Investigating the role of opiorphin genes in prostate cancer and the possible genetic mechanisms by which they modulate tumor growth and androgen-sensitivity.

Background The opiorphin family of genes (represented in humans by ProL1, hSMR3A and hSMR3B) encode peptides which act as potent neutral endopeptidase (NEP) inhibitors. Because modulated NEP activity is associated with cancer development it has been suggested that dysregulated opiorphin expression may be involved in oncogenesis and recent reports have associated upregulated opiorphin gene expression with breast and oropharyngeal cancer. These observations prompted the present studies to determine if opiorphin genes play a role in prostate cancer (PrCa). Methods Publicly available data bases were screened for evidence associating opiorphin gene expression with PrCa and the �ndings con�rmed using PrCa tissue arrays. Androgen-insensitive (PC3) and -sensitive (LNCaP) PrCa cells were engineered to overexpress ProL1, referred to as LNCaP-ProL1+ and PC3-ProL1+, respectively. Xenografted tumor growth of ProL1-overexpressing and parental cell-lines were compared in male, castrated-male and female nude mice. Changes in global gene expression resulting from overexpression of ProL1 in these cell-lines was determined by RNA-Seq. Results Publicly available datasets supported an association between overexpression of opiorphin genes and PrCa, which was con�rmed using tissue arrays. Xenografted tumors derived from PC3-ProL1+ had an initial growth advantage over parent cell-lines in male mice, although at later time points there was no difference. Xenografted tumors derived from LNCaP-ProL1+ were able to grow in castrated male mice (in contrast to the parent cell-lines), and had impaired growth in female mice. Global gene expression analysis demonstrated that overexpression of ProL1 causes modulated expression of genes involved in signaling, angiogenesis and steroid response pathways. Conclusions This is the �rst report associating upregulated opiorphin gene expression and PrCa. Xengrafted tumors derived from the androgen-sensitive LNCaP cell-line engineered to overexpress ProL1 exhibit a more androgen-insensitive phenotype. Previous reports demonstrate opiorphins act as master regulators of the hypoxic response in smooth muscle cells. RNAseq data not only supports a similar role for ProL1 in PrCa (regulating genes involved in angiogenesis)


Background
Prostate cancer (PrCa) is the most prevalent of all cancers in men in the United States and the second most common cause of all cancer deaths.Despite high long-term survival rates with localized PrCa, metastatic PrCa remains largely incurable, even after intensive multimodal therapy [1].Identifying novel molecular markers of PrCa and their involvement in genetic mechanisms leading to tumor growth and progression may lead to new therapeutic strategies to prevent and treat PrCa.Based on recent reports that dysregulated expression of opiorphin-encoding genes are associated with some cancers, we initiated the present studies to determine if they play a role in PrCa.
In humans, the opiorphin family genes are represented by ProL1, hSMR3A and hSMR3B, which are highly homologous genes that have neighboring genomic locations on chromosome 4 (cytogenetic location: 4q13.3) and have coregulated expression.Of the family of opiorphin-encoding genes, ProL1 has been the most studied in the determination of their molecular and biochemical functions.The genes encode preproteins that are post-translationally processed to generate a bioactive pentapeptide, opiorphin, which acts as a potent endogenous neutral endopeptidase (NEP) inhibitor [2].In addition to their activity as NEP inhibitors, members of opiorphin-encoding gene family have also been shown to play a role in the hypoxic response.For example, prior work from our laboratory has demonstrated that the rat homologue of ProL1, vcsa1, is upregulated in corporal smooth muscle cells in response to hypoxia, where it acts as a "master regulator" of the hypoxic response, regulating the expression of hypoxia-inducible factor 1A (HIF1A) and vascular endothelial growth factor A (VEGFA) [3].
