LOXL1 and LOXL4 Are Epigenetically Silenced and Can Inhibit Ras/Extracellular Signal-Regulated Kinase Signaling Pathway in Human Bladder Cancer

Published OnlineFirst April 24, 2007; DOI: 10.1158/0008-5472.CAN-07-0012

Research Article

Guojun Wu,1,3 Zhongmin Guo,1,2 Xiaofei Chang,1 Myoung Sook Kim,1 Jatin K. Nagpal,1 Junwei Liu,1 Joni M. Maki,4 Kari I. Kivirikko,4 Stephen P. Ethier,3 Barry Trink,1 and David Sidransky1

1Department of Otolaryngology–Head and Neck Surgery, The Johns Hopkins University School of Medicine, and 2Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland; 3Karmanos Cancer Institute, Department of Pathology, Wayne State University, Detroit, Michigan; and 4The Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry, University of Oulu, Oulu, Finland

Abstract important molecular mechanism contributing to TSG inactivation Promoter hypermethylation is one of the common mecha- along with other genetic alternations in human cancer (4–6). nisms leading to gene silencing in various human cancers. The significance in identifying novel gene promoter methylation Using a combination of pharmacologic unmasking and targets is 2-fold. First, methylated genes are potential candidate microarray techniques, we identified 59 candidate hyper- TSGs. Subsequent investigation of the function of these methylated methylated genes, including LOXL1, a lysyl oxidase-like gene, genes could increase our understanding of tumor initiation and in human bladder cancer cells. We further showed that LOXL1 progression. Second, the methylation genes could serve as and LOXL4 are commonly silenced genes in human bladder potential DNA markers for cancer diagnosis or even early detection cancer cells, and this silence is predominantly related to of cancer (6). Specifically for bladder cancer, the gene methylation promoter methylation. We also found LOXL1 and LOXL4 gene pattern, combined with the use of urine or serum samples, can be methylation and loss of expression in primary bladder tumors. an alternative method for cancer diagnosis and have profound In addition, somatic mutations were identified in LOXL4, but clinical applications (6–10). not in LOXL1 in bladder cancer. Moreover, reintroduction of There are numerous methods for large-scale screening for the LOXL1 and LOXL4 genes into human bladder cancer cells detection of methylated genes in the human genome. However, leads to a decrease of colony formation ability. Further studies most of them are either technically complex (restriction landmark indicated that the overexpression of LOXL1 and LOXL4 could genomic scanning, methylation-sensitive representational differ- antagonize Ras in activating the extracellular signal-regulated ence analysis; refs. 11, 12), or they can only screen a small number kinase (ERK) signaling pathway. Thus, our current study of genes (CGI array, arbitrarily primed PCR; refs. 13–16). Our suggests for the first time that lysyl oxidase-like genes can act laboratory has developed a simple technique that combines as tumor suppressor genes and exert their functions through microarray and pharmacologic