Molecular Basis For The Concentration-Dependent Tumor Suppressing Effects Of Klk6 In Breast Cancer

Human kallikrein-related peptidase 6 (KLK6) is a serine protease exerting versatile (patho)physiological functions (J Biol Chem 284:32989-32994, 2009). Recently, we showed that KLK6 acts as a tumor suppressor in human breast cancer. Interestingly, the tumor suppressing effects of KLK6 depend on its expression levels (Cancer Res 69:3779-3797, 2009). Namely, KLK6 re-expression at physiological levels (i.e. levels found in normal mammary cells and tissues) in KLK6-negative MDA-MB-231 breast cancer cells resulted in strong inhibition of tumor cell proliferation, anchorage-independent growth and cell motility, while it remarkably abolished their ability to form tumors in SCID mice. Contrary, when KLK6 was highly overexpressed (>50-fold compared to normal cells) in MDA-MB-231 cells, its tumor suppressor activity in vivo was lost. Importantly, overexpression of KLK6 is a characteristic of a small subgroup of breast tumors. Here, we investigated molecular pathways underlying these concentration-dependent effects of KLK6 in breast cancer. In addition, we tested whether the enzymatic activity of KLK6 is required for the observed phenotypes or they may result from non-proteolysis-related protein-protein interactions. To address these questions we carried out genome-wide differential transcriptomic profiling using the Illumina bead microarrays. The profiles of KLK6-negative MDA-MB-231 parental cells was compared in parallel with three different MDA-MB-231 clones stably transfected with wt or mutant KLK6 cDNA, namely C28WT (expresses KLK6 at normal levels), C5WT (highly overexpresses KLK6) and C22MS (expresses a mutant form of KLK6 with a point substitution at the active site serine residue, i.e. S192A, that completely inhibits its protease activity). Detailed proteomic profiles were determined for all the aforementioned MDA-MB-231 clones using one-dimensional (1D) SDS-PAGE analysis of cell lysates, combined with in gel trypsin digestion and LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry). In all cases, we were able to identify in total 3779 cellular proteins with 2487 having ≥2 peptides distributed as: 1776 in parental, 2020 in C28WT, 1228 in C5WT and 1593 in C22MS. Hierarchical clustering proteome analysis clustered C22MS together with parental. Sorting the proteins relative to ≥4 fold-expression over parental resulted in identification of 377 proteins differentially expressed in C28WT, 449 in C5WT and 169 C22MS. The derived datasets were used to infer potential networks using Ingenuity Pathway Analysis (IPA). Important pathways (including NF-kB, TP53 and apoptosis-related) were found differentially deregulated upon KLK6 expression. For the first time, we describe KLK6-associated gene expression signatures and proteomes that were integrated in an effort to derive putative pathways underlying the emerging role(s) of KLK6 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4916. doi:1538-7445.AM2012-4916