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Inflammopharmacol DOI 10.1007/s10787-010-0073-1




Lysyl oxidase: a potential target for cancer therapy

Siddikuzzaman V. M. Berlin Grace C. Guruvayoorappan

Received: 3 September 2010 / Accepted: 2 November 2010 Springer Basel AG 2010

Abstract Lysyl oxidases (LysOX; EC, protein- lysine 6-oxidases) are extracellular copper enzymes that catalyze the cross-linking of collagens or elastin in the extracellular matrix (ECM), thereby regulating the tensile strength of tissues. Recent implication of LysOX in cancer, wound healing, cell motility, chemotaxis, and differentia- tion reflects its remarkable functional diversity and also in the central nervous system pathologies. However, recent reports also demonstrated novel roles for LysOX, including the ability to regulate gene transcription, motility/migra- tion, and cell adhesion. These diverse functions have led researchers to hypothesize that LysOX may have multiple roles affecting both extra- and intracellular cell function(s). Both down and up-regulation of LysOX in tumor tissues and cancer cell lines have been described, suggesting a dual role for LysOX as a tumor suppressor, as well as a metastasis promoter gene. In this review we explain in detail the role of lysyl oxidase in tumor progression and metastasis.


cellular matrix Cancer Tumor suppressor

Metastasis promoter

Lysyl oxidase Copper Extra


The extracellular matrix (ECM) is essential for providing mechanical and physiological properties for various tissues, and it also provides attachment sites for the anchoring of

Siddikuzzaman V. M. B. Grace C. Guruvayoorappan (& ) Department of Biotechnology, Karunya University, Karunya Nagar, Coimbatore 641114, Tamil Nadu, India e-mail:

641114, Tamil Nadu, India e-mail: cells and support for their migration in tissues. It also

cells and support for their migration in tissues. It also sup- ports physical structure formation of tissues during development and tissue maintenance, determines cell– matrix and cell–cell interactions, and provides a structural and signaling environment that is necessary for cell migration, proliferation, and differentiation. The ECM plays a pivotal role in regulation of cellular functions during embryonic development, tissue repair, inflammation, tumor invasion, and metastasis (Hynes 2002; Juliano 2002). The composition of the ECM is unique for each organ. It con- sists of numerous compounds including insoluble fibers, microfibrils, soluble proteins, and glycoproteins. The lysine residues of the ECM molecules, such as of collagen and elastin go through oxidative deamination by the extracellular enzyme lysyl oxidase (LysOX), a copper dependent amine oxidase, which forms reactive aldehyde of its substrates (Kagan 1994; Gibson et al. 1996). The newly formed aldehyde residues then interact spontane- ously to form covalent cross-linkages leading to insoluble extracellular protein matrices in most tissues and organs (Nagan and Kagan 1994; Smith-Mungo and Kagan 1998). The essential function of LysOX in the ECM and LysOX cellular distribution in the skin, lung, and the cardiovas- cular systems have been well characterized. Recently, in addition to its ECM cross-linking activity several studies reported novel roles for LysOX in diverse tumor types and a major role in invasive breast cancer cells and tumors. Although the ECM maturation activity of LysOX has long been thought to be its sole function, more recent evidence implicates the involvement of LysOX in many critical biological functions other than collagen or elastin cross-linking. LysOX has been shown to induce motility and migration in monocytes, vascular smooth muscle cells, and fibroblasts (Nelson et al. 1988; Lazarus et al. 1994; Li et al. 2000). In addition, LysOX expression and activity


Siddikuzzaman et al.

have been observed in the cytoplasm and nucleus (Waka- saki and Ooshima 1990; Li et al. 1997; Nellaiappan et al. 2000; Kagan and Li 2003; Lucero and Kagan 2006; Jansen and Csiszar 2007) and implicated in cell signaling and transcriptional gene regulation, as evidenced by utilization of histone H1 and H2 as substrates (Kagan et al. 1983; Giampuzzi et al. 2003a), altered chromatin condensation (Mello et al. 1995), activation of the collagen III a1 pro- moter through LysOX induced binding of Ku antigen (Giampuzzi et al. 2000), inactivation of the transcription factor NF-jB (Jeay et al. 2003), and regulation of cell adhesion through increased b-catenin and cyclin D1 expression (Giampuzzi et al. 2005). Even more recent are the findings that LysOX protein domains, other than the catalytic domain, can bind to proteins, as with binding to fibronectin (Fogelgren et al. 2005) and placental lactogen (Polgar et al. 2007) and that the cleaved 18 kDa LysOX propeptide (LysOX-PP) is also capable of regulating bio- logical functions (Palamakumbura et al. 2004). Since LysOX protein structure and function are so complex and involve such vital biological processes as cell movement, signal transduction, and gene regulation, it is evident that aberrant regulation of LysOX would lead to tumorigenesis and tumor progression. Indeed, loss of LysOX expression and activity in a number of cancers and oncogene-trans- formed cell models has implicated LysOX as a tumor suppressor gene. Likewise, LysOX expression and activity in a number of cancers has also been observed and has implicated LysOX as a metastasis promoting gene—cre- ating a conundrum within the LysOX research field with regard to LysOX biological function(s) in cancer. This review will summarize the role of LysOX in tumorigenesis and tumor progression with an emphasis on cancer meta- static progression.