Because changes in NEP activity are associated with cancer development, a review article published in 2015 suggested that since opiorphin has biochemical activity as an NEP inhibitor, it may play a role in cancer [22].This has been supported by several recent reports associating dysregulated expression opiorphin-encoding genes with cancer.Upregulated ProL1 expression was observed in invasive multifocal/multicentric compared to unifocal breast cancer [23] and increased hSMR3A expression was associated with unfavorable survival outcomes following surgery for oropharyngeal squamous cell carcinoma [24,25].In addition, in a meta-analysis using a rank aggregation approach to identify changes in gene expression in common between different cancer types, ProL1 was included in the aggregated list of top-50 genes [26].However, with the exception of a brief conference report from our laboratory [27], there are no published reports associating dysregulated expression of opiorphin-encoding genes with PrCa.
The activity of opiorphin as a master regulator of the hypoxic response [3] may also contribute to a role in the development and progression of PrCa.Since the metabolic demands of growing tumors creates a growth limiting hypoxic microenvironment [28][29][30] this might be expected to result in upregulation of opiorphin expression.Based on prior studies [3] the overexpression of opiorphin would in turn activate genetic pathways allowing the tumor to overcome the hypoxic barrier and continue to grow.In addition, given reports suggesting hypoxia may be a driver of malignant progression [31], ProL1 might also be a factor in development of castration-resistant/androgen-insensitive PrCa.
In the present report we demonstrate that upregulated expression of opiorphins are associated with human PrCa and con rmed that ProL1 overexpression modulates the growth and androgen-sensitivity of mouse xenografted PrCa tumors.Global gene expression analysis on the effects of overexpressing ProL1 in PrCa cell-lines suggested possible genetic mechanisms by which ProL1 modulates tumor growth and androgen-sensitivity consistent with its known biochemical and functional activities as an NEP inhibitor and master regulator of the hypoxic response.

Identi cation of upregulated ProL1 expression in prostate cancer
An initial screen was conducted of publicly available gene expression datasets on the Gene Expression Omnibus (GEO) pro les database at NCBI [32] to determine if there was prior but unregognized evidence of an association between modulated expression of opiorphin encoding genes and PrCa.Using the search term "prostate cancer" identi ed 79 datasets.However, only 6 of these datasetss allowed both gene expression comparison between benign and cancerous prostate cancer tissue and included the opiorphin-encoding genes ProL1, hSMR3A or hSMR3B.These 6 datasets were analyzed using the NCBI online data analysis tool (GEO2R) to compare expression of ProL1 (referred to as OPRPN is this datasets), hSMR3A and hSMR3B between cancerous and non-cancerous prostate tissue.A change in expression of the a gene interest was considered signi cant if compared to the control group the gene had a Log2FoldChange of >0.56 or <-0.56 (i.e. a 1.5-fold change) with a p-value <0.05.To provide context on how this analysis might also provide evidence of an association between PrCa and genes previously identi ed as markers of PrCa development and progression [33], we also screened the same 6 datasets to identify if PrCa was associated with changed expression of androgen receptor (AR), breast cancer type 1 susceptibility protein (BRCA1), breast cancer type 2 susceptibility protein (BRCA2), cyclin-dependent kinase inhibitor N1B (CDKN1B), checkpoint kinase 2 (CHEK2), cytochrome P450 1B1 (CYP1B1), zinc phosphodiesterase ELAC protein 2 (ELAC2), tumor protein p53 (TP53), phosphatase and tensin homolog (PTEN) and RAS.
In order to con rm the upregulation of ProL1 in PrCa, a cDNA array from normal and cancerous prostate tissue was purchased from OriGene (HPRT103; TissueScanTM cDNA Arrays; Rockville, MD, USA).This array has 48 samples covering 9-normal and 18-Stage II, 19-III and 2-IV PrCa.Quantitive RT-PCR analysis of the ProL1 transcripts was performed as described below.

