BACKGROUND: Exosomes are extracellular vesicles released by almost all cell types, including cancer cells, into bodily fluids such as saliva, plasma, breast milk, semen, urine, cerebrospinal fluid, amniotic fluid, synovial fluid and sputum. Their key function being intercellular communication with both neighbouring as well as distant cells. Cancer exosomes have been shown to regulate organ-specific metastasis. However, little is known about the functional differences and molecular consequences of normal cells responding to exosomes derived from normal cells compared to those derived from cancer cells.
METHODS: Here, we characterised and compared the transcriptome profiles of primary human normal oral keratinocytes (HNOK) in response to exosomes isolated from either primary HNOK or head and neck squamous cell carcinoma (HNSCC) cell lines.
RESULTS: In recipient HNOK cells, we found that regardless of normal or cancer derived, exosomes altered molecular programmes involved in matrix modulation (MMP9), cytoskeletal remodelling (TUBB6, FEZ1, CCT6A), viral/dsRNA-induced interferon (OAS1, IFI6), anti-inflammatory (TSC22D3), deubiquitin (OTUD1), lipid metabolism and membrane trafficking (BBOX1, LRP11, RAB6A). Interestingly, cancer exosomes, but not normal exosomes, modulated expression of matrix remodelling (EFEMP1, DDK3, SPARC), cell cycle (EEF2K), membrane remodelling (LAMP2, SRPX), differentiation (SPRR2E), apoptosis (CTSC), transcription/translation (KLF6, PUS7). We have also identified CEP55 as a potential cancer exosomal marker.
CONCLUSIONS: In conclusion, both normal and cancer exosomes modulated unique gene expression pathways in normal recipient cells. Cancer cells may exploit exosomes to confer transcriptome reprogramming that leads to cancer-associated pathologies such as angiogenesis, immune evasion/modulation, cell fate alteration and metastasis. Molecular pathways and biomarkers identified in this study may be clinically exploitable for developing novel liquid-biopsy based diagnostics and immunotherapies.

There is growing evidence shown that microRNAs (miRNAs) are associated with cancer and can play a role in human cancers as oncogenes or tumor suppressor genes. MicroRNA-214 (miR-214) shows carcinogenesis in various tumor types, but little is known about biological functions of miR-214 in esophageal squamous cell carcinoma (ESCC). In this study, we observe that the expression of miR-214 is not only increased in human ESCC tissues, but also remarkably increased in cell lines correlates with LZTS1. In addition, the expression of miR-214 inhibited proliferation of ESCC cells in vitro and inhibit the growth of xenograft tumor in vivo. The results show miR-214 serve as a tumor promoter regulating cells migration, invasion and apoptosis in ESCC. Ferthermore, LZTS1 has been proved to as a functional target for miR-214 to regulate cells poliferation and apoptosis. In summary, these results suggest that miR-214 serves as tumor promoter to promote proliferation, migration, invasion and inhibit apoptosis of ESCC cells by targeting LZTS1 via PI3K/AKT/mTOR signaling pathway. The miR-214/LZTS1 pathway provides a new insight into the molecular mechanisms that the occurrence and development of ESCC and it provides a novel therapeutic target for ESCC.

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors (

BACKGROUND: To study the role of LZTS1 in hepatocellular carcinoma (HCC) proliferation and the molecular mechanism involved.
METHODS: LZTS1 expression was studied in 10 HCC cell lines and 1 normal hepatocyte cell line by western blot analysis and qRT-PCR. One HCC cell line was selected and transfected with LZTS1 lentivirus. Cell proliferation and cell cycle were then determined by CCK-8 assay and flow cytometry, respectively. LZTS1, cyclin D1, CDK1, Cdc25C, pS473 Akt, and pT308 Akt mRNA and protein expressions were measured. PS473 Akt and pT308 Akt expression level was also compared with the HCC cells treated with LY294002.
RESULTS: Compared with the normal hepatocyte cells, LZTS1 expression in HCC cells was significantly lower. After the transfection with LZTS1 lentivirus, HCC cell proliferation ability decreased markedly and HCC cells were blocked at G2/M phase. Cyclin D1 and CDK1 expression were both decreased but not significantly. Cdc25C expression was increased significantly. PS473 Akt and pT308 Akt expression level was increased significantly as well, which were almost the same with those transfected with LY294002.
CONCLUSION: LZTS1 could inhibit HCC cell proliferation by impairing PI3K/Akt pathway.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients. Despite the need, there are only a few targeted drug treatment options. Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC. Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis. Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups. Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR. In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines. In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness. Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade. These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness.