The lysyl oxidase protein family

Lysyl oxidase (LysOX; protein-6-oxidase [EC]) is the key enzyme that controls collagen and elastin matura- tion (Pinnell and Martin 1968; Smith-Mungo and Kagan 1998). LysOX is the copper- and quinone-containing amine oxidase that catalyzes the oxidative deamination of peptidyl lysine in elastin and collagen to a-aminoadipic-d-semial- dehyde, the first step in formation of the cross-links that stabilize these structural proteins (Faina and Frederick 2010).The consequent aldehydes lead to a spontaneous condensation forming inter- and intrachain cross-links. This posttranslational modification of ECM molecules plays a very important role in collagen and elastin structural aspects and possibly in triggering still-unknown signal transduction pathways. Several reports have suggested a clear associa- tion between organ fibrosis and increased LysOX activity


(Chanoki et al. 1995; Jourdan-Le Saux et al. 1994; Sommer et al. 1993). The most intriguing aspect regarding LysOX activity relates to its putative cell phenotype control and tumor suppressor activity. LysOX was identified as a ‘‘ras recision gene’’ (rrg), and levels of LysOX were found to be decreased in cells transformed by ras or ras-dependent oncogenes (Contente et al. 1990; Kenyon et al. 1991; Krzyzosiak et al. 1992). Furthermore, Friedman and coworkers showed that ras-transfected NIH 3T3 cells induced to revert by beta or gamma interferon would return to their transformed phenotype upon transfection with an antisense LysOX vector, and retransformation did not affect p21 ras levels (Contente et al. 1990; Kenyon et al. 1991). In many naturally occurring and oncogene-induced tumors, LysOX is down regulated, and LysOX was induced con- comitantly with reversion (Contente et al. 1990; Kenyon et al. 1991; Krzyzosiak et al. 1992; Giampuzzi et al. 2001; Hajnal et al. 1993; Ha¨ma¨la¨inen et al. 1995). However, this oxidative activity does not display high specificity because LysOX can also oxidize additional lysine-rich proteins such as histone-H1, as well as various lysine-rich synthetic peptides (Kagan et al. 1983; Ohkawa et al. 2001). It was observed that LysOX can be translo- cated into cell nuclei and that it can regulate, by an as yet poorly understood mechanism, the expression of collagen- 3A1, indicating that LysOXs may have additional functions (Nellaiappan et al. 2000; Giampuzzi et al. 2000). LysOX is synthesized as an inactive proenzyme that is activated by the products of the BMP-1 gene (Panchenko et al. 1996). Recently, several additional LysOX family members were identified. These new family members are characterized by the presence of a conserved LysOX-like domain containing conserved copper binding and catalytic domains at their COOH termini. These new LysOXL genes include Lys- OXL (Kenyon et al. 1993; Kim et al. 1995), LysOXR-1 or LysOXL2 (Saito et al. 1997), LysOXR-2 or LysOXL3 (Huang et al. 2001), and LysOXC or LysOXL4 (Maki et al. 2001). LysOXR-1, LysOXR-2, and LysOXC differ with respect to LysOX and LysOXL in that they possess a much longer NH2-terminal domain, suggesting that they consti- tute a distinct subclass of LysOXs and that their functions may differ fundamentally from those of LysOXL and LysOX (Csiszar 2001). LysOXR-1 was initially identified as a gene the expression of which is up-regulated in senescent fibroblasts and in adherent tumor-derived cells (Saito et al. 1997). It was also found to be highly expressed in reproductive tissues (Jourdan-Le Saux et al. 1999). It was demonstrated that LysOXL is also processed into its active form by bone morphogenetic protein-1, raising the possibility that the proteins encoded by the other family members may also be activated by proteolytic digestion after secretion. The transition from a localized tumor to an invasive and metastatic tumor represents a landmark in the