Mice xenograft studies
All animal studies were conducted in accordance with the Animal (Scienti c Procedures) Act 1986 and approved by the Institutional Animal Care and Use Committee (IACUC) of Albert Einstein College of Medicine.Athymic nude (homozygous, nu/nu aged 8-12 weeks) male, castrated-male and female mice (Charles River Laboratories, Wilmigton, MA) were housed under standard pathogen-free conditions at a maximum of ve mice per cage.Tumours were established by subcutaneous injection on the upper left ank of nude mice randomly ascribed to groups that received PC3, PC3-ProL1+, LNCaP or LNCaP-ProL1+ cells in a 1:1 mixture of Matrigel (Corning Life Science, Teterboro, NJ) and HBSS using a 27-gauge needle.For PC3 and PC3-ProL1+ cell-lines, 1.5 x 10 6 cells, and for LNCaP and LNCaP-ProL1+ cell-lines, 2.5 × 10 6 cells were injected.The numbers of animals in each group are described in the Figure legends.
Tumors were measured twice a week using Vernier Calipers and volume (mm 3 ) calculated by length × width × height × 0.52.When tumors reached a diameter > 1 cm mice were euthanized through inhalation of CO 2 to effect.

RNA Isolation
To prepare total RNA to be used for quantitive-RT-PCR or RNA-seq analysis from in vitro experimental samples (PC3, PC3-ProL1+, LNCaP and LNCaP cell lines grown in culture), RNA was isolated with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) following the manufacturers' protocol.To prepare total RNA from xenografted tumors for quantitive-RT-PCR analysis, tumors were rst ash-frozen in liquid nitrogen and then ground to a homogenate using a pestle and mortar.RNA was isolated from 30 mg of the homogenate using a combination of the TRIzol (Invitrogen) method and the RNeasy Plus Mini Kit (Qiagen).
Quantitative RT-PCR Generation of cDNA was achieved using the SuperScript III First-Strand Synthesis System (Invitrogen) using 1 µg total RNA as the starting material.For PCR analysis, samples were prepared in a 25 µL reaction mix containing the PowerUp SYBR Green PCR Master Mix (Thermo Fisher).Real-time PCR was performed by the Applied Biosystems 7300 Real Time PCR System (Thermo Fisher) using the standard cycling mode recommended by the PowerUp SYBR protocol (50 ºC for 2 minutes, 95 ºC for 2 minutes, and then 40 cycles of 95 ºC for 15 seconds along with 60 ºC for 1 minute).Commercially available primers were obtained from Qiagen (RPL19, cat.# PCRPPH18637A, ProL1 cat.# PPH10360A and VEGFA cat.# PPH00251C).Quantitative RT-PCR was performed in triplicate for each sample.Data was analyzed using the ∆∆Ct method, with gene expression normalized to 60S ribosomal protein L19 (RPL19) and expressed as the average fold-change relative to controls.

RNA-sequencing (RNA-seq)
Prior to RNA-seq, RNA samples were treated with RNase-free DNase I and then quanti ed using RiboGreen (Thermo Fisher Scienti c, Waltham, MA, USA).RNA integrity number (RIN) was assessed with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) to ensure each sample had a RIN ≥8.RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA kit, with Ribosomal depletion (Illumina, San Diego, CA, USA).Each library was sequenced using the Illumina HiSeq2500 platform and generated 125 nt paired-end (PE) reads at either 30 million or 60 million reads per sample.RNA-seq analysis of gene expression for each cell-line was performed in triplicate (each sample prepared separately).[Raw sequencing datasets will be deposited in the Sequence Read Archive of NCBI (http://www.ncbi.nlm.nih.gov/sra)upon acceptance of this publication with accession number included here)] Bioinformatics analysisThe reads were aligned with STAR (version 2.4.0c)[34] and genes annotated in Gencode v18 were quanti ed with featureCounts (v1.4.3-p1) [35].Normalization and differential expression analyses were performed with the Bioconductor package DESeq2 [36].Differentially expressed genes were assessed using a two-sided t-test and fold-change on log-transformed expression values.All samples were median centered.Cut-off criteria for differentially expressed genes was >1 Log2FoldChange or <-1 Log2FoldChange in gene expression with a p-value <0.01.