Breast invasive micropapillary carcinoma (IMPC) is a rare subtype of breast cancer with a high potential of lymph node metastasis, aggressive clinical behavior, and poor disease-free or overall survival. Expression of leucine zipper putative tumor suppressor 1 (LZTS1) was frequently lost or reduced in breast cancer tissues. This study investigated the expression of LZTS1 protein in breast IMPC tissues using immunohistochemistry. In addition, somatic LZTS1 mutations and promoter methylation were assessed to determine an association with clinicopathological data from IMPC patients. LZTS1 protein was downregulated in 62 (62 %) of 100 IMPC tissue samples and was significantly associated with lymph node metastasis (P < 0.05). A LZTS1 exon mutation occurred in one of the 53 IMPC cases analyzed, whereas a LZTS1 intron mutation occurred in 26 of 53 cases. Moreover, LZTS1 promoter was frequently methylated in IMPC samples and was associated with reduced LZTS1 expression levels in IMPC tissues. These data demonstrated that the loss of LZTS1 expression was associated with lymph node metastasis in patients with IMPC, and LZTS1 promoter methylation could be responsible for the loss of LZTS1 expression.

The Leucine zipper tumor suppressor gene 1 (LZTS1/FEZ1) gene was originally identified as a potential tumor suppressor. However, the expression pattern and the role of LZTS1 in the progression of colorectal cancer (CRC) have not been well characterized. Herein, we reported that LZTS1 was markedly reduced in CRC tissues compared with matched adjacent normal intestine epithelial tissues. In analysis of 160 CRC specimens, we revealed that decreased expression of LZTS1 was correlated to aggressive characteristics and poor survival of patients with CRC. Moreover, we found that expression of LZTS1 in CRC cells significantly inhibited cell proliferation in vitro and prohibited tumor growth in vitro. On the contrary, silence of LZTS1 promoted cell proliferation and tumor growth in CRC cells. Furthermore, we demonstrated that LZTS1 inhibited cell proliferation and tumor growth in CRC in part via suppression of AMT-mTOR, subsequently down-regulating p27Kip and up-regulating cyclin D1. These findings suggest that LZTS1 plays a potential tumor suppressor role in CRC progression and represents a valuable clinical prognostic marker of this disease.

The present study aimed to investigate the possible molecular mechanisms underlying the pathogenesis of metastatic osteosarcoma (OS), by examining the microarray expression profiles of normal samples, and metastatic and non‑metastatic OS samples. The GSE9508 gene expression profile was downloaded from the Gene Expression Omnibus database, which included 11 human metastatic OS samples, seven non‑metastatic OS samples and five normal samples. Pretreatment of the data was performed using the BioConductor package in R language, and the differentially expressed genes (DEGs) were identified by a t‑test. Furthermore, function and pathway enrichment analyses of the DEGs were conducted using a molecule annotation system. A differential co‑expression network was also constructed, and the submodules were screened using MCODE in Cytoscape. A total of 965 genes were identified as DEGs in metastatic OS. The DEGs were shown to participate in the regulation of DNA‑dependent transcription, the composition of the nucleus, cytoplasm and membrane, and protein and nucleotide binding. Furthermore, the screened DEGs were significantly associated with the ribosome, axon guidance and the cytokine‑cytokine receptor interaction pathway. Certain hub genes were identified in the constructed differential co‑expression network, including matrix metalloproteinase 1 (MMP1), smoothened (SMO), ewing sarcoma breakpoint region 1 (EWSR1) and fasciculation and elongation protein ζ‑1 (FEZ1). Brain selective kinase 2 (BRSK2) and aldo‑keto reductase family 1 member B10 (AKRIB10) were present in the screened submodules. The results of the present study suggest that genes, including MMP1, SMO, EWSR1, FEZ1, BRSK2 and AKRIB10, may be potential targets for the diagnosis and treatment of metastatic OS.