Lysyl oxidase: a potential target for cancer therapy

development of malignant disease because it is usually associated with a markedly worse prognosis. The under- standing of the processes that govern this transition is therefore, of prime importance. It was recently observed that, in breast cancer the transition from a localized to an invasive/metastatic tumor is associated in many cases with the formation of fibrotic foci and desmoplasia (the presence of unusually dense collagenous stroma) within the primary tumor (Colpaert et al. 2001; Hasebe et al. 2000). There are also some indications that a similar correlation may exist in other types of cancers such as in colon cancer and in pancreatic cancer (Nishimura et al. 1998; Ellenrieder et al. 2000). These observations represent apparent paradoxes at first glance because invasiveness has long been associated with the destruction of ECM by ECM-degrading enzymes like metalloproteases (Stamenkovic 2000; Duffy et al. 2000) and heparanase (Vlodavsky and Friedmann 2001). However, it is possible that the deposition of excess ECM may stimulate, in turn, expression of matrix-degrading enzymes that will contribute under certain circumstances to tumor invasion. In fact, there is some evidence that an increase in ECM deposition can, indeed, influence the production of ECM-degrading enzymes (Schuppan et al. 2001; Sawada et al. 2001). Two LysOX family members, LysOX and LysOXL, were found to be expressed in areas of fibrogenesis in noninvasive in situ ductal breast carci- nomas (Decitre et al. 1998). However, it was reported that metastatic breast cancer cell lines express LysOX, Lys- OXL, and LysOXR-1, whereas nonmetastatic breast cancer-derived cell lines, such as MCF-7 cells, do not, and that LysOX-expressing MCF-7 cells display increased invasiveness in in vitro invasiveness assays (Kirschmann et al. 2002). It also reported that LysOXR-1 expression in nonmetastatic MCF-7 cells induces massive deposition of dense collagen fibers and the formation of numerous fibrotic foci in tumors that develop after the implantation of these cells in nude mice. These changes were accompanied by an increase in the invasiveness of the MCF-7 cells, although the LysOXR-1-expressing cells were still estro- gen dependent.

Physical and biological properties of LysOX

LysOX is a copper-dependent amine oxidase that initiates the covalent cross-linking of collagens and elastin in extracellular matrices (Smith-Mungo and Kagan 1998; Csiszar 2001). It is secreted as a M r 50,000 glycosylated proenzyme, which is proteolytically processed by procol- lagen C proteinase (bone morphogenic protein-1) into a mature, biologically active M r 32,000 form (Smith-Mungo and Kagan 1998; Csiszar 2001). The formation of collagen/ elastin cross-links by LysOX leads to an increase in tensile

strength and structural integrity and is essential for normal connective tissue function, embryonic development, and wound healing (Smith-Mungo and Kagan 1998; Casey and MacDonald 1997). Consequently, aberrant LysOX expression or enzymatic activity leads to disease. Decrea- ses in LysOX expression or activity have been associated with such heritable connective tissue disorders as Ehler- Danlos syndrome, cutis laxa, and Menkes’ syndrome (Kuivaniemi et al. 1985; Khakoo et al. 1997; Yeowell et al. 1994; Royce et al. 1980; Pinnell 1982). Increases in LysOX expression contribute to the development of fibrotic dis- eases such as arteriosclerosis, scleroderma, and liver cirrhosis, diseases that involve connective tissue remodel- ing (Ooshima and Midorikawa 1977; Kagan et al. 1981; Chanoki et al. 1995; Kagan 1994). Upregulation of lysyl oxidase expression by uric acid leads to increases fibro- nectin synthesis in rat renal tubular epithelial cells (Zhou et al. 2010). Although the ECM maturation activity of LysOX has long been thought to be its sole function, more recent evidence implicates the involvement of LysOX in many important biological functions other than collagen/elastin cross-linking. LysOX has been shown to induce motility and migration in monocytes, vascular smooth muscle cells, and fibroblasts (Lazarus et al. 1994; Li et al. 2000; Nelson

et al. 1988). In addition, LysOX may play a role in cell

growth/differentiation as its expression is up-regulated by a number of growth factors and steroids (Lazarus et al. 1994;

Li et al. 2000; Nelson et al. 1988; Sanada et al. 1978).

