Sox7 negatively regulates prostate-specific membrane antigen (PSMA) expression through PSMA-enhancer

Abstract

Background: PSMA expression in the prostate epithelium is controlled by a cis-element, PSMA enhancer (PSME). PSME contains multiple binding sites for Sox proteins, and in this study, we identified Sox7 protein as a negative regulator of PSMA expression through its interaction with PSME.

Methods: The statistical correlation between Sox7 and PSMA mRNA expression was evaluated using five prostate cancer studies from cBioportal. In vitro and in vivo interaction between Sox7 and PSME was evaluated by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase reporter assay. Synthetic oligonucleotides were generated to define the sites in PSME that interact with Sox7 protein. Sox7 mutants were generated to identify the region of this protein required to regulate PSMA expression. Sox7 was also stably expressed in LNCaP/C4-2 and 22Rv1 cells to validate the regulation of PSMA expression by Sox7 in vivo.

Results: Sox7 mRNA expression negatively correlated with PSMA/FOLH1 and PSMAL/FOLH1B mRNA expression in Broad/Cornell, TCGA and MSKCC studies, but not in two studies containing only metastatic prostate tumors. PC-3 cells mostly expressed the 48.5 KDa isoform 2 of Sox7, and the depletion of this isoform did not restore PSMA expression. Ectopic expression of canonical, wild-type Sox7 in C4-2 and 22Rv1 cells suppressed PSMA protein expression. ChIP assay revealed that canonical Sox7 protein preferentially interacts with PSME in vivo, and EMSA identified the SOX box sites #2 and #4 in PSME as required for its interaction. Sox7 was capable of directly binding to PSME and suppressed PSME-mediated transcription. The NLS regions of Sox7, but not its β-catenin interacting motif, are essential for this suppressing activity. Furthermore, restoration of wild-type Sox7 expression but not Sox7-NLS mutant in Sox7-null prostate cancer cell lines suppressed PSMA expression.

Conclusions: The inactivation of canonical Sox7 is responsible for the upregulated expression of PSMA in non-metastatic prostate cancer.

Keywords: WNT signaling; diagnostic and therapeutic marker; prostate-specific expression; transcriptional factor.

FIGURE 1

Sox7 expression negatively correlates with FOLH1 or FOLH1B expression in prostate cancer studies. RNA expression data was retrieved from cBioPortal. The Pearson Correlation Coefficient (r) and P-value were obtained via PROC CORR in SAS for Broad/Cornell and MSKCC studies. Spearman Rank Correlation Coefficient was obtained for TCGA study because of non-normal data distribution. We also conducted simple linear regression (SLR) analysis to measure the direction and strength of the linear relationship between the expression of Sox7 and FOLH1 as well as Sox7 and FOLH1B. An estimate of the true slope (solid line), P-value, 95% confidence intervals (shaded area) and 95% prediction intervals (dotted line) were obtained via PROC REG in SAS.

FIGURE 2

Sox7 negatively correlates with PSMA expression in prostate cancer cell lines. A. Expression of PSMA mRNA was determined by quantitative real time PCR normalized by GAPDH expression. B. Sox7 and PSMA protein levels were determined by immunoblot using the indicated antibodies. C. Immunoblot analysis of C4–2 or C4–2 cells that constitutively express a mouse wild-type Sox7 protein (lane 2).

FIGURE 3

Sox7 interacts with PSME in vivo and negatively regulates PSME-mediated transcription. A. ChIP analysis of C4–2/Sox7 cell line with anti-Sox7 antibody or IgG control antibody at PSME or SNP28254. Quantitative real-time PCR was carried out in triplicate, and error bars represent one standard deviation. B. Schematic drawing of luciferase reporters for PSMA promoter and enhancer (PSME) activity. PSMA1k only contains a 1-Kb promoter region. PSME-PSMA1k contains both the promoter and enhancer. C. Sox7 suppresses PSME-mediated transcription. Indicated amount of CMV-Tag2B/Sox7 (Sox7) or CMV-Tag2B (V-ctrl) plasmids were transfected into LNCaP cells with 100 ng of PSME-PSMA1k luciferase reporter construct. Control reactions were carried out by transfecting 100 ng of pGL3 or PSMA1K plasmids. Reactions were carried out in duplicates, and error bars represent one standard deviation.

FIGURE 4

Sox7 directly interacts with PSME. A. Sequence of the 331-enhancer core of PSME. This panel was modified from Watt. et al, Genomics 73, 243–254, 2001, and potential Sox7 binding sites (ATTGT) are highlighted by light gray color. Dark gray indicates the DNA sequence used in EMSA analysis. Schematic drawing of probe #1–3 is provided to indicate changes in Sox binding sites. B. Sox7 EMSA analysis with wild-type and mutant PSME probes. 0.6 μg of recombinant Sox7 protein was incubated with 0.1 pmol of biotinylated probes for 30 mins at 4 °C. DNA/protein complex was resolved by 6% native PAGE. Arrow heads indicated unbounded probe, and arrows indicated shifted complex. C. Sox7 EMSA analysis with 200- or 50-fold excess of unlabeled probe #1 or #2.

FIGURE 5

The NLS region but not the β-catenin interacting motif of Sox7 is essential for the regulation of PSMA expression. A. Schematic drawing of wild type and mutant recombinant Sox7 proteins. Areas 1 and 2 represent the bipartite nuclear localization sequence and basic cluster NLS in the HMG domain, respectively. Area 3 denotes the β-catenin interacting motif. Immunoblot analysis of these recombinant proteins is included in the bottom/right of this panel. B. EMSA analysis of wild-type and mutant recombinant Sox7 proteins with probe #1. Reaction conditions were similar to those described in Figure 4B. C. Sox7-Δ but not Sox7-NLS suppresses PSME-mediated transcription. Reaction conditions were similar to those described in Figure 4B. D. Immunoblot analysis of 22Rv1, 22Rv1/Sox7-wt, 22Rv1/Sox7-Δ and 22Rv1/Sox7-NLS cell lines. Cell lysates were harvested and analyzed by Immunoblot for PSMA and Sox7 proteins; actin was used as loading control. E. ChIP analysis of indicated 22Rv1 cell lines with anti-Sox7 antibody or IgG control antibody at PSME locus. Quantitative real-time PCR was carried out in triplicate, and error bars represent one standard deviation.