Previous studies have shown that miR-214 functions either as an oncogene or a tumor suppressor in various human cancer types. The role of this microRNA in osteosarcoma (OS) is presently unclear. Here, we demonstrated that miR-214 is frequently upregulated in OS specimens, compared with noncancerous bone tissues. Bioinformatics analysis further revealed leucine zipper, putative tumor suppressor 1 (LZTS1) as a potential target of miR-214. Expression patterns of miR-214 were inversely correlated with those of LZTS1 mRNA and protein in OS tissues. Data from reporter assays showed that miR-214 directly binds to the 3'-untranslated region (3'-UTR) of LZTS1 mRNA and suppresses expression at both transcriptional and translational levels. In functional assays, miR-214 promoted OS cell proliferation, invasion and tumor growth in nude mice, which could be reversed by overexpression of LZTS1. Taken together, our data provide compelling evidence that miR-214 functions as an onco-miRNA in OS, and its oncogenic effects are mediated chiefly through downregulation of LZTS1.

Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.

The Leucine Zipper Tumor Suppressor 1 (LZTS1) is a tumor suppressor gene, located at chromosome 8p22, which is frequently altered in human cancer. In normal tissue, its ubiquitous expression regulates cell mitosis by the stabilization of microtubule networks. LZTS1-deficient mouse embryonic fibroblasts have been shown to have an accelerated mitotic progression, and a higher resistance to taxanes, microtubule-stabilizing drugs. We investigate the role of Lzts1 in paclitaxel-resistance in breast cancer cells. Downregulation of Lzts1 expression significantly decreases sensitivity to paclitaxel in vitro. We further analyzed Lzts1 expression by immunohistochemistry in 270 primary breast cancer samples and 16 normal breast specimens. Lzts1 was significantly downregulated in breast cancer samples and its deregulation was associated with a higher incidence of tumor recurrence, and to a worse overall survival. Moreover, Lzts1-negative tumors were associated with unfavorable outcome after taxanes-based therapy. Thus our data suggest that Lzts1 deregulation is involved in breast cancer and its immunohistochemical evaluation may serve as a prognostic factor for breast cancer therapy.

Dysregulation of microRNAs has a critical role in cancer progression. Here we identify an intronic microRNA, miR-135b that is upregulated in highly invasive non-small-cell lung cancer cells. Expression of miR-135b enhances cancer cell invasive and migratory abilities in vitro and promotes cancer metastasis in vivo, while specific inhibition of miR-135b by a miR-135b-specific molecular sponge and antagomirs suppresses cancer cell invasion, orthotopic lung tumour growth and metastasis in a mouse model. miR-135b targets multiple key components in the Hippo pathway, including LATS2, β-TrCP and NDR2, as well as LZTS1. Expression of miR-135b, LZTS1, LATS2 and nuclear TAZ predicts poor outcomes of non-small-cell lung cancer. We find that miR-135b is dually regulated by DNA demethylation and nuclear factor-kappaB signalling, implying that abnormal expression of miR-135b in cancer may result from inflammatory and epigenetic modulations. We conclude that miR-135b is an oncogenic microRNA and a potential therapeutic target for non-small-cell lung cancer.

