MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines.

OBJECTIVE: We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC. METHODS: To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively. RESULTS: MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277). CONCLUSION: Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.

Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer.

OBJECTIVE: Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA. METHODS: The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA. RESULTS: Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001). CONCLUSION: Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.

Novel oncogenic function of mesoderm development candidate 1 and its regulation by MiR-574-3p in bladder cancer cell lines.

Our previous studies suggested that microRNA (miR)-574-3p is a candidate tumor suppressor microRNA (miRNA) in human bladder cancer (BC). Among 17 down-regulated miRNAs, miR-574-3p is located on chromosome 4p14 where we had identified a chromosomal loss region by array-CGH in BC cell lines. MiR-574-3p expression was down-regulated in BC cell lines. Gain-of-function analysis revealed that cell proliferation, migration and invasion were significantly inhibited in miR­574­3p-transfected BC cell lines. Flow cytometry analysis showed that cell apoptosis was induced in miR-574-3p transfectants. Oligo microarray analysis suggested that the mesoderm development candidate 1 (MESDC1) gene was a target gene in miR-574-3p transfectants. Luciferase assays revealed that miR­574­3p was directly bound to MESDC1 mRNA. MESDC1 is predicted to be a novel actin-binding protein located on chromosome 15q13. Although the gene is conserved among many species, its functional role is still unknown in both human malignancies and normal tissues. Loss-of-function studies demonstrated that cell proliferation, migration and invasion were significantly inhibited in si-MESDC1-transfected BC cell lines. Flow cytometry analysis showed that apoptosis was induced in si-MESDC1 transfectants. We are the first to demonstrate that miR-574-3p is a miRNA with tumor suppressor function and that MESDC1 (which has a potential oncogenic function in BC) may be targeted by miR-574-3p.

The functional significance of miR-1 and miR-133a in renal cell carcinoma.

PURPOSE: The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. METHODS: We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. RESULTS: The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. CONCLUSIONS: Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma.

The expression of microRNA-375 (miR-375) is significantly reduced in cancer tissues of maxillary sinus squamous cell carcinoma (MSSCC). The aim of this study was to investigate the functional significance of miR-375 and a possible regulatory role in the MSSCC networks. Restoration of miR-375 significantly inhibited cancer cell proliferation and invasion in IMC-3 cells, suggesting that miR-375 functions as a tumor suppressor in MSSCC. Genome-wide gene expression data and luciferase reporter assays indicated that lactate dehydro-genase B (LDHB) was directly regulated by miR-375. Cancer cell proliferation and invasion were significantly inhibited by transfection of si-LDHB into IMC-3 cells, suggesting that LDHB may play a role in MSSCC oncogenic function. In clinical MSSCC specimens, LDHB mRNA levels were up-regulated in cancer tissues, which were inversely correlated with the expression of miR-375. In addition, Kaplan-Meier curves and log-rank tests revealed that the high mRNA expression levels of LDHB had a significant adverse effect on survival rate. The identification of a cancer network regulated by the miR-375 tumor suppressor could provide new insights into the molecular mechanisms of MSSCC oncogenesis.

Tumor suppressive microRNA-375 regulates oncogene AEG-1/MTDH in head and neck squamous cell carcinoma (HNSCC).

Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma.

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Restoration of miR-517a expression induces cell apoptosis in bladder cancer cell lines.

The aim of this study was to find novel tumor suppressor microRNAs through screening genes epigenetically silenced by methylation in bladder cancer (BC) cell lines using microRNA microarrays. Since miR-517a and miR-520g, both located on chromosome 19q13.42, were found to highly up-regulated genes after treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dc), we hypothesized that they are tumor-suppressor microRNAs and performed a gain-of-function study using these mature microRNAs. The miR-517a restoration showed significant inhibition of cell proliferation in the transfectants compared to miR-control-transfected cells (p<0.0001 both in BOY and T24 cells). Furthermore, ectopic overexpression of miR-517a markedly induced apoptosis in the miR-517a-transfected BC cell lines. In addition, we carried out oligo microarray analysis using miR-517a transfectants and miR-control transfectants (BOY and T24), from which 35 down-regulated genes and 19 up-regulated genes were identified. These included amphiregulin (AREG) and BCL2-associated transcription factor 1, transcript variant 1 (BCLAF1), previously reported to be concerned with apoptosis, in both cell lines by miR-517a restoration. These data suggest that miR-517a functions as a tumor suppressor through inhibition of cell proliferation and induction of apoptosis under the regulation of AREG and/or BCLAF1 in BC cells. Anti-apoptotic effects may be maintained by down-regulation of miR-517a due to DNA hypermethylation in human BC cells, suggesting that restoration of miR-517a may be a novel therapeutic strategy for human BC.

