Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

Sonenshein GE (2012) Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide. PLoS ONE 7(3): e33287. doi:10.1371/journal.pone.0033287 Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide Ziyang Yu 0 Seiichi Sato 0 Philip C. Trackman 0 Kathrin H. Kirsch 0 Gail E. Sonenshein 0 Vladimir V. Kalinichenko, Cincinnati Children's Hospital Medical Center, United States of America 0 1 Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts, United States of America, 2 Division of Oral Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, United States of America, 3 Department of Biochemistry, Boston University School of Medicine , Boston, Massachusetts , United States of America B lymphocyte-induced maturation protein 1 (Blimp1) is a master regulator of B cell differentiation, and controls migration of primordial germ cells. Recently we observed aberrant Blimp1 expression in breast cancer cells resulting from an NF-kB RelB to Ras signaling pathway. In order to address the question of whether the unexpected expression of Blimp1 is seen in other epithelial-derived tumors, we selected lung cancers as they are frequently driven by Ras signaling. Blimp1 was detected in all five lung cancer cell lines examined and shown to promote lung cancer cell migration and invasion. Interrogation of microarray datasets demonstrated elevated BLIMP1 RNA expression in lung adenocarcinoma, pancreatic ductal carcinomas, head and neck tumors as well as in glioblastomas. Involvement of Ras and its downstream kinase c-Raf was confirmed using mutant and siRNA strategies. We next addressed the issue of mechanism of Blimp1 activation in lung cancer. Using knockdown and ectopic expression, the role of the Activator Protein (AP)-1 family of transcription factors was demonstrated. Further, chromatin immunoprecipitation assays confirmed binding to identified AP-1 elements in the BLIMP1 promoter of ectopically expressed c-Jun and of endogenous AP-1 subunits following serum stimulation. The propeptide domain of lysyl oxidase (LOX-PP) was identified as a tumor suppressor, with ability to reduce Ras signaling in lung cancer cells. LOX-PP reduced expression of Blimp1 by binding to c-Raf and inhibiting activation of AP-1, thereby attenuating the migratory phenotype of lung cancer cells. Thus, Blimp1 is a mediator of Ras/Raf/AP-1 signaling that promotes cell migration, and is repressed by LOX-PP in lung cancer. - Funding: These studies were supported by National Institutes of Health (NIH) grants R01 CA143108 and PO1 ES011624. 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. . These authors contributed equally to this work. B lymphocyte-induced maturation protein 1 (Blimp1) or Positive-Regulatory Domain I Binding Factor 1 (PRDI-BF1) is a zinc finger protein encoded by the PRDI-BF1 and RIZ domain 1 (PRDM1) or BLIMP1 gene [1,2], which was initially isolated as a transcriptional repressor of the IFNb promoter [3]. Several mechanisms of Blimp1-mediated repression of gene transcription have been elucidated: recruitment of histone methyltransferases (HMTs) [4], histone deacetylases (HDACs) [5] or corepressors [2] or by competition with transcriptional activators [6]. Blimp1 was identified as a master regulator of B cell terminal differentiation [7], which promotes differentiation of B lymphocytes to plasma cells [8]. Several factors have been implicated in the activation of transcription of the Blimp1 gene during the differentiation of B cells, including NF-kB, AP-1, IRF4, STAT3 and STAT5, although, their precise mechanisms of action are not fully understood [9]. Blimp1 was subsequently shown to regulate T cell proliferation and homeostasis [10]. During development, Blimp1 controls primordial germ cell (PGC) specification and migration as Blimp1-deficient mouse embryos generate PGC-like cells which fail to show characteristic PGC migration [11,12]. Somewhat unexpectedly, Blimp1 was detected in non-hematopoietic cancer cells. Our laboratory observed Blimp1 expression in breast cancer cells, and showed it repressed transcription of the ESR1 gene encoding estrogen receptor alpha (ERa), thereby promoting a more migratory phenotype [13]. Transcriptional induction of Bcl-2 levels by the NF-kB RelB subunit recruited Ras to the mitochondria [14]. The resultant Ras signaling led to an aberrant induction of Blimp1 in the breast cancer cells [13]. The exact transcription factor(s) downstream of Ras that mediated the activation of Blimp1 in these cancer cells remained to be identified. However, the involvement of Ras signaling in Blimp1 activation leads us to hypothesize that expression of Blimp1 may be more widespread in cancer than previously realized. Colorectal tumor cells were also found to express Blimp1, which repressed the TP53 gene and thus maintained cell growth [15]. Lung cancer is the leading cause of cancer-related death in Western countries. Approximately two-thirds of patients are diagnosed at an advanced stage, and of the remaining patients who undergo surgery, 3050% develop recurrence with metastatic disease [16,17]. The RAS oncogene is mutated in up to ,30% of lung cancers, with the majority of mutations found in the KRAS gene [16,17]. Oncogenic K-Ras predisposes transgenic mice to lung tumorigenesis [18]. Ras signals via multiple pathways, including mitogen activated protein kinase (MAPK). As nuclear acceptors for MAPK signaling cascades, the activator protein (AP)1 family of transcription factors has been implicated in the highly migratory phenotype of lung cancer cells [19,20,21]. The lysyl oxidase (LOX) gene was isolated as the ras recision gene (rrg) due to its ability to revert Ras-mediated transformation of NIH 3T3 fibroblasts [22]. Our group showed ectopic Pro-LOX expression reduced extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling and activation of NF-kB in Ras-transformed NIH 3T3 cells [23]. Loss of LOX gene expression was seen in many cancerous tissues and derived cell lines including those from lung [24,25,26], colon [27], prostate [28], gastric [29] and head and neck squamous cancers [30]. Ectopic LOX gene expression reduced colony formation of cultured gastric cancer cells and tumor formation in a xenograft model [29]. Lysyl oxidase is synthesized and secreted as a proenzyme (Pro-LOX), and processed to a functional enzyme (LOX) and amino terminal propeptide (LOX-PP) [31]. The rrg activity of Pro-LOX was unexpectedly mapped to the LOX-PP domain, as judged by inhibition of the transformed phenotype of NIH 3T3Ras cells [32]. Subsequently, LOX-PP was shown to reduce the (...truncated)