Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma.

Fibulin-2 (FBLN2) has been identified as a candidate tumor-suppressor gene in nasopharyngeal carcinoma (NPC). Originally identified through a chromosome 3 NotI genomic microarray screen, it shows frequent deletion or methylation in NPC. FBLN2 is located on chromosome 3p25.1 and is associated with tumor development through its important interactions with the extracellular matrix (ECM) proteins. FBLN2 encodes two isoforms. The short isoform (FBLN2S) is expressed abundantly in normal tissues, but is dramatically downregulated in NPC, while the long isoform (FBLN2L) is either not detectable or is expressed only at low levels in both normal and tumor tissues. Reintroduction of this FBLN2S inhibited cell proliferation, migration, invasion and angiogenesis in vitro. Furthermore, in vivo studies in nude mice show its expression is associated with tumor and angiogenesis suppression. FBLN2-associated angiogenesis occurs via concomitant downregulation of vascular endothelial growth factor and matrix metalloproteinase 2. This study provides compelling evidence that FBLN2S has an important tumor-suppressive and anti-angiogenic role in NPC.

Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties.

Alpha B-crystallin (CRYAB) maps within the nasopharyngeal carcinoma (NPC) tumor-suppressive critical region 11q22-23 and its downregulation is significantly associated with the progression of NPC. However, little is known about the functional impact of CRYAB on NPC progression. In this study we evaluated the NPC tumor-suppressive and progression-associated functions of CRYAB. Activation of CRYAB suppressed NPC tumor formation in nude mice. Overexpression of CRYAB affected NPC progression-associated phenotypes such as loss of cell adhesion, invasion, interaction with the tumor microenvironment, invasive protrusion formation in three dimensional Matrigel culture, as well as expression of epithelial-mesenchymal transition-associated markers. CRYAB mediates this ability to suppress cancer progression by inhibition of E-cadherin cytoplasmic internalization and maintenance of ß-catenin in the membrane that subsequently reduces the levels of expression of critical downstream targets such as cyclin-D1 and c-myc. Both ectopically expressed and recombinant CRYAB proteins were associated with endogenous E-cadherin and ß-catenin, and, thus, the cadherin/catenin adherens junction. The CRYAB α-crystallin core domain is responsible for the interaction of CRYAB with both E-cadherin and ß-catenin. Taken together, these results indicate that CRYAB functions to suppress NPC progression by associating with the cadherin/catenin adherens junction and modulating the ß-catenin function.

Methylation pattern of the putative tumor-suppressor gene LRRC3B promoter in clear cell renal cell carcinomas.

The leucine rich repeat containing 3B (LRRC3B) gene is a putative tumor suppressor located on human chromosome 3 in the 3p24 region. LRRC3B is frequently altered in colon and gastric cancers and also in leukaemias. In this study we investigated the promoter region methylation as a possible mechanism of LRRC3B gene inactivation in clear cell renal cell carcinomas. We found that the LRRC3B gene promoter was methylated in 43% of clear cell renal carcinoma samples. However, no correlation between DNA methylation and LRRC3B expression was found.

D-glucuronyl C5-epimerase suppresses small-cell lung cancer cell proliferation in vitro and tumour growth in vivo.

BACKGROUND: D-Glucuronyl C5-epimerase (GLCE) is a key enzyme involved in the biosynthesis of heparan sulphate proteoglycans, which has an important role in cell-cell and cell-matrix interactions and signalling. Decreased GLCE expression in human breast tumours and its anti-proliferative effects in breast cancer cells suggest that it may be a candidate tumour-suppressor gene. The aim of this study was to investigate the involvement of GLCE in lung carcinogenesis. METHODS: D-Glucuronyl C5-epimerase expression in different lung cancer cell lines was determined and the gene was ectopically re-expressed in U2020 small-cell lung cancer cells. Cellular proliferation in vitro and tumour growth in vivo were then examined. RESULTS: Ectopic re-expression of GLCE in U2020 cells did not affect cell viability but did influence morphology. Cellular proliferation in vitro and tumour formation in vivo were both suppressed. These effects were mediated via downregulation of several pro-angiogenic growth factors and their receptors, including VEGF-A, TGFB1, FGFR2, PDGF-A and PDGF-B, and TNFa and its receptors. Expression of matrix metalloproteinase2, MTA1, PLAU, TIMP3, S100A4, SERPINE1 and TWIST1 was also downregulated. CONCLUSION: The anti-tumour effects associated with ectopic GLCE re-expression suggest that it may be a potential tumour-suppressor gene and a possible target for lung cancer diagnosis and treatment.

A novel MECA3 region in human 3p21.3 harboring putative tumor suppressor genes and oncogenes.