Moreover, a role for LysOX in transcriptional gene regu-

lation and cellular transformation has also been implicated

as evidenced by localization of LysOX protein and enzy-

matic activity to cell nuclei (Li et al. 1997), potential utilization of histone H1 as a substrate (Kagan et al. 1983), activation of the collagen III a1 promoter (Giampuzzi et al. 2000), and a putative tumor suppressor in nontumorigenic revertants of ras-transformed fibroblasts (Smith-Mungo and Kagan 1998). LysOX was purified from a variety of tissues and spe-

cies including chick cartilage, bovine aorta and lung, human placenta, and piglet skin (Stassen 1976; Kuivaniemi

et al. 1984; Cronlund and Kagan 1986; Shackleton and

Hulmes 1990). The molecular mass of the enzyme from all these sources was found to be 30 kDa (Panchenko et al.

1996; Kagan and Li 2003). LysOX has been cloned from

rat (Trackman et al. 1990, 1991), human (Hamalainen et al.

1991, 1993; Mariani et al. 1992), chick (Wu et al. 1992), and mouse (Contente et al. 1993) tissues. The mRNA for human LysOX is found as multiple species with sizes of 5.5, 4.3, 2.4, and 2.0 kilobases due to the use of alternate polyadenylation sites and partially to the existence of multiple transcription initiation sites (Mariani et al. 1992; Boyd et al. 1995). The human LysOX gene is located on


Siddikuzzaman et al.

chromosome 5q23.5–31.2 (Hamalainen et al. 1991, 1993; Mariani et al. 1992; Wu et al. 1992; Contente et al. 1993; Boyd et al. 1995) and encodes a 417 amino acid poly- peptide, of which the first 21 residues correspond to the signal peptide (Hamalainen et al. 1991; Mariani et al. 1992). The mouse LysOX gene has been mapped to chromosome 18 (Contente et al. 1993; Boyd et al. 1995; Mock et al. 1992; Chang et al. 1993).

Substrate and activity of LysOX

LysOX is a copper-dependent secreted amine oxidase that oxidatively deaminates specific peptidyl lysine and hydroxylysine residues of collagen and lysine in elastin in the presence of molecular oxygen (Kagan 1986; Pinnell and Martin 1968; Bateman et al. 1986; Reiser et al. 1992). LysOX can oxidize non-ECM lysine rich proteins such as histone-H1 as well as various lysine-rich synthetic peptides including basic fibroblast growth factor (bFGF) (Kagan et al. 1983; Giampuzzi et al. 2003a; Li et al. 2003). As a byproduct of the catalytic reaction, reactive aldehydes and hydrogen peroxide are generated by the LysOX that may contribute to some of the novel roles of LysOX observed in the wound healing, cell migration, motility, proliferation, and differentiation (Li et al. 2000, 2003; Fushida-Takem- ura et al. 1996; Nelson et al. 1988; Palamakumbura et al.


The resulting peptidyl aldehydes condense spontane- ously to form various bi-,tri-,and tetrafunctional intra- and inter molecular cross links (Kagan et al. 1984; Siegel 1974). In collagens, the lysine and hydroxylysine-derived cross-links are essential for providing the tensile strength and mechanical stability of the collagen fibrils and other supramolecular assemblies (Light and Bailey 1980; Bailey 2001) (Fig. 1). Collagen are the most abundant proteins in mammals, with at least 35 members divided into 9 families. This multigen family disperses gene throughout 15 or more chromosomes. Collagen molecules consist of three peptide

chromosomes. Collagen molecules consist of three peptide Fig. 1 The catalytic activity of LysOX: LysOX oxidatively

Fig. 1 The catalytic activity of LysOX: LysOX oxidatively deami- nates a peptidyl lysine to generate a peptidyl allysine which spontaneously reacts with another peptidyl lysine or allysine to form a covalent cross-link between proteins (Kagan 1986; Kagan and Cai



chains called a chains, and contain at least one triple- helical collagenous domain with repeating (Gly-X–Y) n sequences, i.e., a glycine residue as every third amino acid, with the frequent presence of proline and 4-hydroxyproline in the X and Y positions, respectively. After post-transla- tion modification in the extracellular space, the precursor of fibrillar collagen, called procollagen, undergoes prote- olytic conversion to form collagen molecules. Collagen molecules form fibrils and interact with non-collagenous and collagenous protein, while they assemble into supra- molecule structure where the fibrils are stabilized by the formation of intra- and intermolecular cross-links (Byers 2001; Myllyharju and Kivirikko 2001; Koch et al. 2001; Fitzgerald et al. 2001). Elastin is a highly insoluble protein component of the elastic fibers found in the ECM to provide elasticity and resilience to tissues requiring the ability to deform reversibly. Tissues rich in elastin include the aorta and large blood vessels (28–32% of the dry mass), lung (3–7%), elastic ligaments (50%), tendons (4%), and skin (2%) (Vrhovski and Weiss 1998; Debelle and Tamburro 1999). Tropoelastin, the precursor of elastin, is encoded by a single gene located on chromosome 7q11in humans and its alternative splicing results in at least 11 variants (Vrhovski and Weiss 1998). After minor post-translation modifications, tropoelastin is oxidized and covalently cross-linked by LysOX catalytic activity (Gibson et al. 1996; Kagan 1986) after which it is assembled into microfibers (Gibson et al. 1996).

