The LKB 1 Tumor Suppressor as a Biomarker in Mouse and Human Tissues

Germline mutations in the LKB1 gene (also known as STK11) cause the Peutz-Jeghers Syndrome, and somatic loss of LKB1 has emerged as causal event in a wide range of human malignancies, including melanoma, lung cancer, and cervical cancer. The LKB1 protein is a serine-threonine kinase that phosphorylates AMP-activated protein kinase (AMPK) and other downstream targets. Conditional knockout studies in mouse models have consistently shown that LKB1 loss promotes a highly-metastatic phenotype in diverse tissues, and human studies have demonstrated a strong association between LKB1 inactivation and tumor recurrence. Furthermore, LKB1 deficiency confers sensitivity to distinct classes of anticancer drugs. The ability to reliably identify LKB1-deficient tumors is thus likely to have important prognostic and predictive implications. Previous research studies have employed polyclonal antibodies with limited success, and there is no widely-employed immunohistochemical assay for LKB1. Here we report an assay based on a rabbit monoclonal antibody that can reliably detect endogenous LKB1 protein (and its absence) in mouse and human formalin-fixed, paraffin-embedded tissues. LKB1 protein levels determined through this assay correlated strongly with AMPK phosphorylation both in mouse and human tumors, and with mRNA levels in human tumors. Our studies fully validate this immunohistochemical assay for LKB1 in paraffin-embedded formalin tissue sections. This assay should be broadly useful for research studies employing mouse models and also for the development of human tissue-based assays for LKB1 in diverse clinical settings. Citation: Nakada Y, Stewart TG, Peña CG, Zhang S, Zhao N, et al. (2013) The LKB1 Tumor Suppressor as a Biomarker in Mouse and Human Tissues. PLoS ONE 8(9): e73449. doi:10.1371/journal.pone.0073449 Editor: Francisco X. Real, Centro Nacional de Investigaciones Oncológicas (CNIO), Spain Received March 21, 2013; Accepted July 21, 2013; Published September 25, 2013 Copyright: 2013 Nakada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported through National Institutes of Health/National Cancer Institute grants R01CA137181 (DC), RO1CA163896 (NS, KW), U01CA141576 (DC, KW, NB, NS), and Cancer Prevention Research Institute of Texas grant RP100550 (DC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: DC, KW, NB and NS are inventors on an application (United States patent pending) of DNA-based methods to employ LKB1 status for diagnostic and prognostic clinical tests. This invention does not utilize the methods or antibody described in this manuscript. These investigators are paid consultants of Molecular MD (Portland, OR), which has a license agreement with these authors’ respective academic institutions. NH is an inventor on an application (United States patent pending) for the use of LKB1 as a prognostic factor – this invention also does not utilize the methods or antibody described in this manuscript. * E-mail: hayes@med.unc.edu (DNH); diego.castrillon@utsouthwestern.edu (DHC) . These authors contributed equally to this work.

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