PURPOSE: Advanced ovarian clear cell carcinoma (CCC) is one of the most aggressive ovarian malignancies, in part because it tends to be resistant to platinum-based chemotherapy. At present, little is known about the molecular genetic alterations in CCCs except that there are frequent activating mutations in PIK3CA. The purpose of this study is to comprehensively define the genomic changes in CCC based on DNA copy number alterations.
EXPERIMENTAL DESIGN: We performed 250K high-density single nucleotide polymorphism array analysis in 12 affinity-purified CCCs and 10 CCC cell lines. Discrete regions of amplification and deletion were also analyzed in additional 21 affinity-purified CCCs using quantitative real-time PCR.
RESULTS: The level of chromosomal instability in CCC as defined by the extent of DNA copy number changes is similar to those previously reported in low-grade ovarian serous carcinoma but much less than those in high-grade serous carcinoma. The most remarkable region with DNA copy number gain is at chr20, which harbors a potential oncogene, ZNF217. This discrete amplicon is observed in 36% of CCCs but rarely detected in serous carcinomas regardless of grade. In addition, homozygous deletions are detected at the CDKN2A/2B and LZTS1 loci. Interestingly, the DNA copy number changes observed in fresh CCC tissues are rarely detected in the established CCC cell lines.
CONCLUSIONS: This study provides the first high resolution, genome-wide view of DNA copy number alterations in ovarian CCC. The findings provide a genomic landscape for future studies aimed at elucidating the pathogenesis and developing new target-based therapies for CCCs.

Genomic alterations such as chromosomal amplifications, deletions and loss of heterozygosity play an important role in the pathogenesis and progression of cancer. Environmental risk factors contribute to the development and progression of tumors by facilitating the loss of tumor suppressor genes and amplification of oncogenes. In this current study, Affymetrix 10K single nucleotide polymorphism (SNP) arrays were used to evaluate genomic alterations in 20 pairs of matched germ-line and tumor DNA obtained from patients with esophageal squamous cell carcinoma (ESCC) from high-risk area of India where tobacco, betel quid and alcohol use are widespread. Twenty-two amplified regions and 16 deleted regions identified across chromosomal arms were biologically relevant. The candidate genes located at amplified regions of chromosomes or low-level gain regions such as PLA2G5 (1p36-p34), COL11A1 (1p21), KCNK2 (1q41), S100A3 (1q21), ENAH (1q42.12), RGS1 (1q31), KCNH1 (1q32-q41), INSIG2 (2q14.1), FGF12 (3q28), TRIO (5p15.2), RNASEN (5p15.2), FGF10 (5p13-p12), EDN1(6p24.1-p22.3), SULF1 (8q13.2-13.3), TLR4 (9q32-q33), TNC (9q33), NTRK2 (9q22.1), CD44 (11p13), NCAM1 (11q23.1), TRIM29 (11q22-q23), PAK1 (11q13-q14) and RAB27A (15q15-q21.1), are found to be associated with cellular migration and proliferation, tumor cell metastasis and invasion, anchorage independent growth and inhibition of apoptosis. The candidate genes located at deleted regions of chromosomes, such as FBLN2 (3p25.1), WNT7A (3p25), DLC1 (8p22), LZTS1 (8p22), CDKN2A (9p21), COL4A1 (13q34), CDK8 (13q12) and DCC (18q21.3), are found to be associated with the suppression of tumor. The suggested candidate genes were mostly involved in potential signaling pathways such as focal adhesion (COL4A1), tight junction (CLDN10), MAPK signaling pathway (FGF12) and neuroactive ligand receptor interaction pathway (CCKAR). Expression of FGF12 and COL4A1 was validated by tissue microarray. These unique copy number alteration profiles should be taken into consideration when developing biomarkers for the early detection of ESCC in high-risk areas of India in association with tobacco and betel quid use.

