Latexin Is Down-Regulated in Hematopoietic Malignancies and Restoration of Expression Inhibits Lymphoma Growth Yi Liu2, Dianna Howard1, Kyle Rector2, Carol Swiderski1, Jason Brandon3, Lawrence Schook4, Jayesh Mehta5, J. Scott Bryson1,3, Subbarao Bondada3, Ying Liang 1 * 1 Department of Internal Medicine, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 2 Department of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 3 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, United States of America, 4 Department of Animal Sciences, University of Illinois, Urbana, Illinois, United States of America, 5 Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America Abstract Latexin is a negative regulator of hematopoietic stem cell number in mice. Its dysregulated expression in other tumors led us to hypothesize that latexin may have tumor suppressor properties in hematological malignancies. We found that latexin was down-regulated in a variety of leukemia and lymphoma cell lines as well as in CD34+ cells from the blood and marrow of patients with these malignancies. 5-aza-29-deoxycytodine treatment and bisulfite sequencing revealed hypermethylation of latexin promoter in tumor cells. Retrovirus-mediated latexin overexpression in A20 mouse lymphoma cells inhibited their in vitro growth by 16 fold and in vivo tumor volume by 2 fold. Latexin caused growth inhibition of lymphoma cells by significantly increasing apoptosis through the down-regulation of anti-apoptotic genes Bcl-2 and Pim-2. The molecular mechanism underlying latexin-mediated tumor inhibition was not through its canonical carboxypeptidase inhibitor activity. These results are consistent with a tumor suppressor role for latexin and suggest that latexin may have clinical efficacy in the treatment of malignancies. Citation: Liu Y, Howard D, Rector K, Swiderski C, Brandon J, et al. (2012) Latexin Is Down-Regulated in Hematopoietic Malignancies and Restoration of Expression Inhibits Lymphoma Growth. PLoS ONE 7(9): e44979. doi:10.1371/journal.pone.0044979 Editor: Ilya Ulasov, University of Chicago, United States of America Received April 30, 2012; Accepted August 16, 2012; Published September 27, 2012 Copyright: 2012 Liu 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 work was supported by American Cancer Society ACS178898 (to YL) and BD Biosciences Research Grant Award (to YL) and the support of The Edward P. Evans Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ying.liang@uky.edu Introduction Both solid and liquid tumors are now understood to originate from the malignant transformation of resident adult stem and progenitor cells. [1,2] Nowhere is this paradigm better established than in leukemia, yet events causing neoplastic conversion remain poorly understood. [3] We identified latexin (Lxn) as a novel, homeostatic regulator of the size of the hematopoietic stem cell population in mice. [4] The stem cell pool size was inversely related to quantitative Lxn expression at both the levels of transcript and protein. We found that population size was influenced by Lxn in a stem cell-autonomous manner, and acting through the concerted mechanisms of self-renewal and apoptosis, which were decreased and increased, respectively, by Lxn abundance. [4] Thus, Lxn acts as a brake on the expansion of stem cell population. Unrestrained stem cell expansion carries with it the risk of mutations, genomic instability and carcinogenesis. We therefore hypothesized that Lxn expression patterns in stem and progenitor cells may act as a tumor suppressor by inhibiting stem and progenitor cell proliferation, influencing the crucial steps toward malignancy. Lxn was primarily studied in the nervous system, involving the specification of cortical brain regions during development [5,6,7,8,9], as well as the speed of nerve transmission in adult peripheral nervous systems [10]. Lxn was found to be involved in the inflammatory response in macrophages owing to its paired cystatin-like domains [11]. It was detected in mast cells associated with a unique type of intracellular granule distinct from histamine- containing secretory granules and lysosomes [12]. Lxn has about 30% sequence homology, but much greater structural homology, with tazarotene-induced gene 1 (TIG1) (or retinoic acid receptor responder 1, RARRES1), whose expression was down-regulated in an extensive list of tumor types in humans. [11,13,14,15,16]. Lxn and TIG1 are closely linked genetically and may represent members of a family of functionally related genes. Moreover, Lxn was reported to be a TNF-responsive gene in human papillovirus-infected keratinocytes, suggesting that it may contrib- ute to TNF-mediated suppression of cervical cancer development [17]. A recent report revealed that Lxn was down-regulated in patients with gastric carcinomas, and overexpression or knock- down of Lxn inhibited or stimulated tumor growth respectively [18]. Decreased or absent Lxn expression was observed in several human leukemia and lymphoma cell lines [19], as well as in malignant melanoma patients. [20] However, there is still lack of direct evidence for the cause-effect relationship between Lxn and hematopoietic malignancy. Gene silencing, especially of tumor suppressors, and inappro- priate gene activation, especially of oncogenes, are common PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44979