miR-218 on the genomic loss region of chromosome 4p15.31 functions as a tumor suppressor in bladder cancer.

Growing evidence suggests that microRNAs (miRNAs) are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the mechanism of carcinogenesis. However, the genetic or epigenetic regulations of these miRNAs have not yet been fully elucidated in bladder cancer (BC). Chromosomal alterations of cancer cells give us important information for the identification of tumor suppressor genes. Our miRNA array-comparative genomic hybridization (CGH) analysis showed several miRNAs to be candidate tumor suppressors of BC. Our array-CGH analysis revealed that chromosome 4 was lost in all BC cell lines. We selected 19 miRNAs located on chromosome 4 and evaluated their expression levels in cancer cell lines as well as clinical samples. Gain-of-function analysis revealed that miR-218 inhibited BC cell proliferation, migration and invasion. Furthermore, flow cytometry analysis showed that it induced BC cell apoptosis. Genome-wide gene expression analysis showed that it targeted multiple oncogenes in BC. Our study is the first to demonstrate that miR-218 located on chrosomosme 4p15.31 is a tumor suppressive miRNA in BC. The identification of tumor suppressive miRNAs and their target genes on the basis of array-CGH analysis could provide new insights into the mechanisms of BC carcinogenesis.

miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma.

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.

SWAP70, actin-binding protein, function as an oncogene targeting tumor-suppressive miR-145 in prostate cancer.

BACKGROUND: MiR-145 is down-regulated in various human cancers. We previously demonstrated that some actin-binding proteins were targeted by several microRNAs (miRNAs), including miR-145, in bladder and prostate cancer (CaP). The aim of this study is to determine a novel oncogenic gene targeted by miR-145 by focusing on actin-binding proteins in CaP. METHODS: We focused on the SWAP switching B-cell complex 70 kDa subunit (SWAP70), which is an F-actin binding protein involved in activating B-cell transformation. A luciferase reporter assay was used to identify the actual binding sites between miR-145 and SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-SWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS: Molecular target searches of this miRNA and the luciferase reporter assay showed that SWAP70 was directly regulated by miR-145. Silencing of SWAP70 studies demonstrated significant inhibitions of cell migration and invasion in CaP cell lines. The SWAP70 positive-staining was significantly higher in percentage in the CaP than in benign prostate hyperplasia tissue. CONCLUSIONS: Down-regulation of miR-145 was a frequent event in CaP, and it may have a tumor suppressive function. SWAP70 may be a target of miR-145, and it might have a potential oncogenic function. The novel molecular networks though which miR-145 acts, may provide new insights into the underlying molecular mechanisms of CaP.

Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, primarily at the post-transcriptional level. Growing evidence suggests that miRNAs function as oncogenes or tumor suppressors in human cancers. The down-regulation of miR-145 has been reported in many types of human cancer, including prostate cancer (PC), suggesting that miR-145 functions as a tumor suppressor. Using the PC cell lines, PC3 and DU145, gain-of-function assays revealed that miR-145 transfection inhibited cell proliferation, migration and invasion. Fascin homolog 1 (FSCN1), an actin-bundling protein, is a candidate target gene of miR-145 based on genome-wide gene expression analysis. A luciferase reporter assay showed a significantly decreased signal at two miR-145 target sites at the 3'UTR of FSCN1, suggesting that miR-145 directly regulates FSCN1. In FSCN1 loss-of-function assays, cell growth, migration and invasion were all inhibited, implying that FSCN1 is associated with the progression of PC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the potential mechanisms of prostate carcinogenesis.

Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer.

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

MiR-96 and miR-183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology.

A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.

