Poster Presentation 9th General Meeting of the International Proteolysis Society 2015

Functional validation of PSA coding variants associated with prostate cancer risk (#107)

Judith Clements 1 , Srilakshmi Srinivasan 1 , Carsons Stephens 1 , Emilia Marijanovic 2 , Ashley Buckle 2 , Hans Lilja 3 , Jyotsna Batra 1
  1. Australian Prostate Cancer Research Centre, QLD, VIC, Australia
  2. Department of Biochemistry and Molecular Biology, Faculty of Medicine, Biomedical Sciences, Monash University, Clayton
  3. Departments of Laboratory Medicine, Medicine (Genitourinary Oncology), and Surgery (Urology), Memorial Sloan Kettering Cancer Center, New York

Prostate specific antigen (PSA)/Kallikrein-related peptidase 3 (KLK3) is the current biomarker for prostate cancer and has a significant role in the proteolytic cascades involved in seminal clot dissolution and prostate tumour biology. Our recent genetic fine-mapping studies identified two KLK3 non-synonymous single nucleotide polymorphisms (SNPs), rs61752561:G>A (D102N) and rs17632542:T>C (I179T) to be significantly associated with prostate cancer risk and aggressiveness [1]. These two SNPs are associated with low PSA levels at diagnosis [1, 2, 3] and the rs17632542 SNP with reduced tumor volume (p=0.002) [4]. To date, no studies have been undertaken to understand the potential molecular effect of these two non-synonymous SNPs on PSA mRNA expression, protein structure, stability or function.

In-silico analysis suggests an alteration in splicing by creating enhancer motifs for the risk allele. Mini-gene assays verified differential allele-specific mRNA splicing induced by the rs17632542 but not the rs61752561SNP. Differential scanning fluorimetry using recombinant protein isoforms showed an altered thermal stability for both the SNPs as suggested by in-silico analysis. Deglycosylation analysis confirmed the additional glycosylation site (D102N) created for the rs61752561 SNP. Proteomic identification of cleavage specific sites (PICS) assay performed to identify the cleavage site specificity within the active wild-type showed no difference in cleavage specificity. However, peptide substrate activity testing assays using recombinant protein isoforms showed an altered substrate activity for both the SNPs. Proliferation and migration experiments using stable cell lines expressing these active PSA isoforms are being performed using Incucyte.

Our results indicate the rs17632542 and rs61752561 SNPs to have a biological effect on the expression and function of the KLK3/PSA protein suggesting that genetic variation in the PSA gene may be a contributor to the functional role of PSA in prostate cancer pathogenesis. Further cell-based functional assays will provide more insight into their potential role in prostate cancer initiation and progression.

  1. Kote-Jarai Z, Amin Al Olama A, et al. (2011). Hum Genet 129(6): 687-694.
  2. Gudmundsson J, Besenbacher S, et al. (2010). Science Translational Medicine 2(62): 62-92.
  3. Sullivan J, Kopp R, et al. (2015). BJC 113: 166-172.
  4. Reinhardt D, BT Helfand, et al. (2014). J Urol 191(6): 1733-6.