate cancer patients develop treatment resistance with a median time of 18-24 months to disease progression. To identify molecular targets that aid in prostate cancer cell survival and contribute to the androgen independent phenotype, we evaluated changes in LNCaP cell gene expression during 12 months of androgen deprivation. At time points reflecting critical growth and phenotypic changes, we performed Affymetrix expression array analysis to examine the effects of androgen deprivation during the acute response, during the period of apparent quiescence, and during the emergence of highly proliferative, androgen-independent prostate cancer cells (LNCaP-AI). We discovered alterations in gene expression for a host of molecules associated with promoting prostate cancer cell growth and survival, regulating cell cycle progression, apoptosis and adrenal androgen metabolism, in addition to AR co-regulators and markers of neuroendocrine disease. These findings illustrate the complexity and unpredictable nature of cancer cell biology and contribute greatly to our understanding of how prostate cancer cells likely survive AAT. The value of this longitudinal approach lies in the ability to examine gene expression changes throughout the cellular response to androgen deprivation; it provides a more dynamic illustration of those genes which contribute to disease progression in addition to specific genes which constitute a malignant androgen-independent phenotype. In conclusion, it is of great importance that we employ new approaches, such as the one proposed here, to continue exploring the cellular mechanisms of therapy resistance and identify promising targets to improve cancer therapeutics. Experiment Overall Design: To identify molecular targets that aid in prostate cancer cell survival and contribute to the androgen independent phenotype, we evaluated changes in LNCaP cell gene expression during 12 months of androgen deprivation. At time points reflecting critical growth and phenotypic changes, we performed Affymetrix expression array analysis to examine the effects of androgen deprivation during the acute response, during the period of apparent quiescence, and during the emergence of highly proliferative, androgen-independent prostate cancer cells (LNCaP-AI).

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Analyzing The Effects of ProL1 Upregulation On LNCaP Global Gene Expression

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