Glutathione S-transferase P1 (GSTP1) suppresses cell apoptosis and its regulation by miR-133α in head and neck squamous cell carcinoma (HNSCC).

The glutathione S-transferase P1 (GSTP1) protein plays several critical roles in both normal and neoplastic cells, including phase II xenobiotic metabolism, stress responses, signaling and apoptosis. Overexpression of GSTP1 has been observed in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the role of GSTP1 in HNSCC is not well understood. We investigated the role of GSTP1 in two HNSCC cell lines, HSC3 and SAS. Silencing of GSTP1 revealed that cancer cell proliferation was significantly decreased in both cell lines. In addition, the frequency of apoptotic cells increased following si-GSTP1 transfection of HSC3 and SAS cell lines. Growing evidence suggests that microRNAs (miRNAs) negatively regulate gene expression and can function as oncogenes or tumor suppressors in human cancer. Based on the results of web-based searches, miR-133α is a candidate miRNA targeting GSTP1. Down-regulation of miR-133α has been reported in many types of human cancer, including HNSCC. Transient transfection of miR-133α repressed the expression of GSTP1 at both the mRNA and protein levels. The signal from a luciferase reporter was significantly decreased at one miR-133α target site at the 3'UTR of GSTP1, suggesting that miR-133α directly regulates GSTP1. Our data indicate that GSTP1 may have an oncogenic function and may be regulated by miR-133α, a tumor suppressive miRNA in HNSCC. The identification of a novel oncogenic pathway could provide new insights into potential mechanisms of HNSCC carcinogenesis.

Caveolin-1 mediates tumor cell migration and invasion and its regulation by miR-133a in head and neck squamous cell carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that can function as oncogenes or tumor suppressors in human cancer. Down-regulation of the miRNA miR-133a in many type of cancers, and a reduction of cell proliferation, migration, and invasion upon over-expression, suggests that miR-133a is a tumor suppressor. In this study, genome-wide gene expression analysis of HNSCC cells that over-express miR-133a showed that caveolin-1 (CAV1), a multifunctional scaffolding protein, is down-regulated, a result that was confirmed by real-time PCR and Western blot analysis. A luciferase reporter assay revealed that miR-133a is directly bound to CAV1 mRNA. Cancer cell migration and invasion were significantly inhibited in HNSCC cells transfected with si-CAV1. Therefore, CAV1 functions as an oncogene in HNSCC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into potential mechanism of HNSCC carcinogenesis.

CpG hypermethylation of cellular retinol-binding protein 1 contributes to cell proliferation and migration in bladder cancer.

We have previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacological demethylating agent [5-aza-2'-deoxycytidine (5-aza-dC)] to find candidate genes that have tumor suppressive functions. We focused on the cellular retinol-binding protein 1 (CRBP1) gene that was selected by using the microarray data. As CRBP1 regulates intracellular retinoic acid (vitamin A) homeostasis, which is involved in morphogenesis, and cellular proliferation and differentiation, the loss of CRBP1 could cause tumorigenesis in BC. We hypothesized that the inactivation of the CRBP1 gene through CpG methylation contributes to cell viability, including the migration and invasion activity of human BC cells. After the 5-aza-dC treatment, the mRNA and protein expression levels of CRBP1 markedly increased in all BOY and T24 BC cell lines. Combined bisulfite-restriction analysis and bisulfite DNA sequencing revealed that promoter CpG hypermethylation existed in 28 out of the 65 BCs (43%) and in none of the 16 normal bladder epithelia (NBEs). Conversely, CRBP1 mRNA expression in the BCs was significantly lower than that in the NBEs (0.63 ± 0.11 vs. 4.92 ± 0.80, p<0.0001). We found significant inhibition of cell growth (p<0.0001) and migration (p<0.0001) in the CRBP1 stable transfectants compared to the control cell line, in a cell proliferation and wound-healing assay, respectively. In conclusion, the aberrant CpG hypermethylation of the CRBP1 gene promoter could be involved in the development of BC. We demonstrate here for the first time that the CRBP1 gene could have a tumor suppressive function in BC.

CpG hypermethylation of human four-and-a-half LIM domains 1 contributes to migration and invasion activity of human bladder cancer.

