Identification of HERC5 and its potential role in NSCLC progression.

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status (p = 0.020) and occurrence of brain metastases (p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients (p = 0.022) and also for poor overall survival in metastatic lung cancer patients (p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer.

Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells.

BACKGROUND: Disseminated tumor cells (DTCs) can be detected using ultrasensitive immunocytochemical assays and their presence in the bone marrow can predict the subsequent occurrence of overt metastasis formation and metastatic relapse. Using expression profiling on early stage primary breast tumors, low IRX2 expression was previously shown to be associated with the presence of DTCs in the bone marrow, suggesting a possible role of IRX2 in the early steps of metastasis formation. The purpose of this study is to gain insights into the significance of IRX2 protein function in the progression of breast cancer. METHODS: To assess the physiological relevance of IRX2 in breast cancer, we evaluated IRX2 expression in a large breast cancer cohort (n = 1992). Additionally, constitutive IRX2 over expression was established in BT-549 and Hs578T breast cancer cell lines. Subsequently we analyzed whether IRX2 overexpression effects chemokine secretion and cellular motility of these cells. RESULTS: Low IRX2 mRNA expression was found to correlate with high tumor grade, positive lymph node status, negative hormone receptor status, and basal type of primary breast tumors. Also in cell lines low IRX2 expression was associated with mainly basal breast cancer cell lines. The functional studies show that overexpression of the IRX2 transcription factor in basal cell lines suppressed secretion of the pro-metastatic chemokines and inhibited cellular motility but did not influence cell proliferation. CONCLUSION: Our results imply that the IRX2 transcription factor might represent a novel metastasis associated protein that acts as a negative regulator of cellular motility and as a repressor of chemokine expression. Loss of IRX2 expression could therefore contribute to early hematogenous dissemination of breast cancer by sustaining chemokine secretion and enabling mobilization of tumor cells.

Relevance of PTEN loss in brain metastasis formation in breast cancer patients.

INTRODUCTION: With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Therefore, our aim was to identify molecular markers associated with the development of brain metastases in breast cancer. METHODS: Patterns of chromosomal aberrations in primary breast tumors and brain metastases were compared with array-comparative genetic hybridization (CGH). The most significant region was further characterized in more detail by microsatellite and gene-expression analysis, and finally, the possible target gene was screened for mutations. RESULTS: The array CGH results showed that brain metastases, in general, display similar chromosomal aberrations as do primary tumors, but with a notably higher frequency. Statistically significant differences were found at nine different chromosomal loci, with a gain and amplification of EGFR (7p11.2) and a loss of 10q22.3-qter being among the most significant aberrations in brain metastases (P < 0.01; false discovery rate (fdr) < 0.04). Allelic imbalance (AI) patterns at 10q were further verified in 77 unmatched primary tumors and 21 brain metastases. AI at PTEN loci was found significantly more often in brain metastases (52%) and primary tumors with a brain relapse (59%) compared with primary tumors from patients without relapse (18%; P = 0.003) or relapse other than brain tumors (12%; P = 0.006). Loss of PTEN was especially frequent in HER2-negative brain metastases (64%). Furthermore, PTEN mRNA expression was significantly downregulated in brain metastases compared with primary tumors, and PTEN mutations were frequently found in brain metastases. CONCLUSIONS: These results demonstrate that brain metastases often show very complex genomic-aberration patterns, suggesting a potential role of PTEN and EGFR in brain metastasis formation.

MMP7 is a target of the tumour-associated antigen EpCAM.

Epithelial cell adhesion molecule (EpCAM) is a single-transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP7) as a target gene of EpCAM signalling viaEpICD nuclear translocation. EpCAM and MMP7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP7 is a novel target of EpCAM signalling.

A distinct subset of glioma cell lines with stem cell-like properties reflects the transcriptional phenotype of glioblastomas and overexpresses CXCR4 as therapeutic target.

Glioblastomas contain stem-like cells that can be maintained in vitro using specific serum-free conditions. We investigated whether glioblastoma stem-like (GS) cell lines preserve the expression phenotype of human glioblastomas more closely than conventional glioma cell lines. Expression profiling revealed that a distinct subset of GS lines, which displayed a full stem-like phenotype (GSf), mirrored the expression signature of glioblastomas more closely than either other GS lines or cell lines grown in serum. GSf lines are highly tumorigenic and invasive in vivo, express CD133, grow spherically in vitro, are multipotent and display a Proneural gene expression signature, thus recapitulating key functional and transcriptional aspects of human glioblastomas. In contrast, GS lines with a restricted stem-like phenotype exhibited expression signatures more similar to conventional cell lines than to original patient tumors, suggesting that the transcriptional resemblance between GS lines and tumors is associated with different degrees of "stemness". Among markers overexpressed in patient tumors and GSf lines, we identified CXCR4 as a potential therapeutic target. GSf lines contained a minor population of CXCR4(hi) cells, a subfraction of which coexpressed CD133 and was expandable by hypoxia, whereas conventional cell lines contained only CXCR4(lo) cells. Convection-enhanced local treatment with AMD3100, a specific CXCR4 antagonist, inhibited the highly invasive growth of GS xenografts in vivo and cell migration in vitro. We thus demonstrate the utility of GSf lines in testing therapeutic agents and validate CXCR4 as a target to block the growth of invasive tumor-initiating glioma stem cells in vivo.

Inhibition of glioblastoma growth in a highly invasive nude mouse model can be achieved by targeting epidermal growth factor receptor but not vascular endothelial growth factor receptor-2.