Statistical analyses
Statistical analyses were performed using either Microsoft Excel (Microsoft, Seattle, WA) or Prism 8.2.(GraphPad Software, Inc., La Jolla, CA).To determine statistical signi cance of two group comparisons, unpaired, two-tailed t-tests were performed, and results reported in Tables and Figures.Error bars represent standard error or standard deviation of mean (as described in gure legends).

ProL1 is overexpressed in prostate cancer
Of the 6 datasets in the GEO database that were suitable for our analysis, 2 demonstrated a signi cant association between PrCa and overexpression of opiorphin-encoding genes (Table 1 and Supplemental Table 3).In comparison, using the same criteria used to identify signi cant changes in opiorphin gene expression (Log2FoldChage of >0.56 or <-0.56, i.e. a 1.5-fold change, with a p-value <0.05) revealed that out of the group of genes previously identi ed as associated with PrCa, the most commonly overrepresented gene in this analysis was BRAC1 in 4/6 datasets.The remaining genes were represented in only 2/6 datasets.A less stringent analysis datasets (a LogFC of >0.26 or <-0.26 i.e. a 1.2-fold change with a p-value <0.1) associated PrCa with a change in expression of opiorphin genes in 3/6 datasets, BRAC1 in 5/6 datasets, PTEN and CYP1B1 in 4/6 datasets, AR in 3/6 datasets and the remaining genes (BRCA2, CDKN1B, CHEK2, ELAC2, RAS AND TP53) in 2/6 datasets.Therefore, a search of publicly available datasets associating PrCa with changed expression of the opiorphin genes identi ed an association in a similar number of datasets when the same search was applied to several other genes previously identi ed as associated with PrCa.DataSet GSE55945 was generated from a study aimed at identifying novel biomarkers and immunotherapy targets for PrCa [44] and contained 8 normal (benign) prostate tissue samples and 13 prostate cancer tissues (sub-divided into ERG fusion positive (N=6) and ERG fusion negative (N=7) PrCa).
As shown in Table 1, comparing the 8 normal prostate tissue samples and 13 prostate cancer tissues and using a p-value of <0.05 to de ne signi cance, PrCa was associated with overexpression of both ProL1 and hSMR3A.When the group of prostate cancer tissues was sub-divided into ERG fusion positive and ERG fusion negative PrCa, there was a greater signi cance and fold-increase of ProL1 expression in ERG fusion positive PrCa tissue compared to non-cancerous prostate tissue (2.25-fold, p-value 9.7x10 -4 ) than when the same analysis was performed with ERG fusion negative PrCa tissue (2.16-fold, p-value 1.3x10 - 2 ).
Dataset GSE3325 [45] was generated in a study to identify the genetic signatures of metastatic progression and contained 6 samples from normal (benign) prostate tissue samples and 13 PrCa tissues (sub-divided into localized (N=6) and metastatic (N=7) PrCa).As shown in Table 1, PrCa was associated with overexpression of hSMR3B, with a trend for greater overexpression with progression from localized to metastatic disease, from 5-fold (p-value 0.0035) to 9.32-fold, (p-value 0.00038), respectively.
Given that ProL1 has been the most intensily studied memebr of the opiorphin gene family, we focused on this gene as represetative of the opiorphin family.Figure 1A and B show the relative expression levels of Prol1 determined in a tissue cDNA array containing human non-cancerous and cancerous (at different stages) prostate tissue.The data from the tissue array demonstrates that ProL1 is overexpressed in PrCa tissue (at a similar level of overexpression as observed in the dataset analysis) and has a trend for higher levels of expression as the cancer stage increased.Overall, these analysis suggest that PrCa is associated with overexpression of ProL1.