LysOX is a tumor suppressor

The first direct evidence of the tumor suppressor activity of LysOX was demonstrated (Contente et al. 1990), and it identified a markedly down-regulated cDNA species upon transformation of mouse NIH 3T3 cells with LTR-c-H-ras. The expression level of this cDNA was restored when the transformed cell line (RS485) was treated with interferon to obtain a persistent revertant cell line (PR4). This cDNA species was named as ras recision gene (rrg). Contente et al. (1990) were the first to isolate an mRNA (called the ras recision gene) that was downregulated in ras-trans- formed fibroblasts. Persistent treatment of ras-transformed fibroblasts with IFNa/b yielded a revertant of the ras- transformed phenotype and a corresponding re-expression of the ras recision gene. It was later determined that the ras recision gene was, in fact, LysOX (Kenyon et al. 1991). Subsequently, it also demonstrated that the experimental downregulation of LysOX in normal rat kidney fibroblasts (NRKF) led to increased cellular proliferation and anchorage-independent growth, loss of PDGF and IGF-1 regulation, and constitutive activation of ras (Giampuzzi

Lysyl oxidase: a potential target for cancer therapy

et al. 2001). A non-orthotopic injection of LysOX knock out NRKF cells demonstrated increased tumorigenicity and metastasis. These investigators went on to demonstrate that the constitutive activation of ras (by downregulation of LysOX) led to increased expression of b-catenin and cyclin D1 through a noncanonical ras signaling pathway (Gia- mpuzzi et al. 2003b). Moreover, re-expression of LysOX in ras-transformed fibroblasts led to a decrease in the activa- tion of NF-jB, a potent transcription factor capable of regulating cell growth and neoplastic transformation (Jeay et al. 2003). The deactivation of NF-jB was not due to direct interaction with LysOX, but by the inhibition of Akt/ PI3K activation and membrane localization (a ras activated pathway). The most unanticipated results demonstrated that it was not LysOX catalytic activity that mediated the suppression of neoplastic transformation signaling in fibroblasts, but it was the 18-kDa LysOX-PP cleaved from pro-LysOX by BMP-1 (Palamakumbura et al. 2004). Although intracellular activity of the cleaved amino ter- minus of proLysOX is a recent finding, it is not novel as the cleaved procollagen N-propeptide has also been shown to function intracellularly to alter protein synthesis and phosphorylation, as well as cellular adhesion (Oganesian et al. 2006). In addition to oncogene-transformed fibro- blasts, a decrease in LysOX activity has also been observed in fibrosarcoma, choriocarcinoma, and rhabdomyosarcoma cell lines compared with normal fibroblast cell lines, which was subsequently shown to be due to low quantities of LysOX mRNA (Kuivaniemi et al. 1986a, b; Ha¨ma¨la¨inen et al. 1995). With the introduction of microarray analysis, LysOX has been shown to be modulated in various cancer cell lines and their corresponding tumor tissues. Thus, the majority of reports indicating alterations in LysOX expression have been limited to mRNA a transcript level, which does not always correlate with catalytic activity (Uzel et al. 2000). To date, a decrease in LysOX mRNA and/or protein has been observed in basal and squamous cell, bronchogenic, colon, esophageal, gastric, head and neck squamous cell, pancreatic, and prostatic carcinomas, as well as melanoma. However, only two reports have definitively demonstrated a tumor suppressor role for LysOX using in vitro/in vivo model systems. Downregu- lation of LysOX (stable antisense expression) in keratinocytes induced their invasion into the dermis of an in vitro skin equivalent model (Bouez et al. 2006). Interestingly, treatment of normal keratinocytes with bAPN did not induce invasion and suggests a role for LysOX-PP in tumor suppression in this model. Alterna- tively, LysOX protein may be capable of binding to novel target proteins outside of the catalytic domain to alter cell signaling involved in tumor progression. Stable transfec- tion of full-length LysOX cDNA into an intestinal-type gastric cancer cell line decreased proliferation and