unmasking (5-aza-2¶-deoxycytidine the inhibition of the Ras/ERK signaling pathway in human treatment of cancer cells). This technique can screen up to 32,000 bladder cancer. [Cancer Res 2007;67(9):4123–9] genes in one experiment and is now widely used in methylation screening for all different types of cancers (17, 18). Introduction In this study, we used this combination technique in bladder cancer for the first time and identified 59 candidate hyper- With more than 120,000 new cases a year in Europe and the methylated genes. Among these genes, LOXL1, a lysyl oxidase-like United States, bladder cancer remains the second most common gene, has been further studied. We showed that LOXL1 and LOXL4, genitourinary neoplasm and the fifth most common cancer in the but not other lysyl oxidase genes, are frequently silenced in human United States (1–3). Like other human cancers, loss of function of bladder cancer cell lines, and that this silence is predominantly tumor suppressor genes (TSG) and activation of oncogenes are two related to promoter methylation. We further confirmed promoter major alternations that promote bladder cancer initiation and methylation and loss of expression of LOXL1 and LOXL4 genes in progression. Gene mutations and loss of heterozygosity (LOH) are human bladder tumors. We also found that in bladder tumors, well-known genetic changes leading to the inactivation of TSGs. In LOXL4 but not LOXL1 harbored somatic mutations and poly- recent years, the epigenetic change of gene promoter hyper- morphisms, which are clustered in exon 8 that encodes for the methylation has been intensively studied and confirmed to be an scavenger receptor cysteine-rich (SRCR) domain. Moreover, reintroduction of LOXL1 and LOXL4 could antagonize the oncogene Ras in activating the extracellular signal-regulated kinase (ERK) Note: Supplementary data for this article are available at Cancer Research Online signaling pathway in bladder cancer cells and NIH3T3 cells (http://cancerres.aacrjournals.org/). LOXL1 G. Wu, Z. Guo, and X. Chang contributed equally to this work. containing an active Ras mutation. Finally, we found that Requests for reprints: David Sidransky and Barry Trink, Department of and LOXL4 genes predominantly localized in the cytoplasm, and Otolaryngology–Head and Neck Surgery, Head and Neck Cancer Research Division, overexpression of LOXL1 and LOXL4 genes leads to a dramatic 1550 Orleans Street 5N.03, Baltimore, MD 21231. Phone: 410-502-5153; Fax: 410-614- 1411; E-mail: [email protected] and [email protected] and Guojun Wu, Karmanos decrease of colony formation in human bladder cancer cells. All Cancer Institute, Department of Pathology, Wayne State University, HWCRC Room together, our data indicate that LOXL1 and LOXL4 are potential 831, 4100 John R Street, Detroit, MI 48201. E-mail: [email protected]. I2007 American Association for Cancer Research. TSGs, and that their expressions are predominantly silenced doi:10.1158/0008-5472.CAN-07-0012 through epigenetic mechanisms in human bladder cancer. www.aacrjournals.org 4123 Cancer Res 2007; 67: (9). May 1, 2007