Xenografted tumors derived from PC3 cell show a tumor-environment, time-dependent increase in the expression of ProL1 and VEGFA The metabolic demands of developing tumors create a hypoxic microenvironment that must be overcome for continued growth [28][29][30].Because of our prior published studies suggesting that opiorphins are upregulated in smooth muscle cells in response to hypoxia, and then act as "master regulators" of the hypoxic response through the regulation of genes involved in the hypoxic response (such as VEGFA) [3], we determined if the microenvironment of a xenografted tumor would induce genes (ProL1 and VEGFA) involved in hypoxic response pathways.
The relative expression of ProL1 and VEGFA was compared between PC3 cells (isolated from castrationresistant/androgen-insensitive PrCa), xengrafted tumors derived from this cell-line at 2-and 4-week timepoints, and cells isolated from the xengrafted tumors and maintained under normoxic cell culture conditions.The PC3-derived tumors showed a progressive and signi cant increase in both ProL1 and VEGFA expression (Figure 2A and B, respectively), such that after 4-weeks, tumors had approximately a 20-fold increase in ProL1 expression, and approximately a 3.2-fold increase in VEGFA expression, compared to the levels of these genes in the parent PC3 cell-line.In cells isolated from these tumors and cutured under normoxic conditions, the expression both ProL1 and VEGFA returned to levels that were not signi cantly different from the parent PC3 cells.Overall, these data suggest that the hypoxic tumor microenvironment results in overexpression of ProL1 and VEGFA in PC3 tumor xenograft.
Xenografted tumors derived from a PC3 cell-line engineered to over-express ProL1 (PC3-ProL1+) initially exhibit accelerated growth compared to its parent cell-line in male nude mice In order to determine if overexpression of ProL1 might directly affect PrCa tumor growth we compared the growth of xenografted tumors in male and female nude mice derived from a PC3 cell-line genetically engineered to overexpress ProL1 (PC3-ProL1+) with its parent cell-line (PC3).As can be seen in Figure 3A, in male nude mice at early stages of tumor detection (at 10-and 13-days post-implantation), there was a signi cantly greater rate of tumor growth in mice implanted with PC3-ProL1+ compared to PC3.However, at later time-points (from 17-days onwards) in the male mice, and at any time-point in female nude mice, there was no signi cant size difference in the tumors derived from PC3-ProL1+ or the parental PC3 cells (Figure 3B and C).
Overexpression of ProL1 in PC3 cells modulates expression of genes associated with the hypoxia and steroid response pathways.
In order to identify possible genetic mechanisms by which upregulated ProL1 expression in PC3 cells might modulate tumor growth, we compared global gene expression between the PC3 and PC3-ProL1+ cells grown in culture.This analysis identi ed 1,698 differentially expressed genes between the two celllines (Supplemental Table 1) with ProL1 being the most differentially expressed gene (5,995-fold, p-value 2.82 x 10 -26 ).To identify biological functions that may be regulated through overexpression of ProL1 in PC3 cells, the list of differentially expressed genes (minus ProL1) was submitted to the DAVID, GOC and KEGG databases, which identi ed 1,250, 1,366 and 480 unique genes within each database, respectively, which were then used for ontological analysis.The most signi cant, broadly de ned, functional ontologic groups with over-representation of differentially expressed genes were involved in morphogenesis and signal transduction (Supplemental Tables 4 and 5).For example, there was over-representation of differentially expressed genes in the ontological groups "anatomical structure development/morphogenesis" (GOC: GO:0048856 and GO:0009653, p-value 2.05x10 -22 and 2.21x10 -20 , respectively, and more speci cally "prostate gland morphogenesis" GOC: GO:0060512, p-value 6.39x10 -4 ), "extracellular matrix organization" (DAVID: GO:0030198, 1.54x10 -9 ), "signaling" (GOC: GO:0023052, pvalue 3.35 x 10 -14 ) and "signal transduction" (DAVID: GO:0007165, p-value 4.29x10 -5 ).In Table 2 we present the more de ned ontologic groups where there was signi cant over-representation of differentially expressed genes.Both DAVID and GOC analysis identi ed differentially expressed genes in ontological groups involved in vascularization (such as genes involved in angiogenesis, blood vessel development, organ growth and morphogenesis) and also groups that function in hypoxia and direct modulation of blood ow (such as genes involved in over-coming hypoxia, vasodilation, blood vessel diameter and regulation of smooth muscle contraction).Analysis of the list of differentially expressed genes using the KEGG database identi ed the ontologic group "pathways in cancer" as having the greatest number of differentially expressed genes (41 genes, p-value 8.96x10 -3 ) (Supplemental Table 6).Differentially expressed genes were also identi ed with signi cant overrepresentation in speci c biochemical pathways, such as the P13K-Akt, VEGF and MAPK signaling pathways and steroid metabolic pathways (Table 2).