BACKGROUND: Human chromosome arm 3p is often affected in various epithelial tumors, and several tumor suppressor genes were recently identified in this region. The most affected is 3p21 region that is 50-100% rearranged in more than 30 types of malignancies, mostly in epithelial cancers: lung, breast, ovarian, cervical, kidney, head and neck, nasopharyngeal, colon etc. These cancers are responsible for 90% of cancer deaths. AIM: To perform the detailed analysis of 3p (especially 3p21 region) to discover novel potential oncogenes and/or tumor suppressors. METHODS: To find novel "hot spots" and genes involved in major cancers, dense 3p microsatellite markers (altogether 24 ) were allelotyped in four epithelial carcinomas (272 patients in total): breast (BC), renal cell (RCC), non-small cell lung (NSCLC) and epithelial ovarian (EOC) cancers. RESULTS: As a main result, a novel region, frequently affected in BC, RCC, NSCLC and EOC was localized between markers D3S2409 and D3S3667 in the 3p21.3. This region (MECA3, major epithelial cancers affected region No. 3) covers numerous UniGene clusters, including genes involved in vital cell functions and carcinogenesis (e.g. MST1, MSTR1/RON, GPX1 and RHOA). The homozygous deletions were detected in the GPX1 in RCC (12%, 6 of 50 cases) and BC (1 of 37 cases). At the same time, amplifications and multiplications within the RHOA putative oncogene were identified in BC and RCC. CONCLUSIONS: The data suggest that genes with potential oncogenic features are located in the close proximity to putative tumor suppressor gene(s) (TSG(s)) in the MECA3. Multiplication of the RHOA was not reported before. Significant correlation of allelic alterations in the, AP20, MECA3 and LUCA regions with tumor progression was found for some common histological tumor subtypes (e.g. clear cell RCC, and serous EOC).

Genetic and epigenetic changes of NKIRAS1 gene in human renal cell carcinomas.

UNLABELLED: Renal cell carcinoma (RCC) is the most common malignant tumor of kidney associated with the worst clinical outcome. No molecular markers for RCC diagnostics and prognosis that could be applied in clinics were described yet. Large-scale screening of 3p human chromosome genes/loci in RCC and histologically normal tissues surrounding the tumors using NotI-microarray approach demonstrated that NKIRAS1 gene contained the largest percent of genetic/epigenetic changes in RCC tumor cells. AIM: To validate the results of NotI microarray analysis and study genetic, epigenetic changes, and the expression level of NKIRAS1 gene in human RCC samples. METHODS: DNA and RNA were isolated from freshly-frozen renal tumors' samples (n = 12) and from normal tissues surrounding the tumors. Epigenetic changes (methylation status) of NKIRAS1 were detected by bisulfite sequencing. Genetic changes and expression level were analyzed by Quantitative real-time PCR (qPCR) with SYBR Green. For relative quantification 2-(DeltaDeltaCP) method was used. Nonparametric tests (Wilcoxon, Kruskal - Wallis and Mann - Whitney) were applied for statistical data analysis using the BioStat software. RESULTS: NKIRAS1 expression was downregulated in 75% of RCC samples (9 of 12) compared with surrounding normal tissue. High grade tumors (3 and 4) showed lower expression of NKIRAS1 at the mRNA level than tumors of low grade (1 and 2). No significant association was found between gene expression level and gender or age. Analysis of NKIRAS1 gene copy number was performed in 19 tumor samples. Changes in the copy number of NKIRAS1 gene were observed in 64% (9 of 14) of cRCC samples. 9 samples displayed ratio (< 0.85 and >or= 0.35), thus were considered as hemizygous deletions. 3 samples showed ratio (> 0.85) and were considered as normal copy number. Changes in NKIRAS1 gene copy number were detected in all 3 benign oncocytomas, 1 papillary cancer and 1 sarcoma, where hemizygous deletion was observed. No changes in methylation status of NKIRAS1 were found in RCC. CONCLUSIONS: We have validated the results of NotI microarray analysis of NKIRAS1 gene in RCC. It was shown the decreased expression level of NKIRAS1 in this type of tumor.

The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-kappaB signaling pathways.

The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1-6. Recently, four novel family members, RASSF7-10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1-6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell-cell adhesion, co-localizing with the adherens junction (AJ) component beta-catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and E-cadherin is lost from the cell membrane. The AJ components beta-catenin and p65 are also lost from sites of cell-cell contact and are relocalized to the nucleus with a concomitant increase in beta-catenin-dependent and nuclear factor-kappaB (NF-kappaB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.

Screening of epigenetic and genetic disturbances of human chromosome 3 genes in colorectal cancer.

DNA microarray technology comprising NotI-linking clones was used in a large-scale study of genetic and epigenetic changes in colorectal cancer. Analysis of samples from 24 patients revealed methylation, deletions, and amplifications in 137 of 181 NotI clones. For 27 genes/loci, these changes occurred in more than 30% of the tumor samples, suggesting that these genes are involved in the development of colorectal cancer. An analysis of the methylation status of CpG island of the ITGA9 gene/loci by bisulfite sequencing confirmed the NotI microarray data on the gene/loci methylation in colorectal cancer. Aberrations in 19 genes/loci were unknown previously. Their characterization may help ascertain the mechanisms responsible for colorectal cancer development and identify novel diagnostic and prognostic markers.

Epigenetic regulation of the ras effector/tumour suppressor RASSF2 in breast and lung cancer.

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.

Hypermethylation of Ron proximal promoter associates with lack of full-length Ron and transcription of oncogenic short-Ron from an internal promoter.

The gene for tyrosine-kinase receptor Ron (MST1R) resides in the chromosome 3p21.3 region, frequently affected in common human malignancies. The gene generates two transcripts, 5 and 2 kb-long, full-length Ron (flRon) and short-form Ron (sfRon), respectively. Here, we show for the first time that the variegated Ron expression is associated with variations in the methylation patterns of two distinct CpG islands in Ron proximal promoter. Widespread hypermethylation associates with lack of flRon whereas hypermethylation of the distal island associates with transcription of sfRon, a constitutively active tyrosine-kinase that drives cell proliferation. sfRon inhibition with kinase-dead transgenes decreases cancer cell growth and induces cellular differentiation. sfRon could be a new drug target in cancer types in which it contributes to tumor progression.