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Materials and Methods in gene expression were analyzed using microarray chips contain- Tissue sample and cell lines. Seven different bladder cancer cell lines ing 12599 genes (Affymetrix U95Av2). Treatment with 5-Aza-dC and one immortalized bladder cell, HUC1, were purchased from American resulted in the up-regulation (defined as a 3.0-fold increase) of Type Culture Collection and cultured in recommended media. All bladder more than 500 unique genes in each of the three cell lines. Among tumor samples and matched adjacent normal tissues were from the Johns them, 132 genes were reactivated by demethylation treatment in at Hopkins Hospital. The tissues were collected immediately after surgery and least two cell lines. We further examined the expression status of kept in À80jC for long-term storage. The DNA was extracted using regular these 132 genes in the expression profiles from normal bladder phenol-chloroform and ethanol precipitation. The RNA was extracted using tissue and the immortalized uroepithelial cell line, HUC-1. Fifty- A TRIzol (Invitrogen), and 2 g RNA was used for reversal transcription nine genes which showed abundant expression in normal bladder reaction. tissue but were commonly silenced in at least two bladder cancer DNA bisulfite modification and bisulfite sequencing. DNA bisulfite cell lines were thus identified as specifically silenced genes in modification was done using a protocol described previously (17). Briefly, 2 Ag DNA was denatured in 0.2 mol/L of NaOH for 20 min at 50jC. The bladder cancer (Supplementary Data S1 and S2). LOXL1 LOX denatured DNA was then diluted in 500 AL of freshly prepared solution of Among these 59 genes, is of particular interest. 10 mmol/L hydroquinone and 3 mol/L of sodium bisulfite and incubated for was the first gene identified in this family and was confirmed to 3 h at 50jC. The DNA was then purified through a column and suspended be related to the Ras-induced cell reversion procession (20–22). in 100 AL TE buffer. Bisulfite-modified DNA was amplified using 10Â PCR Recently, several family genes (LOXL1, LOXL2, LOXL3, and buffer [166 mmol/L (NH4)2SO4, 670 mmol/L Tris (pH 8.8), 67 mmol/L LOXL4) were cloned and have been shown to possess various MgCL2, 0.7% 2 mercaptoethanol, 1% DMSO] and primer sets that were functions, including developmental regulation, tumor suppres- designed according to sequence change after bisulfite treatment. The sion, cell motility, and cellular senescence (23, 24). We thus sequences of the primers are shown as Supplementary Data S4. All PCR hypothesized that LOXL1 and possibly other LOX gene family products were sequenced with an internal primer using ABI big-dye cycle members might be epigenetically silenced and act as TSGs in sequencing kit (Applied Biosystems). 5-Aza-2¶-deoxycytidine treatment and reverse transcription-PCR. bladder cancer. LOXL1 LOXL4 Bladder cancer cells were treated with 5 Amol/L 5-aza-2¶-deoxycytidine and are the most frequently silenced genes in (5-Aza-dc) every 24 h for 3 days, TSA (300 nmol/L) was added for the final the lysyl oxidase gene family in bladder cancer cells. To 24 h. RNA was extracted using the TRIzol reagent. Reverse-transcription confirm our hypothesis, we first did reverse transcription-PCR to reaction was done using single-strand cDNA Synthesis Kit (Invitrogen). check the expression pattern of all family members in a panel of Sequences of PCR primers are provided as Supplementary Data S4. bladder cancer cells. As shown in Fig. 1A, the loss of expression was Exon-PCR and mutation analysis. Mutation screenings of 7 exons of seen in six out of eight bladder cell lines for LOXL1 (75%), five out LOXL1 LOXL4 and 15 exons of , including intron-exon boundaries, were of eight cell lines for LOXL4 (62.5%), and four out of eight cell lines done by PCR amplification and direct sequencing of both strands for DNA samples of all cells and tumors. The primers used for PCR and sequencing analysis are described as Supplementary Data S5. Plasmids construction and immunofluorescence. LOXL1 and LOXL4 full-length open reading frame sequence were amplified using cDNA with high-fidelity enzyme. The PCR products were purified with gel purification kit and directly cloned into pcDNA3.1D/V5-His-Topo vector using pcDNA3.1 Directional Topo Expression kit (Invitrogen). Positive clones were confirmed by direct sequencing and Western blotting with anti-V5 antibody (1:2,500; Invitrogen). Immunofluorescence assay was done using the protocol described previously (19). Anti-V5 antibody (1:5,000) was used as primary antibody, and Alex 548 anti-mouse antibody (1:800; Invitrogen) was used as secondary antibody. Approximately 1 Ag/mL Hoechst 33342 was used for nucleus staining. The images were taken using a Zeiss confocal microscope. Colony formation assay. Cells seeded in six-well plates were transfected with 1 Ag LOXL1, LOXL4, or pcDNA3.1D/V5-His-Topo vector. After 24 h, cells were reseeded in 10-cm dishes with a density of 2,000 cells per dish and 5,000 cells per dish in the presence of G418 (500 Ag/mL) for 2 to 3 weeks. Transfection and Western blotting. Cells seeded in 10-cm dishes were transfected with 5 Ag LOXL1, LOXL4, and pcDNA3.1D/V5-His-Topo vector. Cell lysates were collected after 48 h and blotting with either anti-V5 antibody to confirm the expression of LOXL1 or LOXL4 or anti–p-ERK antibody. For 5637 cells, transfection was done with the same amount of LOXL1, LOXL4, and HRAS G12V constructs. The enhanced chemilumines- cence kit (Amersham Biosciences) was used for the detection of signals. Statistical analysis. Statistical analysis was done using Student’s t test.