Xenografted tumors derived from LNCaP cells engineered to over-express ProL1 (LNCaP-ProL1+) have gender-speci c modulated growth compared to its parent cell-line.
We expanded the studies described above for the androgen insensitive PrCa cell-line, PC3, to determine if upregulated ProL1 expression might also play a role in tumor development in an androgen sensitive cellline (LNCaP).We compared the growth between xenografted tumors derived from LNCaP cells engineered to over-express ProL1 (LNCaP-ProL1+) and their parental cells in male, castrated-male and female nude mice (Figure 4).Although overexpression of ProL1 in LNCaP cells did not affect tumor growth in male mice (Figure 4A) it signi cantly modulated tumor growth in both castrated-male and female mice (Figure 4B and C).As expected for an androgen sensitive cell-line, no tumors developed when LNCaP cells were xenografted into castrated-male mice (Figure 4B).Remarkably, although detection of tumors was delayed from 3 to 7 weeks, xenografted LNCaP-ProL1+ cells were able to develop tumors in castrated-male mice.In addition, in at least one of the 8 castrated-male mice xenografted with LNCaP-ProL1+ cells, several secondary tumors were observed.Similar to previous studies [46], xenografted tumors derived from LNCaP did develop tumors in female mice, albeit at a delayed time for detection compared to male nude mice.However, once LNCaP tumors are established, their growth rate is not signi cantly different between male and female mice.Interestingly, in female mice there was signi cantly impaired growth of tumors derived from LNCaP-ProL1+ compared to tumors derived from LNCaP (Figure 4C).Overall these observations suggest that overexpressing ProL1 in the androgen sensitive LNCaP cell-line modulates the hormonal sensitivity of xenografted tumors towards a more androgen-insensitive phenotype.

Overexpression of ProL1 in LNCaP cells modulates expression of genes associated with steroid metabolism and the androgen response
In order to determine possible mechanisms for the difference in xenografted LNCaP tumor growth when ProL1 is overexpressed, we compared global gene expression between the LNCaP-ProL1+ and LNCaP (its parent cell-line) growing in culture, identifying 1,110 differentially expressed genes (Supplemental Table 2).Of these 1, differentially expressed genes, 209 were in common with PC3 when ProL1 was overexpressed (Supplemental Table 2).ProL1 was the most differentially expressed gene (28,560-fold, pvalue 24.36 x 10 -36 ).To identify biological functions that may be regulated through overexpression of ProL1 in LNCaP cells, the list of differentially expressed genes (minus ProL1) was submitted to the DAVID, GOC and KEGG databases, identifying 665, 812 and 287 unique genes within each database, respectively, which were then used for ontological analysis.Although the analysis identifed fewer ontologic groups with signi cant overrepresentation of differentially expressed genes caused by ProL1 overexpression in LNCaP compared to PC3 cells, many these ontologic groups were the same, or served similar functions (Supplemental Tables 7, 8 and 9).In common with PC3, ProL1 overexpression regulated gene expression in ontological groups involved in morphogenesis and signal transduction (Supplemental Tables 7 and 8).For example, there was over-representation of genes in the ontological groups "anatomical structure development/morphogenesis" (GOC: GO:0048856/GO:0009653, p-value 2.76x10 -8 and 2.09x10 -8 , respectively), "cell-cell signaling" (GOC: GO:0007267, p-value 1.46x10 -10 and DAVID: GO 0007267, p-value 6.00x10 -3 ) and "signal transduction" (GO:0007165, p-value 2.66x10 -4 ).Differentially expressed genes were also overrepresented in more de nded ontological groups potentially involved in vascularization (angiogenesis, circulatory system development and organ morphogenesis) and regulation of blood ow (smooth muscle contraction and blood pressure) (Table 3).Analysis for overrepresentation of differentially expressed genes in biochemical pathways using the KEGG database, also identi ed similar effects caused by overexpression of ProL1 in PC3 and LNCaP cells (Supplemental Table 9).For example, overexpressing ProL1 regulated genes that were signi cantly over-represented in the ontologic groups "pathways in cancer" (p-value 5.97x10 -2 ,), steroid metabolism and the regulation of MAPK signaling (Table 3).
