Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma.

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5'-aza-2' deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC-cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored.

D-glucuronyl C5-epimerase cell type specifically affects angiogenesis pathway in different prostate cancer cells.

D-glucuronyl C5-epimerase (GLCE) is involved in breast and lung carcinogenesis as a potential tumor suppressor gene, acting through inhibition of tumor angiogenesis and invasion/metastasis pathways. However, in prostate tumors, increased GLCE expression is associated with advanced disease, suggesting versatile effects of GLCE in different cancers. To investigate further the potential cancer-promoting effect of GLCE in prostate cancer, GLCE was ectopically re-expressed in morphologically different LNCaP and PC3 prostate cancer cells. Transcriptional profiles of normal PNT2 prostate cells, LNCaP, PC3 and DU145 prostate cancer cells, and GLCE-expressing LNCaP and PC3 cells were determined. Comparative analysis revealed the genes whose expression was changed in prostate cancer cells compared with normal PNT2 cells, and those differently expressed between the cancer cell lines (ACTA2, IL6, SERPINE1, TAGLN, SEMA3A, and CDH2). GLCE re-expression influenced mainly angiogenesis-involved genes (ANGPT1, SERPINE1, IGF1, PDGFB, TNF, IL8, TEK, IFNA1, and IFNB1) but in a cell type-specific manner (from basic deregulation of angiogenesis in LNCaP cells to significant activation in PC3 cells). Invasion/metastasis pathway was also affected (MMP1, MMP2, MMP9, S100A4, ITGA1, ITGB3, ERBB2, and FAS). The obtained results suggest activation of angiogenesis as a main molecular mechanism of pro-oncogenic effect of GLCE in prostate cancer. GLCE up-regulation plus expression pattern of a panel of six genes, discriminating morphologically different prostate cancer cell sub-types, is suggested as a potential marker of aggressive prostate cancer.

Novel tumor suppressor candidates on chromosome 3 revealed by NotI-microarrays in cervical cancer.

Genetic and epigenetic alterations in cervical carcinomas were investigated using NotI-microarrays containing 180 cloned sequences flanking all NotI-sites associated with genes on chromosome 3. In total, 48 paired normal/tumor DNA samples, specifically enriched in NotI-sites, were hybridized to NotI-microarrays. Thirty genes, including tumor suppressors or candidates (for example, VHL, RBSP3/CTDSPL, ITGA9, LRRC3B, ALDH1L1, EPHB1) and genes previously unknown as cancer-associated (ABHD5, C3orf77, PRL32, LOC285375, FGD5 and others), showed methylation/deletion in 21-44% of tumors. The genes were more frequently altered in squamous cell carcinomas (SCC) than in adenocarcinomas (ADC, p<0.01). A set of seven potential markers (LRRN1, PRICKLE2, VHL, BHLHE40, RBSP3, CGGBP1 and SOX14) is promising for discrimination of ADC and SCC. Alterations of more than 20 genes simultaneously were revealed in 23% of SCC. Bisulfite sequencing analysis confirmed methylation as a frequent event in SCC. High down-regulation frequency was shown for RBSP3, ITGA9, VILL, APRG1/C3orf35 and RASSF1 (isoform A) genes (3p21.3 locus) in SCC. Both frequency and extent of RASSF1A and RBSP3 mRNA level decrease were more pronounced in tumors with lymph node metastases compared with non-metastatic ones (p ≤ 0.05). We confirmed by bisulfite sequencing that RASSF1 promoter methylation was a rare event in SCC and, for the first time, demonstrated RASSF1A down-regulation at both the mRNA and protein levels without promoter methylation in tumors of this histological type. Thus, our data revealed novel tumor suppressor candidates located on chromosome 3 and a frequent loss of epigenetic stability of 3p21.3 locus in combination with down-regulation of genes in cervical cancer.

Heterogeneity of d-glucuronyl C5-epimerase expression and epigenetic regulation in prostate cancer.