anchorage-independent cell growth, as well as tumorigen- esis in non-orthotopically injected nude mice (Kaneda et al. 2004a, b). These studies have shown that LysOX acts as a potent tumor suppressor gene in fibroblasts, basal, and squamous cell and gastric carcinomas that occur through inhibiting intracellular signaling pathways known to induce neoplastic transformation. It remains to be determined whether the tumor suppressor activity of LysOX is as intimately involved in other cancers where down regulation of LysOX mRNA has been observed. Altered expression of other LysOX family members has been identified as well. LysOXL mRNA expression is downregulated in renal cell carcinoma cell lines in which the Von Hippel-Lindau (VHL) gene has been mutated, suggesting that loss of LysOXL expression is associated with oncogenesis of type 2B VHL disease (mutations in the elongin-binding region) (Tsuchiya et al. 2005). In addition, LysOXL expression was upregulated in wild-type p53 reconstituted lung ade- nocarcinoma cell lines (Kannan et al. 2001). Down- regulation of LysOXL mRNAwas observed in head and neck squamous cell carcinoma cell lines; however, more in-depth analyses are required to establish a tumor sup- pressor role for LysOXL and LysOXL2 in these cancers. The tumor suppressor activity of LysOX has been mapped to the pro-peptide region, and LysOX-PP inhibits tumor formation by breast cancer cells in mice (Paola et al. 2008) (Table 1).

LysOX is a metastasis promoter

The upregulation of LysOX mRNA in various cancer cell lines and their corresponding tumor tissues was demon- strated by microarray technology. To date, an increase in LysOX mRNA and/or protein has been observed in breast, central nervous system cancer cell lines, head and neck squamous cell, prostatic, clear cell renal cell, and lung carcinomas, and in melanoma and osteosarcoma cell lines, compared with their normal or non-aggressive neoplastic counterparts. Statistically significant clinical correlations between LysOX expression and tumor progression have been observed in breast (Erler et al. 2006), head and neck squamous cell (Erler et al. 2006), prostatic (Lapointe et al. 2004), and clear cell renal cell carcinomas (Stassar et al. 2001). LysOXL1 gene regulation is affected by an epige- netic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity (Romain et al. 2010).The expression of high levels of LysOX mRNA and/or protein was a poor prognostic factor and was associated with poorly differentiated, high-grade tumors, increased recur- rence rates, and decreased overall survival. The role of LysOX in tumor progression has been most extensively studied in breast cancer using in vitro models of migration/


Siddikuzzaman et al.

Table 1 LysOX as a tumor suppressor gene: in vitro and in vivo studies

In vivo

In vitro

Inhibition of ras transformation (Di Donato et al. 1997) Stromal reactions in cancer (Bouez et al. 2006) Inhibition of breast cancer (Urashima et al. 2008) Inhibition of Choriocarcinoma (Ha¨ma¨la¨inen et al. 1995) In Esophageal cancer (He et al. 2002) Gastric cancer (Kaneda et al. 2004a, b) Lung cancer (Wu et al. 2007) Multiple endocrine neoplasia (MEN) (Watanabe et al. 2002) Ovarian cancer (Kaneda et al. 2004a, b) Prostate cancer (Ren et al. 1998) Rhabdomyosarcoma (Ha¨ma¨la¨inen et al. 1995)

Inhibition of ras (Csiszar et al. 1996) Inhibition of basal and squamous cell carcinoma (Bouez et al. 2006) Inhibition of bone cancer (Buchinger et al. 2008) Choriocarcinoma (Ha¨ma¨la¨inen et al. 1995) Inhibition of Colon Cancer (Csiszar et al. 2002) Fibrosarcoma (Ha¨ma¨la¨inen et al. 1995) Head and neck squamous cell carcinoma (HNSCC) (Rost et al. 2003) Lung cancer (Shames et al. 2006) Pancreatic cancer (Wu et al. 2007)

invasion and in in vivo tumorigenesis and metastasis mouse models. In malignant human breast carcinomas, LysOX was highly expressed in myofibroblasts and myo- epithelial cells surrounding the in situ tumor and in the reactive fibrosis facing the invasion front of infiltrating tumors (Peyrol et al. 1997). Upregulation of LysOXL2 mRNA and/or protein has been reported in breast, esoph- ageal, head and neck squamous cell, pancreatic, and prostatic carcinomas, melanoma, and E1A-immortalized kidney epithelial cell lines, compared with normal or poorly aggressive neoplastic counterparts. Chung and col- leagues demonstrated that increased LysOXL2 expression (along with the expression of 74 other genes) was signifi- cantly associated with high-risk head and neck squamous cell carcinoma and could be used as a predictive biomarker for high-risk patients (Chung et al. 2006). It has been demonstrated that stable expression of LysOXL2 in poorly invasive/non-metastatic MCF-7 breast cancer cells pro- duced estrogen-dependent tumors in orthotopically injected nude mice with many fibrotic foci and cells that were capable of invading the tumor pseudocapsule and into surrounding blood vessels, nerves, and muscle tissue (Akiri et al. 2003). Taken together, these reports demonstrate the potential of LysOXL2 to promote metastatic tumor pro- gression; however, more in-depth analyses are required to determine the mechanism in these cancers. In contrast, very few reports have demonstrated altered expression of Lys- OXL3 and LysOXL4 in cancers. At this time, very little is known about the biological function of these LysOX family members in normal cell processes (Fig. 2; Table 2).

