The Propeptide Domain of Lysyl Oxidase Induces Phenotypic Reversion of Ras-transformed Cells

Abstract

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. ␤-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Aktdependent induction of NF-B activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.Lysyl oxidase catalyzes oxidative deamination of peptidyl lysine and hydroxylysine residues in collagens, and peptidyl lysine residues in elastin. The resulting peptidyl aldehydes spontaneously condense and undergo oxidation reactions to form the lysine-derived covalent cross-links required for the normal structural integrity of the extracellular matrix (1-3). Lysyl oxidase is synthesized as a 48 -50 kDa proenzyme, secreted into the extracellular environment where it is then processed by proteolytic cleavage to a functional 30 kDa enzyme and an 18 kDa propeptide (4). Evidence supports that 30 kDa lysyl oxidase is active whereas the 50 kDa proenzyme is enzymatically inactive (5-7). Procollagen C-proteinases are active in processing pro-lysyl oxidase and are products of the Bmp1 gene and the structurally related Tll1 and Tll2 genes (6 -8).Lysyl oxidase gene expression was found to inhibit the transforming activity of ras and was hence named the "ras recision gene" (rrg) (9, 10). Lysyl oxidase is down-regulated in rastransformed cells and in many cancer cell lines. Reduced lysyl oxidase levels are also observed in human cancers (9, 11-15), whereas in spontaneous revertants or upon induced phenotypic reversion higher normal levels of lysyl oxidase are again seen (9,14). Conversely stable phenotypic revertants of ras-transfected NIH3T3 cells return to a transformed phenotype upon transfection with an antisense lysyl oxidase vector (9, 10, 16). Antisense lysyl oxidase t...

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

“…LOX is also a tumor suppressor and it inhibits ras-dependent transformation of fibroblasts [4,8,14]. The tumor suppressor activity of LOX has been mapped to the pro-peptide region [24] and LOX-PP inhibits tumor formation by breast cancer cells in mice [19]. In normal osteoblasts, LOX-PP accumulates in cultures [9] and recombinant LOX-PP is taken up by cells and accumulates over time associated with microtubules [7].…”

“…Primary vascular SMCs were grown with LOX-PP (10 μg/ml) or vehicle was then the number of viable cells per culture determined after 48 hours. The concentration of LOX-PP was based on preliminary dose response-and published studies [19,24,28]. Figure 1A shows that LOX-PP significantly inhibits the growth of SMC cultures.…”

“…The LOX propeptide (LOX-PP) is unique in structure and has little sequence identity to LOXL1 and no sequence similarity to the other LOXL proteins [5]. LOX-PP contains the tumor suppressor functionality of LOX and it inhibits ras-dependent signaling, including Erk1/2 Map kinase activation [10,24]. LOX is expressed by vascular SMCs, and LOX deficient mice die at parturition and exhibit major cardiovascular defects [18].…”

Transforming Growth Factor-α Inhibits the Intrinsic Pathway of c-Myc-Induced Apoptosis through Activation of Nuclear Factor-κB in Murine Hepatocellular Carcinomas

“…Likewise, Ha-Ras-mediated transformation of NIH 3T3 fibroblasts and rat liver epithelial cells resulted in IKK complex activation through multiple pathways involving mitogen-activated protein kinases as well as PI(3)K/Akt (7,41). Furthermore, in breast cancer cells, the PI(3)K/Akt axis played a role during NF-nB activation in response to EGFR or HER-2/ neu signaling (31, 42), which could be impeded by the PTEN tumor suppressor (31) or by the pro-peptide domain of lysil oxidase (43,44). Consistent with a role of the PI(3)K/Akt axis in IKK activation, we report that TGF-a/c-Myc -derived hepatocellular carcinomas display constitutive phosphorylation Another important finding of our study is the antagonistic effect of NF-nB on c-Myc-induced apoptosis.…”

Lysyl oxidase-like-1 enhances lung metastasis when lactate accumulation and monocarboxylate transporter expression are involved

“…LOXL-1, similarly to LOX, plays a critical role in the formation and repair of the ECM by oxidizing lysine residues in elastin and collagen, thereby stabilizing the fibrous proteins (18,27). In addition to its fibrogenic function, LOX expression is associated with tumor suppression and tumor progression, and its role in tumorigenesis appears to be dependent on cellular location, cell type and transformation status (27)(28)(29)(30)(31)(32). Microarray studies have shown that the expression of LOX is elevated in hypoxic human tumor cells (33), suggesting that the LOX expression is regulated by hypoxiainducible factor (HIF) and is associated with hypoxia in human breast, head and neck tumors (28).…”