However, there were differences in the regulation of speci c genes related to the androgen response between LNCaP and PC3.Unlike PC3, where overexpression of ProL1 caused a signi cant increase in expression of the androgen receptor gene (AR, 7.46-fold, p-value 3.16x10-197) and a decrease in expression of the estrogen receptor (ESR1, 2.7-fold reduced, p-value 3.38x10 -49 ), in LNCaP neither of these genes were changed in expression (Supplemental Table 1 and 2).In contrast, overexpression of ProL1 in LNCaP reduced expression of the progesterone receptor gene (PGR, 2.17-fold reduced, p-value 4.41x10-3), whereas PGR expression was not changed in PC3 cells.

Discussion
This is the rst report associating elevated expression levels of opiorphin-encoding genes with the development of human PrCa.An initial screen of publicly available databases identi ed an association between expression levels of the opiorphin-encoding genes and PrCa.As shown in Table 1 there is a much greater signi cance of elevated ProL1 expression in ERG-fusion positive PrCa (which is generally considered to represent a more advanced stage PrCa [47]) compared to ERG-fusion negative PrCa (Table 1).There was overexpression of the ProL1 homologue, hSMR3B, in localized PrCa compared to noncancerous prostate tissue (5-fold, p-value 0.0035), with greater overexpression in metastatic PrCa compared to non-cancerous prostate tissue (9.32-fold, p-value 0.00038).Although only 2 of the 6 datasets publicly available on GEO associated an upregulation of opiorphin genes with cancer development, a similar search for an association with several generally well-accepted genes involved in PrCa revealed associations in similar numbers of datasets (Supplemental Table 3).In a meta-analysis of 20 microrarray data sets using a rank aggregation approach to identify changes in gene expression in common between different cancer types, the maximum number of lists that any gene appeared in was 3 [26].In this study, of the aggregated list of top-50 genes, 36 had been previously implicated in cancer (often multiple cancers) and the authors suggested that the other 14 genes "may contain some novel cancer genes that may deserve further scrutiny in the future".It is interesting to that ProL1, the gene encoding opiorphin, was one of these 14 genes.
The apparent low level representation in datasets of the differentially expressed opiorphin genes, as well as other markers of PrCa, may represent the highly heterogenous nature of samples used in gene expression studies [48].Not only is PrCa in general highly heterogeneous in nature, but even in the same patient there are multiple distinct cancer foci at primary diagnosis.It has also been shown that many PrCa datasets have signi cant stromal contamination which has a tendency to mask epithelial gene expression across PrCa pro ling studies [49].Subsequently, PrCa tissue arrays con rmed that ProL1 is signi cantly upregulated in PrCa, with a trend for higher levels of overexpression with stage (Figure 1).
Our data (presented in Figure 2) suggests that overexpression of opiorphin genes is a response to the tumor microenvironment.Because our prior published studies have demonstrated that opiorphins are upregulated in smooth muscle cells in response to hypoxia [3], and that the metabolic demands of developing tumors create a hypoxic microenvironment [28-30], our hypothesis is that it is the hypoxic environment of the developing tumor that results in opiorphin overexpression.