PURPOSE: Major shortcomings of traditional mouse models based on xenografted human glioblastoma cell lines are that tumor cells do not invade and that genetic alterations, such as amplification of the epidermal growth factor receptor (EGFR) gene, are not maintained. Such models are thus of limited value for preclinical studies. We established a highly invasive model to evaluate the effect of antibodies against EGFR (cetuximab) and vascular endothelial growth factor receptor-2 (antibody DC101). EXPERIMENTAL DESIGN: After short-term culture, glioblastoma spheroids were implanted into the brains of nude mice. Animals were treated either i.c. with cetuximab or i.p. with DC101. Tumor burden was determined histologically using image analysis of 36 different landmark points on serial brain sections. RESULTS: Invasive xenografts were obtained from nine different glioblastomas. Three of seven cases treated with cetuximab responded with significant tumor growth inhibition, whereas four did not. All responsive tumors were derived from glioblastomas exhibiting EGFR amplification and expression of the truncated EGFRvIII variant, which were maintained in the xenografts. All nonresponsive tumors lacked EGFR amplification and EGFRvIII expression. The proportion of apoptotic cells was increased, whereas proliferation and invasion were decreased in responsive tumors. None of four xenograft cases treated with DC101 responded to treatment, and the diffusely invading tumors grew independent of angiogenesis. CONCLUSIONS: Inhibition of glioblastoma growth and invasion can be achieved using i.c. delivery of an anti-EGFR antibody, but tumor response depends on the presence of amplified and/or mutated EGFR. Antiangiogenic treatment with DC101 is not effective against diffusely invading tumors.

Early placenta insulin-like growth factor (pro-EPIL) is overexpressed and secreted by c-erbB-2-positive cells with high invasion potential.

By differential-display PCR a subclone of the SKBR3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of the INSL-4 gene. This new member of the insulin-like growth factor family encodes for a peptide, designated early placenta insulin-like (EPIL), being expressed in the so-called "invasive" phase of the placental development. Immunohistochemistry on tissue microarrays revealed a heterogeneous expression of EPIL in breast cancer tissue and no expression in the surrounding stroma cells. A coexpression of pro-EPIL and c-erbB-2 could be observed predominantly in cell clusters at the infiltrating edge of the tumor. Our results give new suggestions for the presence of a signaling network of receptor tyrosine kinases underlying breast cancer invasion and metastasis.

Suppression of early hematogenous dissemination of human breast cancer cells to bone marrow by retinoic Acid-induced 2.

UNLABELLED: Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.

An activated 5' cryptic splice site in the human ALG3 gene generates a premature termination codon insensitive to nonsense-mediated mRNA decay in a new case of congenital disorder of glycosylation type Id (CDG-Id).

A defect of the dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl mannosyltransferase encoded by the ALG3 gene (alias NOT56L) causes congenital disorder of glycosylation type Id (CDG-Id). In this work, a new mutation in the ALG3 gene causing atypical splicing is described with characterization of expression levels and transcript stabilities of the different splice products. A silent mutation in exon 1 of the ALG3 gene (c.165C

Loss of CADM1 expression is associated with poor prognosis and brain metastasis in breast cancer patients.

Breast cancer brain metastases (BCBM) are detected with increasing incidence. In order to detect potential genes involved in BCBM, we first screened for genes down-regulated by methylation in cell lines with site-specific metastatic ability. The expression of five genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which were also found down-regulated in gene expression profiling analyses of BCBM tissue samples, was verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. A higher incidence of CADM1 methylation, correlating with lower expression levels, was found in BCBM as compared to primary BC. Loss of CADM1 protein expression was detected most commonly among BCBM samples as well as among primary tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in four large independent breast cancer cohorts (n=2136). Loss of CADM1 protein expression was associated with disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between loss of CADM1 and shorter survival. In multivariate analyses, survival was significantly shorter among patients with CADM1-negative tumors. Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients.

Quantitative high-resolution genomic analysis of single cancer cells.

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

TOB1 is regulated by EGF-dependent HER2 and EGFR signaling, is highly phosphorylated, and indicates poor prognosis in node-negative breast cancer.

Clinical and animal studies have shown that coexpression of the receptor tyrosine kinases HER2 and epidermal growth factor (EGF) receptor (EGFR) indicates a highly metastatic phenotype of breast cancer. In a cellular model of this phenotype using differential gene expression analysis, we identified TOB1 to be up-regulated depending on EGF stimulation and transduction through phosphorylation of HER2 tyrosine 1248. mRNA expression analysis of breast cancers from a cohort of node-negative patients showed significantly shortened distant metastasis-free survival for patients with high TOB1 expression. In subsequent tissue microarray studies of 725 clinical samples, high HER2 and EGF protein levels were significantly correlated with TOB1 expression in breast cancer, whereas EGFR and EGF levels correlated with TOB1 phosphorylation. We did not observe a correlation between TOB1 expression and cyclin D1, which was previously suggested to mediate the antiproliferative effect of unphosphorylated TOB1. A positive correlation of TOB1 phosphorylation status with proliferation marker Ki67 suggests that elevated TOB1 phosphorylation might abrogate the antiproliferative effect of TOB1 in breast cancer. This suggests a new regulatory role for TOB1 in cancer progression with particular significance in HER2- and/or EGFR-positive breast cancers.

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness.

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employed to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.

3D-extravasation model -- selection of highly motile and metastatic cancer cells.

Extravasation has been described as a rate-limiting step in the process of hematogeneous metastasis formation. Thereby, transendothelial migration of tumor cells consists of a complex series of events involving multiple cell-cell and cell-matrix interactions. 3D-extravasation assays are valuable tools for the identification of genes, which are the key players at switchboards of the intracellular signaling pathways. In consequence, the combination of 3D-modeling and whole genome expression analysis lead to unravel molecular parameters which descripe distinct clinical phenotypes of cancer and therefore, work as prognosticators, predictors of therapy and new therapy targets.