Biosynthesis of LysOX

It has been demonstrated that the LysOX protein is known to be secreted into the extracellular space (Byers et al. 1980; Kuivaniemi et al. 1986a; Layman et al. 1972;


1980 ; Kuivaniemi et al. 1986a ; Layman et al. 1972 ; 123 Fig. 2 LysOX

Fig. 2 LysOX regulation

Peltonen et al. 1983; Royce et al. 1980). The transport through the Golgi and to the cell membrane probably takes place in vesicles formed by budding of, and fusion with, the sub-cellular membranes (Kosonen et al. 1997; Rucker

et al. 1998). Human LysOX is synthesized as a 48-kDa

prepro-enzyme with a 21-amino-acid signal sequence at the

N terminus (Trackman et al. 1992). Following the N-ter-

minal glycosylation, the signal peptide is cleaved off and

the copper co-factor incorporates into the protein, resulting

in an intermediary 50 kDa pro-enzyme in the Golgi appa-

ratus which is then secreted to the extracellular space (Trackman et al. 1992; Layman et al. 1972). There the protein can be activated by a proteolytic cleavage between residues G168 and D-169, which is catalyzed by procol- lagen C-proteinase/BMP-1 (bone morphogenetic protein-1) (Cronshaw et al. 1995). The proteinase activation yields a mature enzyme of 30 kDa (Panchenko et al. 1996; Kagan and Li 2003) and 18 kDa N-terminal propeptide fragment.

Lysyl oxidase: a potential target for cancer therapy

Table 2 LysOX as a metastasis promoter gene: in vitro and in vivo studies

In vivo

In vitro

Promotion of tumor progression in breast cancer (reviewed in Payne et al. 2007).

Promotion of tumor progression in brain cancer (Freije et al. 2004)

Promotion of tumor progression in head and neck squamous cell carcinoma (HNSCC) (Erler et al. 2006; Le et al. 2007)

Promotion of tumor progression in lung cancer (Borczuk et al. 2005)

Promotion of tumor progression in renal cell carcinoma (RCC) (Korenaga et al. 2005)

Promotion of tumor progression in bone cancer (Fuchs et al. 2000)

Promotion of tumor progression in brain cancer (Ross et al. 2000)

Promotion of tumor progression in breast cancer (reviewed in Payne et al. 2007)

Promotion of tumor progression in cervical cancer (Erler et al. 2006)

Promotion of tumor progression in prostate cancer (Stewart et al. 2009)

Proteolytic processing and activation of LysOX

Mammalian BMP-1 proteinase family members (BMP-1, mTLD, mTLL-1, mTLL-2) have LysOX pro-enzyme pro- cessing activity (Uzel et al. 2001). These proteins are encoded by two genes. By alternative splicing, BMP1 gene codes BMP-1 and mammalian tolloid (mTLD) while the tolloid-related proteinases, tolloid-like 1 (mTLL-1) and tolloid-like 2 (mTLL-2) are encoded by mTll-1 gene (Uzel et al. 2001; Kessler et al. 1996; Prockop et al. 1998; Takahara et al. 1994). BMP-1 is a multifunctional enzyme as it was also discovered to have procollagen C-proteinase activity, and moreover its amino acid sequence was iden- tical to the one of procollagen C-proteinase (Kessler et al. 1996). In mouse embryonic fibroblasts, all four BMP-1 proteinases show procollagen-C proteinase activity and cleave the 50-kDa LysOX precursor in vitro. Nevertheless, BMP-1, above all members of the family, showed the highest proteinase activity on LysOX (Uzel et al. 2001).