The FEZ1/LZTS1 (FEZ1) gene maps to chromosome 8p22 and is frequently altered in human cancer. FEZ1 has been proposed as a candidate tumour suppressor gene and its loss may contribute to tumour progression. We have analysed the expression of FEZ1 protein in tissues from ovarian carcinomas in relation to clinico-pathological variables, response to chemotherapy and disease-free and overall survival. FEZ1 status was assessed by immunohistochemistry. Cytoplasmic staining for FEZ1 protein was absent or drastically reduced in 38% of tumours. FEZ1 protein expression was not related to tumour grade, histotype, disease-free survival, or overall survival. On the contrary, it was significantly correlated with age and with FIGO stage of disease. This finding indicates that FEZ1 is involved in ovarian carcinogenesis. Moreover, loss of FEZ1 protein significantly predicted a complete treatment response in patients who received taxane-based chemotherapy. In conclusion, the reduction or loss of FEZ1 protein could be an aid to the clinical management of patients affected by ovarian carcinoma.

Apoptosis is a tightly regulated form of cell death and represents an important process during normal development. In the past years, the scientific community has produced remarkable advances in our understanding of cancer biology, realizing that apoptosis and the genes that control it have a profound effect on the malignant phenotype. Recently, a new class of non-coding RNA genes, known as microRNA (miRNA or miR), have been demonstrated to play important roles in diverse biological processes, including development, cell differentiation, proliferation, and apoptosis. This suggests that other oncogenic mechanisms are needed to produce selective pressure to override apoptosis during multistage carcinogenesis. Intriguingly, since most cytotoxic anticancer agents induce apoptosis, it is possible that defects in apoptotic programs may contribute to treatment failure. Several studies strongly suggest a role for microRNAs in modulating sensitive/resistant phenotypes to cytotoxic therapy, calling for further investigation and validation of microRNA functions and targets in order to improve sensitivity to cancer treatments, thus ultimately improving prognosis and survival. Here, we review the current findings about microRNAs focusing on their involvement in the apoptotic process.

FEZ1/LZTS1 is a tumor suppressor gene located in chromosomal band 8p22, and methylation has been identified as a mechanism for its loss of function in tumors. Chromosomal deletion at 8p22 is also frequent in breast cancer. We therefore examined whether LZTS1 plays a role in breast cancer. We analyzed expression of LZTS1 at both the RNA and protein levels, and promoter methylation in a number of primary tumors and cell lines from breast cancer. We also examined the association between LZTS1 expression and different clinicopathological parameters of breast cancer. We found that the expression of LZTS1 mRNA was reduced in 25 of 50 (50%) primary tumors and 29 of 30 (97%) breast cancer cell lines. Immunohistochemical staining showed that LZTS1 protein was absent or down-regulated in 72 (72%) of 100 primary breast carcinomas. Reduced expression of LZTS1 at either the RNA or protein level was significantly correlated with lymph node metastases (P < 0.05). DNA methylation analysis revealed that the LZTS1 gene was frequently methylated in both cell lines and primary tumors from breast cancer, and the extent of DNA methylation was correlated with reduced expression of the gene. These findings suggest that LZTS1 plays a role in the development and progression of breast cancer at least through promoter methylation-mediated transcriptional downregulation.

PURPOSE: To identify genes that modify metastatic risk in uveal melanoma, a type of cancer that is valuable for studying metastasis because of its remarkably consistent metastatic pattern and well-characterized gene expression signature associated with metastasis.
EXPERIMENTAL DESIGN: We analyzed 53 primary uveal melanomas by gene expression profiling, array-based comparative genomic hybridization, array-based global DNA methylation profiling, and single nucleotide polymorphism-based detection of loss of heterozygosity to identify modifiers of metastatic risk. A candidate gene, leucine zipper tumor suppressor-1 (LZTS1), was examined for its effect on proliferation, migration, and motility in cultured uveal melanoma cells.
RESULTS: In metastasizing primary uveal melanomas, deletion of chromosome 8p12-22 and DNA hypermethylation of the corresponding region of the retained hemizygous 8p allele were associated with more rapid metastasis. Among the 11 genes located within the deleted region, LZTS1 was most strongly linked to rapid metastasis. LZTS1 was silenced in rapidly metastasizing and metastatic uveal melanomas but not in slowly metastasizing and nonmetastasizing uveal melanomas. Forced expression of LZTS1 in metastasizing uveal melanoma cells inhibited their motility and invasion, whereas depletion of LZTS1 increased their motility.
CONCLUSIONS: We have described a metastatic modifier locus on chromosome 8p and identified LZTS1 as a potential metastasis suppressor within this region. This study shows the utility of integrative genomic methods for identifying modifiers of metastatic risk in human cancers and may suggest new therapeutic targets in metastasizing tumor cells.

Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenetic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregulated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients.

Both entry and exit from mitosis are driven through the fine modulation of Cdk1 activity by several proteins or protein complexes. It is well established that to entry into the M-phase a cell requires Cdk1 to be fully activated in the nucleus by the Cdc25A, B and C phosphatases. Then, at the onset of anaphase Cdk1 activity suddenly drops mainly due to Cyclin B1 degradation, thus allowing exit from M-phase. Recent data demonstrate that high Cdk1 activity is necessary also for proper chromosome segregation, since its premature drop determines acceleration of the progression from prophase to metaphase eventually with incorrect division of the DNA content. A primary role in maintaining high Cdk1 activity during prophase and metaphase is played by Cdc25C phosphatase. During the M-phase, the activity of Cdc25C is regulated by the FEZ1/LZTS1 (LZTS1) tumor suppressor gene, which is able to prevent Cdc25C degradation in mitotic cells. As a consequence, Lzts1 absence in mice results in accelerated mitotic progression, improper chromosome segregation and, eventually, in increased incidence of both spontaneous and carcinogen-induced cancer formation.

Patients with malignant mesothelioma (MM), an aggressive cancer associated with asbestos exposure, usually present clinically with advanced disease and this greatly reduces the likelihood of curative treatment. MM is difficult to diagnose without invasive techniques; the development of non-invasively detectable molecular markers would therefore be highly beneficial. DNA methylation changes in cancer cells provide powerful markers that are potentially detectable non-invasively in DNA shed into bodily fluids. Here we examined the methylation status of 28 loci in 52 MM tumors to investigate their potential as molecular markers for MM. To exclude candidate MM markers that might be positive in biopsies/pleural fluid due to contaminating surrounding non-tumor lung tissue/DNA, we also examined the methylation of these markers in lung samples (age- or environmentally induced hypermethylation is frequently observed in non-cancerous lung). Statistically significantly increased methylation in MM versus non-tumor lung samples was found for estrogen receptor 1 (ESR1; p = 0.0002), solute carrier family 6 member 20 (SLC6A20; p = 0.0022) and spleen tyrosine kinase (SYK; p=0.0003). Examination of associations between methylation levels of the 28 loci and clinical parameters suggest associations of the methylation status of metallothionein genes with gender, histology, asbestos exposure, and lymph node involvement, and the methylation status of leucine zipper tumor suppressor 1 (LZTS1) and SLC6A20 with survival.

AIM: To study the loss of heterozygosity (LOH) at 8p21-23 locus in diffuse gastric cancer.
METHODS: To evaluate the involvement of this region in gastric cancer, we used eight microsatellite markers covering two Mb of mentioned region, to perform a high-resolution analysis of allele loss in 42 cases of late diffuse gastric adenocarcinoma.
RESULTS: Six of these STS makers: D8S1149, D8S1645, D8S1643, D8S1508, D8S1591, and D8S1145 showed 36%, 28%, 37%, 41%, 44% and 53% LOH, respectively.
CONCLUSION: A critical region of loss, close to the NAT2 locus and relatively far from FEZ1 gene currently postulated as tumor suppressor gene in this region.