We previously reported a simple technique that combines microarray data from clinical bladder cancer (BC) specimens with those from a BC cell line (BOY) treated with a pharmacologic demethylating agent (5-aza-dC). We focused on the human four-and-a-half LIM domains 1 (FHL1) gene which was selected on the basis of previous microarray data analysis. Because LIM domains provide protein-protein binding interfaces, FHL genes play an important role in cellular events, such as focal adhesion and differentiation, by interacting with the target protein as either a repressor or activator. We hypothesized that inactivation of the FHL1 gene through CpG methylation contributes to cell viability including migration and invasion activity of human BC. After 5-aza-dC treatment, the expression levels of FHL1 mRNA transcript markedly increased in all cell lines tested, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR). The methylation index of FHL1 in our samples was significantly higher in 70 BC specimens than in 10 normal bladder epithelium (NBE) specimens (63.9+/-25.5 and 0.3+/-0.2, respectively; p=0.0066). Conversely, FHL1 mRNA expression was significantly lower in the BC specimens than in the NBE ones (0.331+/-0.12 and 2.498+/-0.61, respectively; p=0.0011). In addition, significant inhibitions of wound healing (45.78+/-6.2, and 100+/-0, respectively; p=0.009) and of cell invasion (18.5+/-2.3 and 95.2+/-2.4, respectively; p=0.02) were observed in stable FHL1-transfected cells than in the control BC cells. In conclusion, we found that the mechanism of FHL1 down-regulation in BC is through CpG hypermethylation of the promoter region. FHL1 gene inactivation by CpG hypermethylation may thus contribute to migration and invasion activity of BC.

Oncogenic functions of secreted Frizzled-related protein 2 in human renal cancer.

The secreted Frizzled-related proteins (sFRP) are modulators of the Wnt signaling pathway, which is involved in embryonic development and tumor progression. The functions of sFRP2 have not been studied in renal cancer. Transient transfection of sFRP2 promoted cell growth in renal carcinoma cells, whereby the largest effect was observed in A498 cells. To further study the functions of sFRP2 gene in renal carcinoma cells, we established A498 renal cancer cell lines, which stably expressed sFRP2. Stably expressed sFRP2 significantly promoted cell proliferation in vitro and in vivo tumor growth. The stably expressed sFRP2 cells were also found to have reduced UV-induced apoptosis and increased G(2) phase of the cell cycle. The phosphorylation level at Ser(33/37)/Thr(41) of beta-catenin was lower in the stable sFRP2 cell lines compared with the control cell line. sFRP2 significantly activated T-cell factor/lymphoid enhancer factor transcriptional activity. In the stable sFRP2 cell line, expression of c-Fos, Bcl2, Bcl-w, cyclin B2, and cyclin E2 genes was significantly increased and p53 expression was decreased. This is the first report documenting that sFRP2 activates the canonical Wnt pathway and promotes cell growth by evoking diverse signaling cascades in renal cancer cells. This study may provide better strategies for the management of renal cancer through regulation of sFRP2 pathways.

Functional significance of Wnt inhibitory factor-1 gene in kidney cancer.

Wnt inhibitory factor-1 (WIF-1) has been identified as one of the secreted antagonists that bind Wnt protein. WIF-1 has been described as a tumor suppressor in various types of cancer. However, the molecular function of WIF-1 gene has never been examined in human renal cell carcinoma (RCC). Therefore, we hypothesized that WIF-1 functions as a tumor suppressor gene and overexpression of this gene may induce apoptosis and inhibit tumor growth in RCC cells. Immunohistochemistry and real-time reverse transcription-PCR revealed that WIF-1 was significantly downregulated in RCC samples and RCC cell lines, respectively. Bisulfite sequencing of the WIF-1 promoter region in RCC cell lines showed it to be densely methylated, whereas there was no methylation of WIF-1 promoter in normal kidney. Significant inhibition of cell growth and colony formation in WIF-1-transfected cells compared with controls were observed. WIF-1 transfection significantly induced apoptosis and suppressed in vivo tumor growth. Also, Wnt signaling activity and beta-catenin expression were reduced by WIF-1 transfection. In conclusion, this is the first report documenting that the WIF-1 is downregulated by promoter methylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.