Dirk Kemming

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 CXCR4hi cells, a subfraction of which coexpressed CD133 and was expandable by hypoxia, whereas conventional cell lines contained only CXCR4lo 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.

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

IntroductionWith 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.MethodsPatterns 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.ResultsThe 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.ConclusionsThese results demonstrate that brain metastases often show very complex genomic-aberration patterns, suggesting a potential role of PTEN and EGFR in brain metastasis formation.

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.

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.

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

BackgroundDisseminated 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.MethodsTo 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.ResultsLow 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.ConclusionOur 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.

Abstract 46: Loss of CADM1 expression is associated with poor prognosis and brain metastasis in breast cancer patients

Purpose: Breast cancer brain metastases (BCBM) are detected with increasing incidence. Therefore, identification of genes involved in BCBM formation is of great interest. Eperimental Design: 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, were verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. Results: A higher incidence of CADM1 methylation, correlating with the expression levels, was found in BCBM as compared to non-matched primary BC. Similarly, loss of CADM1 protein expression was detected more commonly among BCBM samples as well as among primary breast tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in two publicly available data sets (n=418) and in two large independent primary breast cancer TMA tumor cohorts (n=1718). The immunohistochemical analyses revealed an association between loss of CADM1 protein expression and risk factors such as disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between CADM1 and shorter survival. In multivariate analyses, survival was significantly longer for patients with CADM1-positive primary tumors for both TMA data sets (p= 0.04 and p=0.01). Conclusions: Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients. Citation Format: Harriet Wikman, Laura Westphal, Felicitas Schmid, Markus Glatzel, Jakob Matschke, Manfred Westphal, Sirkku Pollari, Kristiina Iljin, Luigi Terracciani, Heini Huhtala, Anne Kallioniemi, Guido Sauter, Volkmar Muller, Isabell Witzel, Katrin Lamszus, Dirk Kemming, Klaus Pantel. Loss of CADM1 expression is associated with poor prognosis and brain metastasis in breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 46. doi:10.1158/1538-7445.AM2014-46

Abstract 117: The transcription factor iroquois homeobox 2 (IRX2) is a candidate metastasis suppressor that acts as regulator of cellular motility and chemokine expression in breast cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The onset of the metastatic cascade -the main cause of cancer related death- is the dissemination of single tumor cells into distant organs. The presence of disseminated tumor cells is an independent prognostic factor for the risk of recurrence and overall survival of cancer patients in different cancer entities. We found that low expression of the transcription factor iroquois homeobox 2 (IRX2) in primary breast and lung tumors is associated with the presence of disseminated tumor cells in the bone marrow. Accordingly, IRX2 represents a candidate metastasis suppressor gene. The purpose of this study is to gain insights into the significance of IRX2 expression in the progression of breast cancer. To assess its physiological relevance in breast cancer, we evaluated IRX2 expression in large publically available breast cancer cohorts. Additionally, we established two breast cancer cell lines with ectopic IRX2 protein expression and subjected those cell lines to a variety of functional assays. IRX2 mRNA expression was found to correlate with tumor grading and estrogen receptor status in primary breast tumors. Western blot analysis of IRX2 protein expression in breast cancer cells lines revealed that luminal and HER2 positive cell lines expressed IRX2, whereas cell lines belonging to the basal molecular subtype did not express the IRX2 transcription factor. SAM-analysis in combination with gene ontology analysis revealed that 6 out of the 40 genes, whose expression is most strongly correlated with IRX2 expression belong to the chemokine signaling pathway. Coincidently, in BT-549 and Hs578T with ectopic IRX2 expression we found markedly reduced levels of CCL5 and CXCL10 mRNA expression. A gene reporter assay also revealed a decreased trans-activation of the CCL5 proximal promoter in BT-549 cells in the presence of ectopic IRX2 expression. Furthermore, we could detect significantly lower CCL5 protein levels in cell culture supernatants obtained from this cell line. When used as chemoattractant, conditioned cell culture medium from this cell line also diminished migration of parental MDA-MD-231 and BT-549 breast cancer cells. BT-549 cells with IRX2 over expressing also showed clearly reduced motility in both wound healing as well as Boyden chamber assays. Our results imply that the IRX2 transcription factor might represent a novel metastasis suppressor protein that also acts a repressor of CCL5 gene transcription. Loss of IRX2 expression could contribute in particular to early hematogenous dissemination of breast and lung cancer cells by sustaining CCL5 secretion and enabling autocrine stimulated cell motility. Citation Format: Stefan Werner, Hauke Stamm, Mutiha Pandjaitan, Dirk Kemming, Klaus Pantel, Harriet Wikman. The transcription factor iroquois homeobox 2 (IRX2) is a candidate metastasis suppressor that acts as regulator of cellular motility and chemokine expression in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2014-117

Abstract 3099: Role of the transcription factor iroquois homeobox 2 (IRX2)in breast cancer progression and metastasis

The onset of the metastatic cascade -the main cause of cancer-related death- is the dissemination of single tumor cells into distant organs. The presence of disseminated tumor cells (DTC) in the bone marrow (BM) of cancer patients is an independent prognostic factor for the occurrence of metastatic relapse in breast cancer and other epithelial tumors. Our current results suggest that the transcription factor IRX2 might represent a novel tumor suppressor gene which might in particular contribute to early hematogenous dissemination. The purpose of this study is to gain insights into the role of the transcription factor IRX2 during breast cancer progression and metastasis. In order to identify genes that could account for early hematogenous dissemination we compared expression profiles of primary breast tumors with or without detectable DTC in the BM. We further analyzed the transcript expression by quantitative RT-PCR analysis in an independent set of primary breast tumor samples. With the purpose to provide further evidence about the clinical relevance and the biological function of the IRX2 transcription factor we also performed different computer-based analyses using a large publicly available patient data set. Potential transcriptional targets of the IRX2 protein were moreover validated in IRX2-knock-down model systems and by gene-reporter-assays. We were able to discover that low IRX2 expression in primary breast tumors is associated with BM status. In addition, we could show that low IRX2 expression was associated with decreased long time survival of breast cancer patients. Furthermore, by using stringent thresholds in a bioinformatical approach we could identify 22 potential transcriptional targets of the IRX2 transcription factor. Most interestingly, we found that LRIG1 might be a crucial target of the IRX2 gene product. We could reveal a positive correlation between IRX2 and LRIG1 expression in primary breast tumors and we could identify a IRX2 binding site in the proximal LRIG1-promotor which is effectively transactivated by the IRX2 protein in vitro. Additionally we could show that shRNA-mediated knock-down of IRX2 expression in cultured breast cancer cell lines is attended by decreased LRIG1 expression. Our present results imply that the IRX2 gene product might represent a novel tumor suppressor protein. Loss of IRX2 expression could contribute in particular to early hematogenous dissemination. In addition, our findings show a direct transcriptional regulation of LRIG1 by the IRX2 transcription factor. Since LRIG1 is an important negative regulator of receptor tyrosine kinases, we assume that decreased IRX2 expression may led to a more aggressive phenotype by sustaining the over expression of receptor tyrosine kinases, like EGFR and ERBB2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3099. doi:10.1158/1538-7445.AM2011-3099