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBN1, and LZTS1, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4L1, and TBC1D5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (>85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Bladder cancer is one of the most common cancers in the world, leading to approximately 145,000 deaths annually. Bladder cancer is typically managed by surgical removal of the tumor; however, the recurrence rate is disappointingly very high, often requiring systemic chemotherapy. Improvement in the diagnosis and prognosis of bladder cancer will only come from a comprehensive understanding of the genetic factors that lead to its development. In this review, we focus on the chromosomal deletions that contribute to the downregulation of tumor suppressor pathways in bladder cancer. Chromosomal deletions are not a random event, since bladder cancer progression has been associated with specific chromosomal deletions and this progression correlates with specific stages of tumor development. The most commonly found chromosomal deletion in all stages of bladder cancer involves deletions in chromosome 9, resulting in the loss of three genes encoding proteins that activate the Rb and p53 tumor suppressors. Additionally, chromosome 9 harbors the TSC1 tumor suppressor which downregulates the well-known anti-apoptotic Akt/mTOR pathway. Hence, deletions on one chromosome may have a crucial influence on the initial steps in tumor development. Other deletions targeting the tumor suppressors Rb, p53, FHIT and LZTS1 occur at later stages of tumor development. Considering the central importance of these tumor suppressor pathways in the formation and evolution of tumors, the time has come to evaluate available drugs in bladder cancer that target the positive regulators of these pathways.

Transitional cell carcinoma of the bladder is a common tumor. While most patients presenting superficial disease can be expected to do well following treatment, still many patients will return to our office with muscle invasive and metastatic disease. Survival in advanced bladder cancer is less than 50%. Tumors of similar histologic grade and stage have variable behavior, suggesting that genetic alterations must be present to explain the diverse behavior of bladder cancer. It is hoped that through the study of the subtle genetic alterations in bladder cancer, important prognostic and therapeutic targets can be exploited. Many new diagnostic tests and gene therapy approaches rely on the identification and targeting of these unique genetic alterations. A review of literature published on the molecular genetics of bladder cancer from 1970 to the present was conducted. A variety of molecular genetic alterations have been identified in bladder cancer. Oncogenes (H-ras, erbB-2, EGFR, MDM2, C-MYC, CCND1), tumor suppressor genes (p53, Rb, p21, p27/KIP1, p16, PTEN, STK15, FHIT, FEZ1/LZTS1, bc10), telomerase, and methylation have all been studied in bladder cancer. Several have proven to be potentially useful clinical targets in the prognosis and therapy of bladder cancer such as staining for p53 and gene therapy strategies such as p53 and fez1. Clinical trials targeting HER2/neu and the EGFR pathways are underway. The UroVysion bladder cancer assay relies on FISH to detect genetic alterations in this disease. Continuing identification of the molecular genetic alterations in bladder cancer will enhance future diagnostic and therapeutic approaches to bladder cancer. Capitalizing on these alterations will allow early detection, providing important prognostic information and unique targets for gene therapy and other therapeutic approaches.

Members of the leucine zipper tumor suppressor (LZTS) protein family are thought to play roles in cell growth modulation. The two currently known members were identified by analyzing genomic and chromosomal alterations reported to be either involved or deleted in various types of cancer, suggesting a causative relationship. By means of computational biology, we have now identified a novel member of the LZTS protein family named LZTS3. The corresponding gene was localized to chromosome 20p13 and consisted of three exons. The novel LZTS3 protein demonstrated a high similarity to LAPSER1/LZTS2 and FEZ1/LZTS1, two members of the LZTS family. The conserved FEZ1 domain contains a leucine zipper motif similar to the cAMP-responsive activating transcription factor 5. As FEZ1 inhibits cancer cell growth through the regulation of mitosis and its alteration resulted in abnormal cell growth, the LZTS3 protein was expected to have similar functions during cancer differentiation. Furthermore, the protein was conserved in vertebrates, as orthologs could be identified in mouse, rat, and zebrafish. The intracellular localization of the LZTS3 protein was predicted to be nuclear by means of Reinhardt's neural network and the k-nearest neighbor algorithm. An RT-PCR-based expression profile available from the human unidentified gene-encoded (HUGE) database demonstrated the highest expression in the brain and kidney, accompanied by lower expression in multiple other tissues.

