Supplementary Materialsoncotarget-09-37733-s001. invasion, and active LOX and LOXL2 as tumor promoters

Supplementary Materialsoncotarget-09-37733-s001. invasion, and active LOX and LOXL2 as tumor promoters in human melanoma cells by promoting their invasive growth. mice [22]. ODC-induced transformation was associated with constitutive c-Jun activation [23], and induced expression of the transactivation domain deletion mutant of c-Jun (TAM67) was found to reverse the transformed morphology and reduce their invasive growth [24]. Similar results were obtained with RAS-transformed mouse fibroblasts (E4 cells) [24]. Lysyl oxidase (LOX) is Gata3 a secreted copper-dependent amine oxidase that plays an important role especially in the crosslinking of collagen and elastin in the extracellular matrix [25]. LOX is synthesized and secreted as a AC220 inhibition 50-kDa inactive glycosylated proenzyme (pro-LOX), which is then cleaved extracellularly into a functional 32-kDa enzyme (LOX) and an 18-kDa propeptide (LOX-PP) by bone morphogenetic protein 1 (BMP-1) and related proteases (Tolloid-like 1 and 2) [26]. LOX-PP can further exist in differentially glycosylated forms of higher molecular weight up to 35 kDa [27]. LOX has been reported to control cell phenotype and regulate many cellular processes, including cell adhesion, migration, and invasion [28C31], as well as epithelial-mesenchymal transition in hypoxic conditions [32, 33]. Paradoxically, LOX has been reported to function both as a tumor suppressor and a promoter in human cancer cells, depending on tumor type and AC220 inhibition stage of progression. Originally, (first named the [48], we additionally studied the expression levels of all LOX family genes in different melanoma cell lines. In contrast to that in ODC-transformed fibroblasts, we found a general increase in the expression of the LOX family members in melanoma cells. To resolve this paradox, we further studied the functions of the encoded proteins by using a universal LOX inhibitor -aminopropionitrile (BAPN) and knocking down of LOX and LOXL2 in melanoma cells. Our data suggest that inactive pro-LOX functions as a tumor suppressor in ODC- and RAS-transformed mouse fibroblasts by inhibiting cell growth and invasion, and that the mature, active LOX and AC220 inhibition LOXL2 act as tumor promoters in human melanoma cells by promoting their invasive growth. Further, we show that high LOXL2 mRNA expression may be correlated with metastasis and poor survival in melanoma. RESULTS LOX expression is downregulated in ODC-transformed mouse fibroblasts in a c-Jun-regulated manner In this study, we first set out to identify ODC-induced transformation-associated genes downregulated by c-Jun. By using gene expression microarray AC220 inhibition analyses, we searched for genes that are both downregulated in ODC-transformed cells (Odc cells) compared to parental N1 fibroblasts as well as upregulated in Odc cells transfected with a tetracycline-inducible TAM67 vector (Odc-pLRT-TAM67) after induction of TAM67 expression. Using two different microarray platforms, only three genes – fibulin 5 (has been proposed to be a tumor suppressor and also to be downregulated in HRAS-transformed mouse cells [34, 35], we selected it to be studied in more detail. First, we verified by RT-PCR the downregulation of in Odc cells, and the upregulation of in Odc-pLRT-TAM67 cells, after TAM67 induction (Figure 1A and 1B). We further studied the expression of in the RAS-transformed (E4) cells and found its expression to be downregulated compared to N1 cells (Figure ?(Figure1A),1A), consistent with previous findings [34, 35]. The downregulation of expression in Odc cells was also seen at the protein level. Immunoblotting with a LOX antibody recognizing both pro-LOX and mature LOX revealed that the normal N1 cells contained high levels of pro-LOX but no detectable amounts of cleaved/mature LOX, and that the transformed Odc cells showed a marked decrease in pro-LOX expression (Figure ?(Figure1C).1C). Analysis of the secreted proteins from the cells with the same antibody showed that pro-LOX was secreted and cleaved to mature/active LOX, roughly proportionally to the cellular levels of pro-LOX (Figure 1D and 1E). The cellular protein levels of the LOX-propeptide region, detected by LOX-PP antibody (Figure ?(Figure1F,1F, left panel), showed no clear difference between the N1 and Odc cells. However, when analyzing the secreted proteins, a 26 kDa protein band was detected in N1 cells, but not in Odc cells (Figure AC220 inhibition ?(Figure1F,1F, right panel). The 26 kDa band may well represent glycosylated LOX propeptide [49]. The 18 kDa protein band seen in the cell extracts equally expressed in the N1 and Odc cells (Figure ?(Figure1F,1F, left panel) is unlikely to be LOX-PP, but represents a protein non-specifically binding the antibody in NIH3T3 cells [50]. Table 1 Identification of genes downregulated in ODC-transformed NIH3T3 cells (Odc) compared to parental N1 cells and upregulated in Odc cells expressing a tetracycline-inducible TAM67 vector (Odc-pLRT-TAM67).