Heparansulfate proteoglycans (HSPG) play an important role in cell-cell and cell-matrix interactions and signaling, and one of the key enzymes in heparansulfate biosynthesis is d-glucuronyl C5-epimerase (GLCE). A tumor suppressor function has been demonstrated for GLCE in breast and lung carcinogenesis; however, no data are available as to the expression and regulation of the gene in prostate cancer. In this study, decreased GLCE expression was observed in 10% of benign prostate hyperplasia (BPH) tissues and 53% of prostate tumors, and increased GLCE mRNA levels were detected in 49% of BPH tissues and 21% of tumors. Statistical analysis showed a positive correlation between increased GLCE expression and Gleason score, TNM staging, and prostate-specific antigen (PSA) level in the prostate tumors (Pearson correlation coefficients GLCE/Gleason = 0.56, P < 0.05; GLCE/TNM = 0.62, P < 0.05; and GLCE/PSA = 0.88, P < 0.01), suggesting GLCE as a candidate molecular marker for advanced prostate cancer. Immunohistochemical analysis revealed an intratumoral heterogeneity of GLCE protein levels both in BPH and prostate cancer cells, resulting in a mixed population of GLCE-expressing and nonexpressing epithelial cells in vivo. A model experiment on normal (PNT2) and prostate cancer (LNCaP, PC3, DU145) cell lines in vitro showed a 1.5- to 2.5-fold difference in GLCE expression levels between the cancer cell lines and an overall decrease in GLCE expression in cancer cells. Methyl-specific polymerase chain reaction (PCR), bisulfite sequencing, and deoxy-azacytidin (aza-dC) treatment identified differential GLCE promoter methylation (LNCaP 70-72%, PC3 32-35%, DU145, and PNT2 no methylation), which seems to contribute to heterogeneous GLCE expression in prostate tumors. The obtained results reveal the complex deregulation of GLCE expression in prostatic diseases compared with normal prostate tissue and suggest that GLCE may be used as a potential model to study the functional role of intratumor cell heterogeneity in prostate cancer progression.

Alterations of the WNT7A gene in clear cell renal cell carcinomas.

WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell renal cell carcinomas (RCCs) using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5'-CpG island of the WNT7A gene. Methylation analysis revealed positive correlations between tumor stage, Fuhrman nuclear grade and WNT7A hypermethylation. Additionally, restoration of WNT7A gene expression in the A498 cell line by 5-aza-2'-deoxycytidine treatment confirmed a direct contribution of hypermethylation in silencing of the WNT7A gene. High frequency of loss of heterozygosity (LOH) was demonstrated on chromosome 3p25 in regions surrounding the WNT7A gene. The frequent down-regulation of WNT7A gene expression was detected in 88% (15/17) of clear cell RCCs. We have also shown that the WNT7A gene possesses tumor suppression function by colony-formation and cell proliferation assays in RCC cell lines. In summary, the WNT7A gene is inactivated by genetic/epigenetic alterations in clear cell RCC and demonstrates tumor suppressor properties.

miRNA-218 contributes to the regulation of D-glucuronyl C5-epimerase expression in normal and tumor breast tissues.

microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218 expression was shown in primary breast tumors. Exogenous miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between miRNA-218 and GLCE mRNA, and negative correlation between miRNA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested.

The TCF4/ß-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer.

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/ß-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/ß-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/ß-catenin mRNA levels and activated TCF4/ß-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/ß-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and ß-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/ß-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/ß-catenin complex activity.

LTBP-2 confers pleiotropic suppression and promotes dormancy in a growth factor permissive microenvironment in nasopharyngeal carcinoma.

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays.

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non-small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA 9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80-100%.

LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication.

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.

Tumor suppressor dual-specificity phosphatase 6 (DUSP6) impairs cell invasion and epithelial-mesenchymal transition (EMT)-associated phenotype.

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.

Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors.

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5'-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.

Differential expression of CHL1 gene during development of major human cancers.

BACKGROUND: CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases)--in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. CONCLUSIONS/SIGNIFICANCE: Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer.

Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma.

The association of Matrix metalloproteinase-19 (MMP19) in the development of nasopharyngeal carcinoma (NPC) was identified from differential gene profiling, which showed MMP19 was one of the candidate genes down-regulated in the NPC cell lines. In this study, quantitative RT-PCR and Western blot analysis showed MMP19 was down-regulated in all seven NPC cell lines. By tissue microarray immunohistochemical staining, MMP19 appears down-regulated in 69.7% of primary NPC specimens. Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. We also clearly demonstrate that the catalytic activity of MMP19 plays an important role in antitumor and antiangiogenesis activities in comparative studies of the wild-type and the catalytically inactive mutant MMP19. In the in vivo tumorigenicity assay, only the wild-type (WT), but not mutant, MMP19 transfectants suppress tumor formation in nude mice. In the in vitro colony formation assay, WT MMP19 dramatically reduces colony-forming ability of NPC cell lines, when compared to the inactive mutant. In the tube formation assay of human umbilical vein endothelial cells and human microvascular endothelial cells (HMEC-1), secreted WT MMP19, but not mutant MMP19, induces reduction of tube-forming ability in endothelial cells with decreased vascular endothelial growth factor (VEGF) in conditioned media detected by enzyme-linked immunosorbent assay (ELISA). The anti-angiogenic activity of WT MMP19 is correlated with suppression of tumor formation. These results now clearly show that catalytic activity of MMP19 is essential for its tumor suppressive and anti-angiogenic functions in NPC.