Transcriptional regulation of LysOX

LysOX mRNA expression and protein activity are highly responsive to a variety of cytokines, growth factors, and intercellular messengers (Csiszar 2001). LysOX mRNA was down-regulated by bFGF in human gingival fibroblasts (Hong and Trackman 2002), and LysOX mRNA in rabbit retinal pigment epithelial cells (Feres-Filho et al. 1996; Omori et al. 2002), and in mouse osteoblastic cells (Feres- Filho et al. 1996). It was also down-regulated by inter- feron- (IF-) in rat aortic smooth muscle cells (Tan et al. 1996; Song et al. 2000), and by prostaglandin E2 (PGE2) in human embryonic smooth muscle cells (Roy et al. 1996), and from neonatal rat lung fibroblasts (Boak et al. 1994; Choung et al. 1998). Transforming growth factor-b1 (TGF- b1) up-regulated both LysOX mRNA and protein levels in human embryonic lung fibroblasts (Roy et al. 1996; Choung et al. 1998), human gingival fibroblasts (Hong

et al. 1999), and myofibroblast-like cells in the flexor reticulum of carpal tunnel syndrome patients (Bose et al. 2000). In rodents TGF-b1 induced up-regulation of LysOX mRNA and protein levels was found in neonatal rat lung fibroblasts (Boak et al. 1994; Koslowski et al. 2003), the osteoblastic mouse cell line MC3T3-E1 (Shibanuma et al. 1993; Feres-Filho et al. 1995). TGF-b regulation of LysOX activity was also reported in rat aortic smooth muscle cells (Shanley et al. 1997; Gacheru et al. 1997). Platelet-derived growth factor (PDGF) also up-regulated LysOX mRNA levels in rabbit retinal pigment epithelial cells (Omori et al. 2002) and rat vascular smooth muscle cells (Smith-Mungo and Kagan 2002; Green et al. 1995). Application of insulin- like growth factor-I (IGF-I) in rat oral tissue increased LysOX protein level (Trackman et al. 1998). Amongst hormones, follicle-stimulating hormone (FSH) decreased LysOX mRNA expression and activity in rat granulose cells, but testosterone increased both LysOX mRNA level and activity (Bronson et al. 1987; Harlow et al. 2003; Slee et al. 2001).

Dietary copper and LysOX production

LysOX activity showed that it is influenced directly by the amount of dietary copper over a wide physiologic range of intakes in growing animals (Opsahl et al. 1982). It also reported that lysyl oxidase activity varies by as much as five- to sixfold in response to dietary copper, ranging from 0 added Cu to 25 mg Cu/g diet (Opsahl et al. 1982). This observation has intrigued us because there is no compelling reason why an excess of dietary copper per se should increase LysOX activity above what might be needed for normal cross-linking (Opsahl et al. 1982; Romero-Chap- man et al. 1990; Rucker et al. 1996). For example, for optimal growth in chickens the copper requirement is 5–10 mg Cu/g diet (Opsahl et al. 1982). Decreased colla- gen cross-linking does not occur until the copper intake is substantially \1 mg Cu/g diet. Copper deficiency also has


Siddikuzzaman et al.

little or no effect on LysOX protein or LysOX mRNA concentrations in tissues (Rucker et al. 1996). LysOX expression is regulated by hypoxia-inducible factors (HIFs), and, hence, LysOX expression is often up- regulated in hypoxic breast and head and neck tumors and also in cardiovascular diseases (Cristina et al. 2008) and regulation of LysOX by statins could contribute to vascular protection and to the cardiovascular risk reduction (Cristina et al. 2009). Patients with high LysOX-expressing tumors have poor overall survival. Furthermore, inhibition of LysOX has been demonstrated to eliminate metastases in mice. Targeting LysOXL2 with an inhibitory monoclonal antibody (AB0023) has been reported efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models (Barry-Hamilton et al. 2010) and also pro-BAPNs showed good levels of in vitro hypoxia-selective inhibition of LysOX activity (Carlotta et al. 2009). The LysOXL1 genetic predisposition is lim- ited to exfoliation glaucoma and does not include normal tension glaucoma (Wolf 2010). In conclusion, our present review reveals that Lysyl oxidase plays a significant role in cancer therapy. Several studies using plant products and natural products show that targeting LysOX could significantly reduce tumor pro- gression, metastasis, and angiogenesis. Some of the natural products that have shown significant results involve Bio- phytum sensitivum, amentoflavone, and b-carotene (Guruvayoorappan and Kuttan 2007, 2008a, b). The dis- covery of new drugs that can inhibit of the LysOX enzyme may be useful in preventing tumor progression and metastasis (Erler et al. 2009), and it can lead to open new approaches and directions for cancer therapy.

Acknowledgments The Valuable support of Dr. Patrick Gomez, Director, School of Biotechnology and Health Sciences, Karunya University is greatfully acknowledged.


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