The Ras signaling inhibitor LOX-PP interacts with Hsp70 and c-Raf to reduce Erk activation and transformed phenotype of breast cancer cells.

Department of Biochemistry, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 07/2011; 31(13):2683-95. DOI: 10.1128/MCB.01148-10
Source: PubMed


The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox(-/-) mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-κB, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.

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    • "Please cite this article in press as : Ozdener , G . B . , et al . , Determination of Cell Uptake Pathways for tumor inhibitor lysyl oxidase propeptide , Molecular Oncology ( 2015 ) , http : / / dx . doi . org / 10 . 1016 / j . molonc . 2015 . 07 . 005 with functional consequences ( Bais et al . , 2015b ; Sanchez - Morgan et al . , 2011 ; Sato et al . , 2011 , 2013 ) . Moreover , nuclear localization of rLOX - PP has been demonstrated in cell confocal microscopy and cell fractionation studies ( Bais et al . , 2014 Q2 ; Guo et al . , 2007 ) ."
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    ABSTRACT: The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    Molecular Oncology 08/2015; DOI:10.1016/j.molonc.2015.07.005 · 5.33 Impact Factor
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    • "LOX-PP-P111A and -R116A were unable to efficiently displace endogenous c-Cbl from GFP-CIN85 in contrast to the effects of LOX-PP-WT (Fig. 4B, C). Lastly, the specificity of the changes in binding due to these mutations in LOX-PP was examined by comparing their effects on interaction with c-Raf, which was previously mapped to aa 26-100 [28]. While LOX-PP mutants P111A and R116A have lost the ability to interact with CIN85, they retained the ability to interact with c-Raf essentially as well as WT LOX-PP (Fig. 4D). "
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    ABSTRACT: The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.
    PLoS ONE 10/2013; 8(10):e77288. DOI:10.1371/journal.pone.0077288 · 3.23 Impact Factor
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    • "We recently noted that LOX-PP can physically interact with c-Raf and Hsp70 in breast cancer cells [61]. Given the role of c-Raf in activation of Blimp1, their physical association in lung cancer cells was next tested. "
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    ABSTRACT: 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-κB 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.
    PLoS ONE 03/2012; 7(3):e33287. DOI:10.1371/journal.pone.0033287 · 3.23 Impact Factor
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