ANO7 is associated with aggressive prostate cancer

Abstract

Prostate cancer is one of the most common and heritable human cancers. Our aim was to find germline biomarkers that can predict disease outcome. We previously detected predisposing signals at 2q37, the location of the prostate specific ANO7 gene. To investigate, in detail, the associations between the ANO7 gene and PrCa risk and disease aggressiveness, ANO7 was sequenced in castration resistant tumors together with samples from unselected PrCa patients and unaffected males. Two pathogenic variants were discovered and genotyped in 1769 patients and 1711 unaffected males. Expression of ANO7 vs. PrCa aggressiveness was investigated. Different databases along with Swedish and Norwegian cohorts were used for validation. Case-control and aggressive vs. nonaggressive association analyses were performed against risk and/or cancer aggressiveness. The ANO7 mRNA level and patient survival were analyzed using expression data from databases. Variant rs77559646 showed both risk (OR 1.40; p = 0.009, 95% CI 1.09-1.78) and association with aggressive PrCa (Genotype test p = 0.04). It was found to be an eQTL for ANO7 (Linear model p-values for Finnish patients p = 0.009; Camcap prostate tumor p = 2.53E-06; Stockholm prostate tumor cohort p = 1.53E-13). rs148609049 was not associated with risk, but was related to shorter survival (HR 1.56; 95% CI 1.03-2.36). High ANO7 expression was independently linked to poor survival (HR 18.4; 95% CI 1.43-237). ANO7 genotypes correlate with expression and biochemical relapse, suggesting that ANO7 is a potential PrCa susceptibility gene and that its elevated expression correlates with disease severity and outcome.

Keywords: ANO7; CRPC; expression; prostate cancer; survival.

Figure 1

Flow chart of initial ANO7 screening, variant selection and results validation. Blood DNA from 50 PrCa cases, tumor DNA from 22 CRPC tumors and blood DNA from 14 healthy males were sequenced via Illumina Miseq. Libraries were prepared with the Illumina TruSeq Custom amplicon kit (Illumina, San Diego, CA) following the manufacturer's instructions. The TruSeq Custom amplicon kit included all 25 exons (hg19) with 97% coverage of the gene. Variant calling and quality control were conducted using an in‐house pipeline (Supporting Information). The ANO7 variants of interest that were selected were verified from the same samples with Sanger sequencing. The three selected SNPs were further genotyped using TaqMan® SNP Genotyping Assay primers and probes in the validation step. All genotyping rounds were validated in two ways: five positive randomly selected samples were validated with Sanger sequencing and, in addition, 10% of all DNA samples were reanalyzed with TaqMan®. Swedish and Norwegian validation sets are described in the Oncoarray data (http://practical.icr.ac.uk). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

The detected ANO7 mutations. Modified figure from http://atlasgeneticsoncology.org/Genes/GC_ANO7.html. The locations of rs148609049 (orange arrow), rs77559646 (purple arrow) and rs181722382 (green arrow) variants in ANO7 transcripts NM_001001666 and NM_001001891 (hg19).

Figure 3

Kaplan–Meier overall survival plots of rs148609049 (a, c), rs77559646 (b, d) variant carriers (red curve) and noncarriers (blue curve) and Cox regression analysis (e). Figures A and B show PrCa specific survival while C and D represent overall survival. PrCa specific survival does not show a statistically significant trend, i.e., carriers of either variant would have a shortened survival time (a, b). Because of missing cause of death information, the overall survival was also analyzed. Variant rs77559646 AG or A/A genotypes (d) do not have an effect on survival, while the rs148609049 C/T or T/T genotype (c) significantly reduce survival. In the Cox regression analysis, Gleason score and rs148609049 were the most significant factors explaining overall survival (e).

Figure 4

Prostate‐lineage‐specific expression of ANO7. The mRNA level of ANO7 is significantly upregulated in the prostate gland (a) and prostate tumors (b) compared to other organs and cancers, respectively. The p values were examined using Student's t‐test. ANO7 expression intensity is shown as log2 median‐centred as downloaded from the licensed Oncomine database19 and displayed by boxplots in R (version 3.2.2). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

eQTL analysis of the association between rs77559646 and ANO7 gene expression. The risk genotype G/A of rs77559646 was significantly associated with increased mRNA expression of ANO7. The ANO7 mRNA levels were examined with RNA‐seq and displayed as RPKM values in a collection of 22 prostate cancer samples (Turku Prostate Cancer Consortium treatment cohort) (a), and by Illumina Expression BeadChip‐based transcriptional profiling in a collection of 119 (b) and 94 (c) human prostate tissue samples17 (levels are log2 transformed and quantile normalized). The ANO7 mRNA expression data in prostate tumors tissues were displayed as a five‐number distribution (minimum, first quartile, median, third quartile, and maximum) according to the rs77559646 genotypes. The p values were assessed via linear regression model. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

ANO7 is highly expressed in prostate cancer and is associated with poor overall survival in patients with prostate cancer. The ANO7 transcript levels were strikingly increased in prostate cancer tissues compared to normal tissues in a collection of cohorts consisting of 550 (a) and 21 (b) samples, respectively.18, 24 The p values were calculated with the Mann–Whitney U test. In (a), ANO7 expression was determined by RNA sequencing; in (b) ANO7 expression intensity is shown as log2 median‐centred intensity as reported in the Oncomine database 19. In (c) the Kaplan–Meier curve and the estimate of the risk for overall survival in a large cohort of PrCa patients with higher (n = 115) or lower (n = 174) expression levels of ANO7 are shown. Patients with tumors expressing higher levels of ANO7 show decreased overall survival. The number of patients in each group at 20‐month intervals is indicated. The p values were calculated by the Log‐rank test and Cox regression model.