PROL1 is essential for xenograft tumor development in mice injected with the human prostate cancer cell-line, LNCaP, and modulates cell migration and invasion.

Background and objective: A growing body of literature suggests modulated expression of members of the opiorphin family of genes (PROL1, SMR3A and SMR3B) is associated with cancer. Recently, overexpression of PROL1 was shown to be associated with prostate cancer, with evidence of a role in overcoming the hypoxic barrier that develops as tumors grow. The primary goal of the present studies was to support and expand evidence for a role of PROL1 in the development and progression of prostate cancer. Material and methods: We engineered knock-out of the opiorphin gene, PROL1, in LNCaP, an androgen-sensitive, human prostate cancer derived, cell-line. Using xenograft assays, we compared the ability of injected LNCaP PROL1 knock-out cell-lines to develop tumors in both castrated and intact male mice with the parental LNCaP and LNCaP PROL1 overexpressing cell-lines. We used RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines. Wound closure and 3D spheroid invasion assays were used to compare cell motility and migration between parental LNCaP cells and LNCaP cells overexpressing of PROL1. Results: The present studies demonstrate that LNCaP cell-lines with consisitutive knock-out of PROL1 fail to develop tumors when injected into both castrated and intact male mice. Using RNAseq to compare global gene expression between the parental and LNCaP PROL1 knock-out cell-lines, we confirmed a role for PROL1 in regulating molecular pathways associated with angiogenesis and tumor blood supply, and also identified a potential role in pathways related to cell motility and migration. Through the use of wound closure and 3D spheroid invasion assays, we confirmed that overexpression of PROL1 in LNCaP cells leads to greater cell motility and migration compared to parental cells, suggesting that PROL1 overexpression results in a more invasive phenotype. Conclusion: Overall, our studies add to the growing body of evidence that opiorphin-encoding genes play a role in cancer development and progression. PROL1 is essential for establishment and growth of tumors in mice injected with LNCaP cells, and we provide evidence that PROL1 has a possible role in progression towards a more invasive, metastatic and castration resistant prostate cancer (PrCa).

Erectile dysfunction resulting from pelvic surgery is associated with changes in cavernosal gene expression indicative of cavernous nerve injury.

Pelvic surgery, even without direct cavernous nerve injury, carries a high risk of post-operative erectile dysfunction. The present studies were aimed at identifying molecular mechanisms by which pelvic surgery results in erectile dysfunction. As a model of pelvic surgery, male Sprague-Dawley rats underwent pelvic laparotomy, avoiding direct cavernous nerve injury. A second group of animals, serving as a model of direct cavernous nerve injury, underwent bilateral transection of the cavernous nerve. Cavernosometry demonstrated, that even in the absence of direct nerve injury, the pelvic surgery model exhibited significant erectile dysfunction 3 days post-operatively. Gene expression profiling also demonstrated that even in this animal model of nerve-sparing pelvic surgery, the profile of differentially expressed genes in cavernosal tissue was indicative of cavernous nerve injury. In addition, although 6 hr after surgery there were significant changes in circulating cytokine/chemokine levels, an inflammatory response in the major pelvic ganglion, cavernous nerve and cavernosal tissue was only observed 3 days post-surgery. Our results validate a rat model of pelvic surgery exhibiting erectile dysfunction and suggest systemic release of cytokines/chemokines following surgical trauma might mediate a pathological inflammatory response in tissues distal to the site of surgical trauma, indirectly resulting in cavernous nerve injury and erectile dysfunction.

Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells.

Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM "relaxant" pathways; excessive activation of these pathways results in priapism.

The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway.

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.

The role of opiorphins (endogenous neutral endopeptidase inhibitors) in urogenital smooth muscle biology.

INTRODUCTION: The opiorphins are a newly characterized class of peptides that act as potent endogenous neutral endopeptidase (NEP) inhibitors. Recent reports have suggested that they play an important role in erectile physiology. AIM: This article reviews recent developments that increase our understanding of the role of the opiorphin family of peptides in erectile physiology. METHODS: During a microarray screen of gene changes that occur in a rat diabetic model of erectile dysfunction (ED), Vcsa1 was one of the most down-regulated genes in the rat corpora. Quantitative real-time polymerase chain reaction demonstrated that in at least three models of diseases that result in ED (diabetes, aging, and cavernous nerve [CN] transection), Vcsa1 was down-regulated in the rat corpora. The human opiorphin family of genes (hSMR3A/B and ProL1) also acts as markers of erectile function in patients with ED. MAIN OUTCOME MEASURES: The reader will be informed of the most current research regarding the role of opiorphins in urogenital smooth muscle biology. RESULTS: These observations led to the suggestion that genes encoding opiorphins (and potentially their peptide products) can act as markers of ED. Gene transfer of plasmids overexpressing Vcsa1 in aging rats, as well as intracorporal injection of sialorphin, led to an improvement in erectile function. In organ bath studies, we demonstrated that sialorphin can cause increased rates of relaxation of corporal smooth muscle (CSM). We have also demonstrated that in vitro, Vcsa1 causes changes in the expression of G-protein-coupled receptors (GPCRs). This has led us to suggest that the action of Vcsa1 on erectile physiology may act through relaxation of CSM by its ability to act as an inhibitor of NEP, therefore prolonging the action of peptide agonists at their GPCRs. CONCLUSIONS: Overall, there is a growing body of evidence that the opiorphins play a role in regulating CSM tone and thereby erectile function.