We also demonstrate that engineered constitutive overexpression of ProL1 modulates the growth of xenografted tumors.The effect on tumor growth caused by overexpression of ProL1 is highly pronounced in LNCaP cells (isolated from castration-sensitive/androgen-sensitive PrCa) (Figure 4).Remarkably, in contrast to the parent cells, xenografted LNCaP-ProL1+ cells were able to develop tumors in castratedmale mice and in female mice overexpression of ProL1 signi cantly impaired growth of tumors.In PC3 cells (a castration-resistant/ androgen-insensitive PrCa cell-line) although overexpression of ProL1 increases the growth rate of xenografted tumors in nude male mice at early time-points there is no signi cant difference at later time points (Figure 3).These experiments provide strong evidence ProL1 overexpression modulates the androgen sensitivity of tumors, such that the xenografted tumor exhibits a more androgen-insensitive phenotype.
Global analysis of the changes in gene expression caused by overexpression of ProL1 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-ow to tissues through their modulation of smooth muscle tone [11,14,18], 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 (Table 2 and 3); 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 (Table 2 and 3), 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 [50, 51] and the reduced expression of PGR in LNCaP-ProL1 cells might therefore explain the negative effect on tumor growth in female mice.In PC3-ProL1+ cells we observed a signi cant upregulation of the AR (7.46-fold, p-value These mechanisms are summarized in Figure 5.The hypoxic barrier to growth developed when tumors reach about 2mm 3 would be expected to upregulate opiorphin gene expression.As master regulators of the hypoxic response, overexpression of opiorphin would both directly relax vascular smooth muscle cells neighboring the tumor, and activate pathways involved in the vascularization and regulation of blood supply to the tumor, allowing tumors to overcome the hypoxic barrier and PrCa growth.In addition, overexpression of ProL1 in response to hypoxia may activate genes involved in steroid metabolism and androgen response, modulating the androgen sensitivity of the tumor and potentially driving the development of castration resistance/androgen-insensitivity.

Conclusion
In conclusion, this is the rst report associating upregulated opiorphin gene expression and PrCa.

Figure 2 Relative
Figure 2

Figure 4 Growth
Figure 4

Table 1 .
The publicly available datasets GSE55945 and GSE3325 were analyzed using the 3.16x10 -197 , Supplemental Table1).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 signi cant over-representation of differentially expressed genes in PC3-ProL1+ cells.The PI3K-Akt signaling pathway is considered one of the most commonly dysregulated pathways in PrCa with approximately 40% of early stage, and 70-100% of advanced stage PrCa, exhibiting dysregulation of this pathway, suggesting PI3K-Akt dysregulation is associated with PrCa progression[52, 53]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 androgen-insensitivity.
Overexpression of ProL1 in PrCa cell-lines modulates tumor growth and, in the androgen-sensitive LNCaP cell-line, xenografted tumors exhibit a more androgen-insensitive phenotype.Global analysis of the changes in gene expression caused by overexpression of ProL1 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.Therefore, opiorphins may play a role in PrCa development by activating pathways that overcome the hypoxic environment of the developing tumor and modulate its androgen sensitivity.Strategies that target opiorphin expression may represent a novel strategy preventing the development and progression of PrCa.potential biomarker and immunotherapy target in prostate cancer.Clinical cancer research : an o cial journal of the American Association for Cancer Research 2009, 15(18):5794-5802.45.Varambally S, Yu J, Laxman B, Rhodes DR, Mehra R, Tomlins SA, Shah RB, Chandran U, Monzon FA, Becich MJ et al: Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression.Cancer cell 2005, 8(5):393-406.Rhodes DR, Cao X, Wang L, Dhanasekaran SM, Kalyana-Sundaram S, Wei JT, Rubin MA, Pienta KJ et al: Integrative molecular concept modeling of prostate cancer progression.Nature genetics 2007, 39(1):41-51.50.Bonkhoff H: Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression.The Prostate 2018, 78(1):2-10.