Cloning and Initial Functional Characterization of Mlk4α and Mlk4ß.

We have cloned a novel human mixed-lineage kinase gene, MLK4. Two alternatively spliced forms, MLK4α (580 aa) and MLK4ß (1036 aa), have been identified and mapped to chromosomal band 1q42. MLK4 shows high amino acid homology to the kinase catalytic domain of MLK3 (72%), MLK1 (71%) and MLK2 (69%). Strong expression of MLK4 was detected in the human pancreas and kidneys. pCMV-MLK4ß c-myc-tagged protein (human) was expressed in the cytoplasm and nucleus of transiently transfected COS-1 cells, while pCMV-MLK4α c-myc-tagged protein (human) was expressed in cytoplasm only. Both MLK4 isoforms reduced the colony formation ability of MCF7 cells by 85%-95% and almost totally suppressed cell proliferation in the CyQUANT cell proliferation assay. Human pCMV-MLK4ß transgenic mice expressed the MLK4ß in all tissues examined but no phenotypic abnormalities were observed. Thus, in this work, we present the cloning and sequencing of MLK4α and MLK4ß for the first time; the data obtained suggest that MLK4 may function as a MAP kinase.

Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells.

BACKGROUND: D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in the biosynthesis of heparansulfate proteoglycans. Down-regulation of GLCE expression in human breast tumours suggests a possible involvement of the gene in carcinogenesis. In this study, an effect of GLCE ectopic expression on cell proliferation and viability of breast carcinoma cells MCF7 in vitro and its potential molecular mechanisms were investigated. RESULTS: D-glucuronyl C5-epimerase expression was significantly decreased in MCF7 cells compared to normal human breast tissue. Re-expression of GLCE inhibited proliferative activity of MCF7 cells according to CyQUANT NF Cell Proliferation Assay, while it did not affect their viability in Colony Formation Test. According to Cancer PathFinder RT Profiler PCR Array, antiproliferative effect of GLCE in vitro could be related to the enhanced expression of tumour suppressor genes р53 (+3.3 fold), E2F1 (+3.00 fold), BRCA1 (+3.5 fold), SYK (+8.1 fold) and apoptosis-related genes BCL2 (+4.2 fold) and NFKB1 (+2.6 fold). Also, GLCE re-expression in MCF7 cells considerably changed the expression of some genes involved in angiogenesis (IL8, +4.6 fold; IFNB1, +3.9 fold; TNF, +4.6 fold and TGFB1, -5.7 fold) and invasion/metastasis (SYK, +8.1 fold; NME1, +3.96 fold; S100A4, -4.6 fold). CONCLUSIONS: The ability of D-glucuronyl С5-epimerase to suppress proliferation of breast cancer cells MCF7 through the attenuated expression of different key genes involved in cell cycle regulation, angiogenesis and metastasis molecular pathways supports the idea on the involvement of the gene in regulation of breast cancer cell proliferation.

Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis.

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC.

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: clinical and prognostic significance.

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin alpha RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion/promoter methylation/expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion/methylation) of hMLH1, ITGA9, and RBSP3 were high (45-55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT-PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5-aza-2'-deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less-active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low/intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco-addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck.

Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21 and RASSF1A in primary non-small cell lung cancer.

BACKGROUND: The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely RBSP3 (AP20 region),NPRL2 and RASSF1A (LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10-6). METHODS: We estimated the quantity of RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 mRNA and RBSP3 DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification. RESULTS: We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (> or =2) was found for all three genes early in tumor development: in 85% of cases for RBSP3; 73% for NPRL2 and 67% for RASSF1A (P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, NPRL2 and RBSP3 was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of RASSF1A correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of RBSP3 in non-small cell lung cancers as promoter methylation of RBSP3 according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (NPRL2, RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05). CONCLUSION: Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of RBSP3, NPRL2 and RASSF1A was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).

RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic significance.

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4-9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN --> Stage I/II and for ITGA9 from CIN --> Stage I/II and also from Stage I/II --> Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (>or=5)/early sexual debut (