Correlation of B7-H3 with androgen receptor, immune pathways and poor outcome in prostate cancer: an expression-based analysis

Abstract

Background: B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer.

Methods: Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson's correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease.

Results: B7-H3 mRNA expression was positively associated with higher Gleason score (P<0.001), tumor stage (P<0.001), and castrate resistant metastatic disease (P<0.0001). High B7-H3 expression correlated with the development of metastasis and prostate cancer specific mortality, but this was not significant on multi-variable analysis. B7-H3 expression correlated with ERG-positive disease (r=0.99) and AR expression (r=0.36). ChIP revealed an AR-binding site upstream of B7-H3, and the presence of androgens decreased B7-H3 expression in LNCaP suggesting potential direct AR regulation. Gene set enrichment analysis demonstrated an association of B7-H3 with androgen signaling as well as immune regulatory pathways.

Conclusions: Higher B7-H3 expression correlates with Gleason grade, prostate cancer stage and poor oncologic outcomes in prostatectomy cohorts. B7-H3 expression appears to be related to androgen signaling as well as the immune reactome.

Figure 1

(a) Expression distributions of B7-H3, B7-H4, PD-L1 and PD-L2 in a prospective radical prostatectomy (RP) cohort (n = 2111) (b) Dot plots showing B7-H3 expression is higher in more aggressive tumors as it is significantly associated with pathologic Gleason grade and tumor stage. (c) Box plots showing B7-H3 expression is associated with metastasis as expression is higher in metastatic tumors vs to primary tumors. Data for this comparison were derived from Oncomine consisting of 59 primary tumor samples and 34 metastatic castration resistant prostate cancer tumor samples (P <0.0001). GS, Gleason score.

Figure 2

(a) Kaplan–Meier curves for biochemical recurrence (BCR), clinical metastasis (METS), prostate cancer specific mortality (PCSM) and overall survival (OS) in a natural history prostate cancer radical prostatectomy (RP) cohort. (b) Kaplan–Meier curves in a natural history RP cohort of patients that developed BCR after RP.

Figure 3

(a) B7-H3 expression trends with the molecular subtype in samples from a prospective RP cohort (n = 2111) (b) B7-H3 expression positively correlates with AR expression when measured in a prospective cohort (n = 2111, R = 0.36) (c) AR is among the most correlated genes with B7-H3 (85th percentile) (d) B7-H3 is among the most correlated genes with androgen receptor (AR; 79th percentile). RP, radical prostatectomy.

Figure 4

Gene set enrichment analysis in the prospective cohort (left) and Johns Hopkins Medical Institute (JHMI) natural history cohorts (right) shows the androgen receptor signaling pathway is positively correlated with B7-H3 expression.

Figure 5

Androgen-induced binding of the androgen receptor (AR) to a putative regulatory element upstream of B7-H3/CD276. (a) Schematic of primer locations (bars indicate primer positions relative to transcriptional start sites (TSS)). (b) AR binding sites were identified by mining previously published ChIP-Seq data. Chromatin precipitation was performed using LNCaP cells grown in the absence of androgens and treated for 8 h with 100 n_M DHT or solvent control. Cell lysates were incubated with anti-AR or IgG control antibodies and precipitated DNA was analyzed using primers specific to an upstream region of B7-H3/CD276 or the B7-H3/CD276 promoter (TSS). Enrichment at the KLK3/PSA enhancer is shown as a positive control. Data are shown normalized to total DNA input for each amplicon. (c) Expression of B7-H3 as assayed by quantitative reverse transcription (qRT-PCR) in LNCaP cells grown in RPMI supplemented with FBS (which contains androgens), RPMI supplemented with charcoal stripped FBS (deprived of androgens), or RPMI with charcoal stripped FBS, which was supplemented with DHT. FBS, fetal bovine serum.

Figure 6

Pearson’s correlation analysis shows positive correlations between B7-H3 expression and SMAD2, IL17RA and RORC in the prospective cohort.