Genomic deletions of the short arm of chromosome 8 are common in many human cancers and are frequently associated with a more aggressive tumour phenotype. One of the regions of loss of heterozygosity (LOH) on 8p22 identified in bladder cancer contains two genes, LZTS1 (FEZ1) and DBC2 (RHOBTB2) that have been shown to be mutated at low frequency in other cancers. We screened a panel of bladder tumours and bladder tumour-derived cell lines for mutations in these genes. Forty two percent of the tumours were found to have LOH in the 8p22 region and many of the cell lines have known loss of 8p. Several known polymorphisms and novel polymorphisms were detected. One possible mutation of LZTS1 (G374S) was found in a cell line. The functional significance of this is unknown but the novel serine residue created may represent a novel phosphorylation site. In DBC2, we found a single somatic mutation in a tumour (E349D) that lies in a highly conserved region of the protein. mRNA levels for both genes were reduced in the majority of bladder cancer cell lines. We conclude that neither LZTS1 nor DBC2 is commonly mutated in bladder cancer. However, neither can yet be excluded as the target of 8p22 LOH. The finding of a somatic mutation of DBC2 in a tumour sample and the down-regulation of both gene transcripts in bladder tumour cell lines may indicate that an alternative mechanism of inactivation of the second allele, for example promoter hypermethylation, is more common than mutation and this must now be examined.

LZTS1 has been shown to have tumour suppressor activities against prostate and breast cancer and is located within a region of frequent loss of heterozygosity (LOH) at 8p22 in ovarian cancer. We have analysed the expression of LZTS1 in ovarian cancer and found no evidence of loss of expression relative to normal ovarian surface epithelial cells. We have also analysed the coding region of the LZTS1 gene in 87 primary ovarian adenocarcinomas by DHPLC and detected a single silent somatic mutation. These data indicate that LZTS1 is not the target of LOH at 8p22 in ovarian cancer.

Chromosomal deletions are often observed in lung cancers suggesting that inactivation of tumor suppressor genes plays an important role in the development of this neoplasm. The region around chromosome 8p22 is a frequent and early target of these deletions and has therefore been investigated for the presence of candidate genes. The FEZ1/LZTS1 gene, located at 8p22, is inactivated in many cancers with 8p deletions, including prostate, esophageal, gastric, bladder, and breast cancer and the Fez1 protein has been shown to suppress growth of cancer cells and to regulate mitosis. To elucidate the role of FEZ1 in lung cancer, we have analyzed its expression by immunohistochemistry in 103 primary lung cancer specimens including 98 non-small cell lung cancers (57 adenocarcinomas, 32 squamous cell carcinomas, 7 large cell carcinomas, and 2 others) and five small cell carcinomas. Absence of Fez1 protein expression was observed in 27 cases (26%) and additional 43 cases (42%) showed strong reduction in immunoreactivity. There was a positive association between loss of FEZ1 expression and tumor grading (P = 0.0345) and a tendency toward a reduction in the mortality rate in subjects with strong FEZ1 expression. Overall, these data indicate an important role for FEZ1 in lung cancer and suggest the possibility that it may serve as a novel prognostic indicator.

BACKGROUND: Collecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellini's ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer (FEZ1/LZTS1, FHIT, TP53, P27kip1, and BCL2).
METHODS: We studied eleven patients who underwent radical nephrectomy for primary CDC. All patients had an adequate clinical follow-up and none of them received any systemic therapy before surgery. The expression of the five markers for tumor initiation and/or progression were assessed by immunohistochemistry and correlated to the clinicopathological parameters, and survival by univariate analysis.
RESULTS: Results showed that Fez1 protein expression was undetectable or substantially reduced in 7 of the 11 (64%) cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.
CONCLUSIONS: These findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC.