BACKGROUND: Malignant melanoma cells can rapidly acquire phenotypic properties making them resistant to radiation and mainline chemotherapies such as decarbonize or kinase inhibitors that target RAS-proto-oncogene independent auto-activated mitogen-activated protein kinases (MAPK)/through dual specificity mitogen-activated protein kinase (MEK). Both drug resistance and inherent transition from melanocytic nevi to malignant melanoma involve the overexpression of histone deacetylases (HDACs) and a B-Raf proto-oncogene (BRAF) mutation.
MATERIALS AND METHODS: In this work, the effects of an HDAC class I and II inhibitor trichostatin A (TSA) on the whole transcriptome of SK-MEL-3 cells carrying a BRAF mutation was examined.
RESULTS: The data obtained show that TSA was an extremely potent HDAC inhibitor within SK-MEL-3 nuclear lysates, where TSA was then optimized for appropriate sub-lethal concentrations for in vitro testing. The whole-transcriptome profile shows a basic phenotype dominance in the SK-MEL-3 cell line for i) synthesis of melanin, ii) phagosome acidification, iii) ATP hydrolysis-coupled proton pumps and iv) iron transport systems. While TSA did not affect the aforementioned major systems, it evoked a dramatic change to the transcriptome: reflected by a down-regulation of 810 transcripts and up-regulation of 833, with fold-change from -15.27 to +31.1 FC (p<0.00001). Largest differentials were found for the following transcripts: Up-regulated: Tetraspanin 13 (TSPAN13), serpin family i member 1 (SERPINI1), ATPase Na+/K+ transporting subunit beta 2 (ATP1B2), nicotinamide nucleotide adenylyl transferase 2 (NMNAT2), platelet-derived growth factor receptor-like (PDGFRL), cytochrome P450 family 1 subfamily A member 1 (CYP1A1), prostate androgen-regulated mucin-like protein 1 (PARM1), secretogranin II (SCG2), SYT11 (synaptotagmin 11), rhophilin associated tail protein 1 like (ROPN1L); down-regulated: polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), carbonic anhydrase 14 (CAXIV), BCL2-related protein A1 (BCL2A1), protein kinase C delta (PRKCD), transient receptor potential cation channel subfamily M member 1 (TRPM1), ubiquitin associated protein 1 like (UBAP1L), glutathione peroxidase 8 (GPX8), interleukin 16 (IL16), tumor protein p53 (TP53), and serpin family H member 1 (SERPINH1). There was no change to any of the HDAC transcripts (class I, II and IV), the sirtuin HDAC family (1-6) or the BRAF proto-oncogene v 599 transcripts. However, the data showed that TSA down-regulated influential transcripts that drive the BRAF-extracellular signal-regulated kinase (ERK)1/2 oncogenic pathway (namely PRKCD and MYC proto-oncogene which negatively affected the cell-cycle distribution. Mitotic inhibition was corroborated by functional pathway analysis and flow cytometry confirming halt at the G
CONCLUSION: TSA does not alter HDAC transcripts nor BRAF itself, but down-regulates critical components of the MAPK/MEK/BRAF oncogenic pathway, initiating a mitotic arrest.

Previous studies have shown that c-MET is overexpressed in cases of aggressive bladder cancer (BCa). Identification of crosstalk between c-MET and other RTKs such as AXL and PDGFR suggest that c-MET network genes (c-MET-AXL-PDGFR) may be clinically relevant to BCa. Here, we examine whether expression of c-MET network genes can be used to identify BCa patients at increased risk of developing aggressive disease. In vitro analysis, c-MET knockdown suppressed cell proliferation, invasion, and migration, and increased sensitivity to cisplatin-induced apoptosis. In addition, c-MET network gene (c-MET, AXL, and PDGFR) expression allowed discrimination of BCa tissues from normal control tissues and appeared to predict poor disease progression in non-muscle invasive BCa patients and poor overall survival in muscle invasive BCa patients. These results suggest that c-MET network gene expression is a novel prognostic marker for predicting which BCa patients have an increased risk of developing aggressive disease. These genes might be a useful marker for co-targeting therapy, and are expected to play an important role in improving both response to treatment and survival of BCa patients.

Neurofibromatosis type-1 (NF1) is associated with the growth of benign and malignant tumors. Approximately 15% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), underlining the need to identify specific diagnostic/prognostic biomarkers associated with MPNST development. The Affymetrix Genome-Wide Human single-nucleotide polymorphism (SNP) Array 6.0 was used to perform SNP genotyping and copy number alteration (CNA), loss-of-heterozygosity (LOH), and copy number neutral-LOH (CNN-LOH) analyses of DNA isolated from 15 MPNSTs, five benign plexiform neurofibromas (PNFs), and patient-matched lymphocyte DNAs. MPNSTs exhibited high-level CNN-LOH, with recurrent changes occurring in MPNSTs but not PNFs. CNN-LOH was evident in MPNSTs but occurred less frequently than genomic deletions. CNAs involving the ITGB8, PDGFA, Ras-related C3 botulinum toxin substrate 1 (RAC1) (7p21-p22), PDGFRL (8p22-p21.3), and matrix metallopeptidase 12 (MMP12) (11q22.3) genes were specific to MPNSTs. Pathway analysis revealed the MPNST-specific amplification of seven Rho-GTPase pathway genes and several cytoskeletal remodeling/cell adhesion genes. In knockdown experiments employing short-hairpin RAC1, ROCK2, PTK2, and LIMK1 RNAs to transfect both control and MPNST-derived cell lines, cell adhesion was significantly increased in the MPNST cell lines, whereas wound healing, cell migration, and invasiveness were reduced, consistent with a role for these Rho-GTPase pathway genes in MPNST development and metastasis. These results suggest new targets for therapeutic intervention in relation to MPNSTs.

The occurrence of de novo malignant neoplasms has been shown in post-transplant recipients receiving immunosuppressive treatment. We present a case of a rare extragastrointestinal stromal tumor (EGIST) located in the pelvic cavity of a kidney transplant patient. A 57-year-old female patient was admitted to our department because of non-specific lower abdominal pain 6 months after renal transplantation. An abdominal computed tomography scan showed a 4.5 cm diameter pelvic tumor mass. The tumor was resected en bloc and confirmed as not being connected to the gastrointestinal wall. Microscopically, the tumor consisted of typical spindle cells with 2-3 mitotic figures per 50 high-power fields. Immunohistochemically, the tumor cells were strongly positive for CD117 (c-kit), and negative for CD34, SMA, s-100 protein, and desmin. Genetically, the tumor showed a silent mutation in exon 18 of the PDGFRA gene at codon 824 GTC > GTT (V824V) [rs2228230]. No recurrence was noted 24 months after the operation. This case draws our attention to the importance of considering EGISTs (including GISTs), even though they are extremely uncommon, in the differential diagnosis of mesenchymal neoplasms, especially in transplant patients.

AIM: To investigate the role of platelet-derived growth factor receptor-like gene (PDGFRL) in the anti-cancer therapy for colorectal cancers (CRC).
METHODS: PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in CRC and colorectal normal tissues. PDGFRL prokaryotic expression vector was carried out in Escherichia coli (E. coli), and purified by immobilized metal affinity chromatography. The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), clone counting, cell cycle, and wound healing assay.
RESULTS: Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues. Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E. coli, and the target protein was expressed in the form of inclusion bodies. After purification and refolding, recombinant human PDGFRL (rhPDGFRL) could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT, clone counting and wound healing assay. Moreover, rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase.
CONCLUSION: PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.

BACKGROUND: The most common application of microarray technology in disease research is to identify genes differentially expressed in disease versus normal tissues. However, it is known that, in complex diseases, phenotypes are determined not only by genes, but also by the underlying structure of genetic networks. Often, it is the interaction of many genes that causes phenotypic variations.
RESULTS: In this work, using cancer as an example, we develop graph-based methods to integrate multiple microarray datasets to discover disease-related co-expression network modules. We propose an unsupervised method that take into account both co-expression dynamics and network topological information to simultaneously infer network modules and phenotype conditions in which they are activated or de-activated. Using our method, we have discovered network modules specific to cancer or subtypes of cancers. Many of these modules are consistent with or supported by their functional annotations or their previously known involvement in cancer. In particular, we identified a module that is predominately activated in breast cancer and is involved in tumor suppression. While individual components of this module have been suggested to be associated with tumor suppression, their coordinated function has never been elucidated. Here by adopting a network perspective, we have identified their interrelationships and, particularly, a hub gene PDGFRL that may play an important role in this tumor suppressor network.
CONCLUSION: Using a network-based approach, our method provides new insights into the complex cellular mechanisms that characterize cancer and cancer subtypes. By incorporating co-expression dynamics information, our approach can not only extract more functionally homogeneous modules than those based solely on network topology, but also reveal pathway coordination beyond co-expression.

Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.

BACKGROUND: Loss of heterozygosity on chromosomal band 8p22 is a common event in several epithelial tumors including ovarian carcinoma. So far, no clear evidence for a tumor suppressor gene (TSG) in this region has been found.
METHODS: On the basis of publicly available expression data in ovarian tissues, the authors selected the eight most noteworthy genes from 8p22 (DLC1, N33, ZDHHC2, FLJ32642, PDGFRL, MTSG1, PCM1, and EFA6R) for a detailed expression analysis in 58 primary ovarian carcinoma tissues and in 38 ovarian cancer cell lines by using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Expression data were correlated to various clinicopathologic characteristics and survival.
RESULTS: Two genes showed a significantly (P< 0.05) lower expression in grade 3 tumors compared with tumors of lower grade (N33) or compared with normal controls and tumors with lower grade (EFA6R). Expression of N33 and EFA6R seems to have an impact on survival, in particular when the combined expression of both genes was used as predictive factor (P< 0.003). In addition, N33 and EFA6R showed a complete loss of expression in several ovarian cancer cell lines. Three genes (FLJ32642, MTSG1, and PCM1) had a significantly (P< 0.001, P< 0.004, and P< 0.001) lower expression in primary ovarian carcinoma compared with controls (ovarian tissues and cysts).
CONCLUSIONS: Two to five new potential tumor suppressor or antagonizing gene candidates (N33 and EFA6R with impact on survival, and potentially FLJ32642, MTSG1, and PCM1) for ovarian carcinoma, were identified from the chromosomal band 8p22 and are promising candidates for further functional analysis in ovarian carcinoma.

To provide new insights into the molecular mechanisms underlying the effect of irradiation on esophageal squamous cell carcinomas (ESCCs), we used a cDNA microarray screening of more than 4,000 genes with known functions to identify genes involved in the early response to ionizing irradiation. Two human ESCC cell lines, one each of well (TE-1) and poorly (TE-2) differentiated phenotypes were screened. Subconfluent cells of each phenotype were treated with single doses of 2.0 Gy or 8.0 Gy irradiations. After a 15 min incubation time-point, the cells were collected and analyzed. Compared with non-irradiated cells, many genes revealed at least 2-fold upregulation or downregulation at both doses in well or poorly differentiated ESCC cells. The common upregulated genes in well and poorly differentiated cell types at both irradiation doses included SCYA5, CYP51, SMARCD2, COX6C, MAPK8, FOS, UBE2M, RPL6, PDGFRL, TRAF2, TNFAIP6, ITGB4, GSTM3, and SP3 and common downregulated genes involved NFIL3, SMARCA2, CAPZA1, MetAP2, CITED2, DAP3, MGAT2, ATRX, CIAO1, and STAT6. Several of these genes were novel and not previously known to be associated with irradiation. Functional annotations of the modulated genes suggested that at the molecular level, irradiation appears to induce a regularizing balance in ESCC cell function. The genes modulated in the early response to irradiation may be useful in our understanding of the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel less hazardous alternative adjuvant therapies.

BACKGROUND AND AIM: In hepatocarcinogenesis, both de novo and multistep pathways have been suggested, and in the latter a dysplastic nodule is the proposed precancerous lesion. But genetic changes involved in the dysplastic nodule are not well understood. In this study, we tried to determine whether allelic loss of the chromosome 8p and/or 11p could be involved in the development of the dysplastic nodule and/or hepatocellular carcinoma. Platelet-derived growth factor-receptor beta-like tumor suppressor gene (PRLTS) and deletion in liver cancer-1 tumor suppressor gene are located at 8p21.3-p22. The hepatitis B virus integration site and WT1 tumor suppressor gene are located at 11p13.
METHODS: We therefore studied loss of heterozygosity (LOH) of chromosome 8p21.3-p22 and 11p13 in 22 dysplastic nodules and 21 hepatocellular carcinomas. The samples, microdissected from paraffin-embedded tissues, were examined using a polymerase chain reaction-based LOH assay using microsatellite markers.
RESULTS: Loss of heterozygosity was detected for chromosome 8p21.3-p22 in nine (40.9%) of 22 dysplastic nodules and in eight (42.1%) of 19 hepatocellular carcinomas. D8S261, located adjacent to PRLTS, showed most frequent LOH: 28.6% in dysplastic nodule and 40.0% in hepatocellular carcinoma. Loss of heterozygosity on chromosome 11p13 was found in three (15.8%) of 19 dysplastic nodules and in six (31.6%) of 19 hepatocellular carcinomas. Loss of heterozygosity of D11S995 and D11S907 was found in 33.3% and 7.1% of dysplastic nodules, and 8.3% and 44.4% of hepatocellular carcinomas, respectively.
CONCLUSION: These results suggest that at least one putative tumor suppressor gene involved in the development and progression of hepatocellular carcinoma may be located on 8p21.3-p22 and 11p13. Particularly, PRLTS might be related to an early genetic event of hepatocarcinogenesis.

In the present study, we used 22 microsatellite markers flanking to or within 13 known or candidate tumor suppressor genes (TSGs) to detect loss of heterozygosity (LOH) in these chromosomal regions among 41 cases of non-small cell lung cancer, including 28 squamous cell carcinoma (SCC) and 13 adenocarcinoma (ADC). The studied TSGs comprised FHIT, VHL, APC, PRLTS, p16, IFNA, PTEN, p57, ATM, p53, BRCA1, DPC4 and DCC. Our data demonstrated frequent allelic losses of FHIT, p53, IFNA, VHL and p16 in both SCC and ADC. PTEN and ATM showed the least frequency of LOH, while no deletion of BRCA1 was detected in all tumor samples. LOH analysis of PRLTS was extended to 26 cases of ADC, which demonstrated significantly higher frequency of LOH than SCC. Our data indicated a possible correlation between specific TSG(s) and either histological type of lung cancer, and more attention should be paid to the PRLTS gene, which might play an important role in the development of ADC.

Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC). Recurrent chromosomal deletions have suggested the presence of tumor-suppressor genes specifically involved in lung carcinogenesis. For one of these, 2 non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The LPL region has been extensively studied in NSCLC and other cancer types. Two genes, N33 and PRLTS, have been identified, but the small number of mutations excludes their involvement in the vast majority of tumors. In order to delineate a reliable region of deletional overlap on chromosome 8p in NSCLC, a series of 77 NSCLC was studied for 34 microsatellite polymorphisms distributed on chromosome 8p, using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, allelic losses on chromosome 8p were observed in 39% of cases. Measurement of DNA index showed that 62% of tumors were hyperploid; allelic losses were more frequent in hyperploid than in diploid tumors (54% vs. 14%; p < 10(-4)). Deletions of part of the short arm were observed in 7 instances. Our data allow definition of an interval of common deletion, flanked by the loci D8S511 and D8S1992, where the putative tumor-suppressor gene might be localized.

Since loss of heterozygosity on 8p22-p21.3 has been found frequently in prostate cancer, the status of a candidate tumor suppressor gene named PRLTS gene, recently cloned from the same region in some human malignancies, was examined in the present study. DNAs were isolated from 69 Japanese prostate cancer patients (37 localized and 32 cancer-death cases). Loss of heterozygosity at this gene locus was observed in 15 of 36 (42%) localized prostate cancer patients and 22 of 32 (69%) cancer-death patients. One cancer-death patient had a missense mutation, ACG-->ATG (Thr-->Met) at codon 64 in metastatic tumor tissues of pelvic lymph node and liver, and these tissues showed loss of the homologous allele, indicating that "two-hit" mutation of the PRLTS gene had occurred in this case. The others did not show any mutation, regardless of the presence or absence of loss of heterozygosity. Although loss of heterozygosity at the PRLTS gene locus is a relatively common abnormality, mutation of this gene is rare in prostate cancer.

Numerous studies have implicated the short arm of chromosome 8 as the site of one or more tumor suppressor genes inactivated in carcinogenesis of the prostate, colon, lung, and liver. Previously, we identified a homozygous deletion on chromosome 8p22 in a metastatic prostate cancer. To map this homozygous deletion physically, long-range restriction mapping was performed using yeast artificial chromosomes (YACs) spanning approximately 2 Mb of chromosome band 8p22. Subcloned genomic DNA and cDNA probes isolated by hybrid capture from these YACs were mapped in relation to one another, reinforcing map integrity. Mapped single-copy probes from the region were then applied to DNA isolated from a metastatic prostate cancer containing a chromosome 8p22 homozygous deletion and indicated that its deletion spans 730-970 kb. Candidate genes PRLTS (PDGF-receptor beta-like tumor suppressor) and CTSB (cathepsin B) are located outside the region of homozygous deletion. Généthon marker D8S549 is located approximately at the center of this region of homozygous deletion. Two new microsatellite polymorphisms, D8S1991 and D8S1992, also located within the region of homozygous deletion on chromosome 8p22, are described. Physical mapping places cosmid CI8-2644 telomeric to MSR (macrophage scavenger receptor), the reverse of a previously published map, altering the interpretation of published deletion studies. This work should prove helpful in the identification of candidate tumor suppressor genes in this region.

We have isolated a candidate tumor suppressor gene from a 600-kb region on chromosome 8p21.3-p22 that is commonly deleted in sporadic hepatocellular carcinomas (HCC), colorectal cancers (CRC), and non-small cell lung cancers (NSCLC). As this gene encodes a protein of 375 amino acids that bears significant sequence similarity to the extracellular (ligand-binding domain of platelet-derived growth factor receptor beta, we have designated it PRLTS (PDGF-receptor beta-like tumor suppressor). Structural rearrangement involving this gene was found in a sporadic NSCLC. In addition, somatic missense and frame-shift mutations were found in two HCCs and one CRC. These findings indicate that inactivation of the PRLTS gene may play a significant role in development of some carcinomas.