ProL1 is overexpressed in prostate cancer
Of the 6 data sets available data sets on the GEO database that were suitable for our analysis, 2 demonstrated a significant associateion between PrCa and opiorphin-encoding genes (Table 1).
DataSet GSE55945 was generated from a study aimed at identifying novel biomarkers and immunotherapy targets for PrCa (22) 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 define significance, 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 significance 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 (23) 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 shows the relative expression levels of Prol1 determined in a tissue cDNA array containing human non-cancerous and cancerous (at different stages) prostate tissue. This data, shown in Figure 1B, is consistent with the microarray analysis and demonstrates that ProL1 is overexpressed in PrCa tissue, with a trend for higher levels of expression as the cancer stage increased.
Xenografted tumors derived from PC3 cell show time-dependent increase in the expression of ProL1 and VEGFA
Expression levels of two markers of the hypoxic response, ProL1 and VEGFA, were compared between the PC3 cell-line (isolated from castration-resistant/androgen-insensitive PrCa) prior to injection and in tumors derived from this cell-line at 2- and 4-weeks post-injection into nude male mice. As shown in Figure 2, the PC3-derived tumors showed a progressive and significant increase in both ProL1 and VEGFA expression, such that the 4-week old 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.
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 3, in male nude mice at early stages of tumor detection (at 10- and 13- days post-implantation), there was a significantly 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 significant size difference in the tumors derived from PC3-ProL1+ or parent PC3 cells (data not shown).
Overexpression of ProL1 in PC3 cells modulates expression of genes associated with the hypoxic response
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 PC3 and PC3-ProL1+ cell-lines. This analysis identified 1 698 DEG’s between the two cell lines (Supplemental Table 1) with ProL1 being the most DEG (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 DEG’s (minus ProL1) was submitted to the DAVID, GOC and KEGG databases, which identified 1 250, 1 366 and 480 unique genes within each database, respectively, which were then used for ontological analysis. The most significant, broadly defined, functional ontologic groups with over-representation of DEG’s were involved in morphogenesis and signal transduction (Supplemental Tables 3 and 4). For example, there was over-representation of DEG’s 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 specifically “prostate gland morphogenesis” GOC: GO:0060512, p-value 6.39x10-4), “extracellular matrix organization” (DAVID: GO:0030198, 1.54x10-9), “signaling” (GOC: GO:0023052, p-value 3.35 x 10-14) and “signal transduction” (DAVID: GO:0007165, p-value 4.29x10-5). In Table 2 we present the more defined ontologic groups where there was significant over-representation of DEG’s. Both DAVID and GOC analysis identified DEG’s 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 flow (such as genes involved in over-coming hypoxia, vasodilation, blood vessel diameter and regulation of smooth muscle contraction). Analysis of the list of DEG’s using the KEGG database identified the ontologic group “pathways in cancer” as having the greatest number of DEG’s (41 genes, p-value 8.96x10-3) (Supplemental Table 5). DEG’s were also identified with significant overrepresentation in specific 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-specific modulated growth compared to its parent cell-line.
The PC3 cell-line used in the preceding experiments was isolated from advanced stage, castration-resistant/androgen-independent PrCa. In order to determine if upregulated ProL1 expression might also play a role in earlier stages of PrCa growth, we compared the growth of xenografted tumors in nude male and female mice derived from LNCaP cells (isolated from androgen-sensitive PrCa) engineered to over-express ProL1 (LNCaP-ProL1+) with tumors derived from its parent cell-line (LNCaP). Similar to previous studies (24), xenografted tumors derived from LNCaP are more rapidly established in male compared to female nude mice (the LNCaP tumor size at 4-weeks post-implantation is significantly greater in male compared to female mice, Figure 4A). We also observed (as previously reported (24)) that once LNCaP tumors are established their growth rate is not significantly different between male and female mice (Figure 4A). However, overexpression of ProL1 in LNCaP cells significantly modulated these gender specific growth characteristics. LNCaP-ProL1+ derived tumors have a significantly impaired growth rate in female compared to male mice (Figure 4B). Although there was no significant difference in growth rate of tumors derived from LNCaP-ProL1+ or LNCaP in male mice (Supplemental Figure 1A), there was significantly impaired growth of tumors derived from LNCaP-ProL1+ compared to tumors derived from LNCaP in female mice (Supplemental Figure 1B). Therefore, the observed gender specific differences in xenografted tumor growth caused by overexpression of ProL1 is primarily mediated by impairing LNCaP tumor growth in female mice, rather than conferring a growth advantage in male mice. This contrasts to our observations in PC3 cells, where overexpression of ProL1 conferred (at least during the early time-points following transplant) a growth advantage to xenografted tumors in male mice, but had no effect on growth of tumors in female mice (Figure 3).
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 its parent cell-line, LNCaP identifying 1 110 DEG’s (Supplemental Table 2). Of these 1 100 DEG’s, 209 were in common with PC3 when ProL1 was overexpressed (Supplemental Table 2). ProL1 was the most DEG (28 560-fold, p-value 24.36 x 10-36). To identify biological functions that may be regulated through overexpression of ProL1 in LNCaP cells, the list of DEG’s (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 significant overrepresentation of DEG’s caused by ProL1 overexpression in LNCaP compared to PC3 cells, many these ontologic groups were the same, or served similar functions (Supplemental Tables 6-8). In common with PC3, ProL1 overexpression regulated gene expression in ontological groups involved in morphogenesis and signal transduction (Supplemental Tables 6 and 7). 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). DEG’s were also overrepresented in more definded ontological groups potentially involved in vascularization (angiogenesis, circulatory system development and organ morphogenesis) and regulation of blood flow (smooth muscle contraction and blood pressure) (Table 3). Analysis for overrepresentation of DEG’s in biochemical pathways using the KEGG database, also identified similar effects caused by overexpression of ProL1 in PC3 and LNCaP cells (Supplemental Table 8). For example, overexpressing ProL1 regulated genes that were significantly 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 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, 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.