Results LOXL1 is one of the candidate methylated genes identified using pharmacologic unmasking and microarray techniques. Three bladder cancer cell lines, SCaBER, T24, and UM-UC-3, were Figure 1. Loss of expression of LOXL1 and LOXL4 in bladder cancer cells. A A, LOXL1 and LOXL4 genes are most frequently down-regulated LOX genes in treated with the demethylating agent 5 Aza-dC (5 mol/L) for bladder cancer cells. B, reactivation of LOXL1 and LOXL4 gene expression in 3 days to reactivate genes that are epigenetically silenced. Changes bladder cancer cells with 5-Aza-dC treatment.

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LOXL1, LOXL4: Potential TSG in Bladder Cancer

methylation pattern of these two genes perfectly matched their expression pattern in human bladder cancer cell lines (Fig. 2B). All together, our data clearly indicate that LOXL1 and LOXL4 genes frequently lose their expression in human bladder cancer cell lines, and that this silence is predominantly related to their promoter hypermethylation status. LOXL1 and LOXL4 are frequently hypermethylated and lose expression in primary bladder tumors. We next investigated LOXL1 and LOXL4 gene methylation in 64 primary bladder tumor samples and 12 normal tissues using bisulfite sequencing. LOXL1 methylation was found in 42 out of 60 primary tumors (70%) and 4 out of 12 (33.3%) normal appearing controls (Student’s t test, P = 0.037; Fig. 3A). LOXL4 was methylated in 26 out of 63 primary tumors (40%), and no methylation was found in normal appearing controls (Fig. 3B). All together, 58 out of 63 (92%) tumors displayed either LOXL1 or LOXL4 methylation in their promoter regions, and 10 out of 58 cases (17%) showed both LOXL1 and LOXL4 methylation (Fig. 3C). We next checked the expression ratio in 14 cases of primary bladder tumor samples and 6 cases of normal appearing tissues. LOXL1 expression was lost or dramatically decreased in 7 out of 14 primary tumor samples (50%). Likewise, LOXL4 was found to have low or no expression in 5 out of 14 cases (35.7%). Lost expression of both of LOXL1 and LOXL4 was found in four cases. In contrast, Figure 2. LOXL1 and LOXL4 are both methylated in bladder cancer cell lines. LOXL1 expression was retained in all six normal appearing tissues, A, representative figures showed position of CpG islands and methylation of and only one case showed lost expression of LOXL4 (Fig. 4A). LOXL1 and LOXL4 genes. Arrows,the methylated CpGs. B, expression and methylation of LOX genes in bladder cancer cell lines. We next checked the expression and methylation pattern of LOXL1 and LOXL4 genes in four paired normal appearing tissues and tumor samples. We found that LOXL1 lost expression in two for LOX (50%). LOXL2 and LOXL3, however, retain their expression tumor samples compared with their normal appearing tissue, in the majority of the cell lines. In contrast, all five genes retained whereas LOXL4 lost expression in one case of tumor, with another expression in normal bladder tissue, and three genes (LOXL1, case having a dramatic decrease in expression (Fig. 4B). We further LOXL2, and LOXL3) also retained expression in HUC1 cells. We compared the methylation profile with the expression pattern. We therefore focused on the LOXL1 and LOXL4 genes because they found that the two paired samples without expression change are were the most frequently silenced in bladder cancer cell lines. both unmethylated, whereas one case (1386) is methylated in We next treated these cells with the demethylating agent 5-Aza- tumor (loss of expression), but not in the normal appearing tissue dC. Both LOXL1 and LOXL4 genes were robustly reactivated in all (with expression). These data further support the notion that the cell lines, which previously showed none or low expression of methylation is the main mechanism leading to the loss of these genes (Fig. 1B). We did not observe additional reactivation expression of LOXL1 and LOXL4 in bladder cancer. effect with TSA (300 nmol/L) on these cell lines (data not shown). Somatic mutations were identified in LOXL4 but not in In addition, we did direct bisulfite DNA sequencing to investigate LOXL1 in bladder cancer. To further evaluate whether there are the promoter hypermethylation status of the LOXL1 and LOXL4 other genetic alterations in LOXL1 and LOXL4 genes in human genes in human bladder cancer cells (Fig. 2A). We found that the bladder cancer, we sequenced each exon of both genes in 8 bladder

Figure 3. Methylation of LOXL1 and LOXL4 in primary bladder tumors and normal appearing samples. A, LOXL1 is methylated in 70% primary bladder tumors and 33.3% normal appearing samples. B, LOXL4 is methylated in 40% primary bladder tumor samples but not in normal appearing samples. C, 92% of primary bladder tumors have either LOXL1 or LOXL4 gene methylation,and 17% tumor have both methylation of LOXL1 and LOXL4.

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mutations, just two base pairs 5¶ of the polymorphism, were identified in bladder tumors. T1212C leads to a nonsense mutation (Asn404Asn), whereas T1212G leads to missense mutation (Asn404Lys; Table 1; Supplementary Data S3). Notably, all the polymorphisms and somatic mutations were located in exon 8 encoding for the SRCR domain. LOXL1 and LOXL4 antagonize Ras activation of the ERK signaling pathway in bladder cancer cells. Although the reverse correlation of the LOX gene and ras oncogene was observed 20 years ago, no further studies were done to investigate the mechanisms behind this phenomenon. Because we have already found that LOXL1 and LOXL4 genes are frequently silenced and methylated in bladder cancer, it was reasonable to hypothesize that the overexpression of LOXL1 and LOXL4 can block Ras-induced cell signaling in bladder cancer. We initially used the mouse fibroblast NIH3T3 cells and ras-transformed NIH3T3 cells as a model to test our hypothesis. As shown in Fig. 5A, ras-transformed NIH3T3 cells have much stronger p-ERK expression than wild-type NIH3T3 cells. We observed significant inhibition of p-ERK activation when we transfected either LOXL1 or LOXL4, but not their vector into ras-transformed NIH3T3 cells (Fig. 5A). We then checked the status of Ras and p-ERK in a panel of bladder cancer cell lines (Fig. 5B). T24 and UM-UC-3 are two cell lines harboring both a Ras mutation and an enhanced ERK Figure 4. Correlation of LOXL1 and LOXL4 expression and methylation in activation. 5637 and SW780 are two cell lines with wild-type Ras bladder tumor and normal appearing samples. A, LOXL1 and LOXL4 gene LOXL1 expression in 14 tumor samples (top) and 6 normal appearing samples (bottom). with a dramatically low ERK activation. To check whether Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading and LOXL4 genes can antagonize the Ras-activating ERK signaling control. T, tumor; N, normal appearing sample. B, LOXL1 and LOXL4 gene pathway in bladder cells, we cotransfected the Ras gene containing expression and methylation pattern in four paired normal appearing and LOXL1 LOXL4 tumor samples. GAPDH was used as a loading control. U, unmethylated; the G12V mutation and or gene into 5637 cells M, methylated. N/A, result is not available. without detectable ERK activation. Mutant Ras alone can significantly activate ERK signaling, and this activation can be cancer cells, 30 cases of normal appearing tissue, and 30 cases of inhibited by both LOXL1 and LOXL4 reintroduction, with LOXL4 tumors. No somatic mutations were identified in all seven exons being less effective than LOXL1 (Fig. 5C). of LOXL1. However, for LOXL4, we found an A1214C (exon 8) Finally, we transfected LOXL1 and LOXL4 into J82 and UM-UC-3 heterogeneous polymorphism in 2 of the 8 cell lines and 10 of cell lines. As shown in Fig. 5D, these two cell lines both have 30 normal and tumor DNA samples. In addition, homogeneous enhanced ERK activation but with a different Ras status. We found polymorphisms in the same position were found in three other cell that LOXL1 can markedly inhibit ERK activation in both cell lines, lines and in five tumors, but not in the normal appearing samples. whereas LOXL4 can only marginally inhibit ERK activation in these Among these five, two cases have paired normal appearing tissues cells. Thus, consistent with the results from 5637 cells, LOXL1 and (Supplementary Data S3). Thus, these homogeneous polymor- LOXL4 have different capabilities in inhibiting the ERK signaling phisms are possibly cancer related. In addition, two somatic pathway.

Table 1. Somatic mutations and polymorphisms of LOXL4 gene in bladder cancer

Exon NT AA Functional domain Normal Tumor Cell lines Total

Heterogeneous polymorphisms Exon 8 A1214C Asp405Ala SRCR* 10 10 2 22 Homogeneous polymorphisms c c Exon 8 A1214C Asp405Ala SRCR 0 5 38 Somatic mutations Exon 8 T1212G Asn404Lys SRCR 0 1 0 1 Exon 8 T1212C Asn404Asn SRCR 0 1 0 1 Number with mutation 0 2 0 Number of samples screened 30 30 8 Percent of cases with mutation (%) 0 6.7 0

*Scavenger receptor cysteine-rich (SRCR) domains. cThese NT changes have also been detected in two paired samples.

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LOXL1, LOXL4: Potential TSG in Bladder Cancer

known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin (23, 26). We have shown here for the first time that two novel members of this amine oxidase family, LOXL1 and LOXL4, are frequently silenced, and this silence is predominantly related to promoter hypermethylation in human bladder cancer. Beyond that, LOXL1 and LOXL4 can inhibit anchoring-dependent colony formation and can antagonize the Ras effect in activating the ERK signaling pathway in bladder cancer cells. Thus, our study indicates that LOXL1 and LOXL4 can serve as TSGs in human bladder cancer. Recently, LOX was reported to be methylated in human gastric cancer (26% by MSP; ref. 27). In contrast to that report, we showed that LOX was not the most frequent silenced LOX gene family member in bladder cancer. Although the same promoter region showed hypermethylation in human bladder cancer cells, we did not observe hypermethylation of LOX in primary bladder tumors (data not shown). Thus, LOX gene methylation might be restricted to some specific tissues. It is of interest that we identified a homogeneous polymorphism (A1214C) of the LOXL4 gene in five tumor cases but not in normal tissue. Further study is needed to clarify whether this kind of polymorphism is related to any pathologic characteristic of bladder cancer. It is necessary to point out that all the polymorphisms and somatic mutations were identified in exon 8 of the LOXL4 gene, which encodes for the SRCR domain. SRCR domains are Figure 5. Antagonizing effect of LOXL1 and LOXL4 on Ras/ERK signaling conservative disulfide-rich extracellular domains shared by LOXL2, pathway in bladder cancer cells. A, top, increased ERK activity in LOXL3, and LOXL4, but not by LOX and LOXL1. They are also Ras-transformed NIH3T3 cells. Bottom, LOXL1 and LOXL4 expression inhibit found in several extracellular receptors and may be involved in the ERK activity in NIH3T3/A6 cell,which is Ras transformed. B, the Ras mutation pattern and p-ERK status in a panel of bladder cancer cells. +, protein-protein interactions. Interestingly, Xu et al. (28) reported mutation; À,wild-type. C, mutant Ras can significantly activate ERK signaling, and this activation is inhibited by both LOXL1 and LOXL4 reintroduction with LOXL4,showing much lower ability than LOXL1 in 5637 cells. D, LOXL1 and LOXL4 gene can differentially inhibit endogenous ERK signaling pathway in J82 and SCaBER cells.

LOXL1 and LOXL4 are predominantly located in the cytoplasm and can inhibit colony formation in bladder cancer cells. Lysyl oxidase has been shown to have both intracellular and intranuclear locations in liver and kidney cells (23, 25). Until now, the cellular locations, as well as the functions of the new lysyl oxidase-like proteins in cancer cells, have not been studied. To clarify whether LOXL1 and LOXL4 gene cellular locations are consistent with their function to inhibit the Ras/ERK signaling pathway, we transfected the LOXL1 and LOXL4 genes into the bladder cancer cell lines J82 and 5637. The immunofluorescence assay shows strong and predominant fluorescent signals in the cytoplasm, but not in the nucleus for both genes in the two different cell lines (Fig. 6A). We next investigated the effect of reintroducing the LOXL1 and LOXL4 genes into the bladder cancer cell line UM-UC-3, which contains a Ras mutation and, accordingly, an enhanced ERK activation (Fig. 6B). We found that both LOXL1 and LOXL4 expression can lead to a 40% to 50% decrease in anchorage- dependent colony formation in UM-UC-3 cells. Similar results were also obtained when we used the J82 cell line, which contains wild- type Ras but enhanced ERK activation (data not shown). Taken together, these data indicate that LOXL1 and LOXL4 could serve as TSGs in bladder cancer.

Figure 6. Tumor suppressor functions of LOXL1 and LOXL4 genes. Discussion A, subcellular localization of LOXL1 and LOXL4 genes in bladder cancer cells. Lysyl oxidase (LOX) is a copper-containing amine oxidase that Immunofluorescence was done in J82 cells. For each figure, left, nucleus staining; middle, gene localization; right, merged signals. Right side of the belongs to a heterogeneous family of enzymes that oxidize primary figures, gene names. B, LOXL1 and LOXL4 expression can lead to 40% to 50% amine substrates to reactive aldehydes. LOX has been traditionally decrease in anchorage-dependent colony formation in UM-UC-3 cells. www.aacrjournals.org 4127 Cancer Res 2007; 67: (9). May 1, 2007

Cancer Research that two out of seven missense mutations located in the SRCR upstream signals. TP53 is a well-known TSG, whereas the family domain of MSR1 (macrophage scavenge receptor 1) gene in member DeltaNp63a is often overexpressed and oncogenic in many families affected with hereditary prostate cancer, suggesting that tumor types (4, 36–40). germ line mutations and sequence variants of the MCR1 gene were LOX was initially known as rrg (ras recision gene; ref. 20). In many associated with prostate cancer risk. Taken together, the hotspot of naturally occurring and oncogene-induced (ras) tumors, LOX is nucleotide change clustered in the SRCR domain might influence down-regulated (21), whereas LOX is one of the main genes induced the primary function of LOXL4 and MSR1 genes and lead to in concomitance with the reversion process (22, 41). H-ras– alterations of downstream signaling pathways in bladder and transfected NIH3T3, induced to revert by IFNh/g, would return to prostate cancer. their transformed phenotype upon transfection with an antisense There is some controversy in the literature regarding the LOX. Until now, it has not been shown that the newly identified lysyl function of the different members of the lysyl oxidase family in oxidase gene family members (LOXL1 and LOXL4) can directly human cancers. It was reported that LOX, LOXL1, and LOXL2 were inhibit the Ras/ERK signaling pathway. Giving the primary overexpressed in metastatic breast cancer. LOX-expressing MCF7 importance of the ras gene in carcinogenesis, the Ras/ERK signaling cells displayed increased invasiveness in an in vitro invasiveness pathway has attracted considerable attention as a target for assay (29). LOXL2 overexpression was also reported to promote anticancer therapy. Several major approaches have been undertaken tumor fibrosis and tumor progression in vivo (30). More recently, thus far: (a) the inhibition of Ras protein expression through LOX was reported to be essential for hypoxia-induced metastasis ribozymes, antisense, or micRNA; (b) the prevention of membrane (31). In contrast to the above, LOX levels are reduced in several localization of Ras; and (c) the inhibition of downstream effectors of cancer cell lines as well as in ras-transformed cell lines (21, 32, 33). Ras function such as the use of Raf kinase inhibitors and mitogen- The LOX gene was reported to be methylated in human gastric activated protein/ERK kinase inhibitors (37, 42–44). Our current cancer (27). Our current study indicates that at least two members study provides new evidence that LOXL1 and LOXL4 can antagonize of the lysyl oxidase family, LOXL1 and LOXL4, can serve as TSGs in Ras function and inhibit the ERK signaling pathway in bladder bladder cancer. We believe that this kind of functional variation in cancer. LOXL1 and LOXL4 status may thus be important in LOX family members is structure dependent and tissue specific. determining sensitivity to the Ras/ERK signaling pathway inhibitors On the one hand, all LOX and LOX-like proteins (LOXL1, LOXL2, in bladder cancer. LOXL3, and LOXL4) have highly conserved copper-binding and lysyl-trosylquinone cofactor sites (23, 24). There are also some Acknowledgments conserved domains such as multiple SRCR domains shared only Received 1/4/2007; revised 2/19/2007; accepted 2/21/2007. in LOXL2, LOXL3, and LOXL4 (34, 35). Thus, these structural Grant support: National Cancer Institute Lung Cancer Specialized Program of similarities and differences might account for their varied Research Excellence grant (CA 58184-01 to D. Sidransky) and National Institute of functions in cells. On the other hand, it is not unusual that Dental and Craniofacial Research grant (RO1-DE 012588-0 to D. Sidransky). The costs of publication of this article were defrayed in part by the payment of page different gene family members play different active roles in charges. This article must therefore be hereby marked advertisement in accordance different tissues due to the specific environment and different with 18 U.S.C. Section 1734 solely to indicate this fact.

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www.aacrjournals.org 4129 Cancer Res 2007; 67: (9). May 1, 2007

LOXL1 and LOXL4 Are Epigenetically Silenced and Can Inhibit Ras/Extracellular Signal-Regulated Kinase Signaling Pathway in Human Bladder Cancer

Guojun Wu, Zhongmin Guo, Xiaofei Chang, et al.

Cancer Res 2007;67:4123-4129. Published OnlineFirst April 24, 2007.

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