CGM2, a Member of the Carcinoembryonic Antigen Gene Family is Down- Regulated in Colorectal Carcinomas

We have determined the precise chromosomal location, the exon structure, and the expression pattern of CGM2, a member of the carcinoembryonic antigen (CEA) gene family. CGM2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT/PCR) from the colon adenocarcinoma cell line, LS174T. A defective exon is missing from this cDNA clone, leading to a novel domain organization for the human CEA family with two immunoglobulin-like domains. The derived C-terminal domain predicts that the CGM2 protein is membrane-bound through a glycosyl phosphatidylinositol anchor. RT/PCR analyses identified CGM2 transcripts in mucinous ovarian and colonic adenocarcinomas as well as in adjacent colonic tissue, but not in other tumors including leukocytes from six chronic myeloid leukemia patients. Thus, unlike several other family members, CGM2 is not expressed in granulocytes but reveals a more CEA-like expression pattern. Northern blot analyses identified a 2.5-kilobase CGM2 mRNA that is strongly down-regulated in colonic adenocarcinomas compared with adjacent colonic mucosa, suggesting a possible tumor suppressor function. In addition, a 3.2- kilobase transcript was observed in a number of colon tumors that is not detectable in normal colonic tissue. This mRNA species could represent a tumor-specific CGM2 splice variant

Investigations on the Usefulness of CEACAMs as Potential Imaging Targets for Molecular Imaging Purposes

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well. In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.Germany. Bundesministerium für Bildung und Forschung (TOMCAT, grant number 01EZ0824

Functional interaction of SCAI with the SWI/SNF complex for transcription and tumor cell invasion.

We have recently characterized SCAI (Suppressor of Cancer Cell Invasion), a transcriptional modulator regulating cancer cell motility through suppression of MAL/SRF dependent gene transcription. We show here that SCAI is expressed in a wide range of normal human tissues and its expression is diminished in a large array of primary human breast cancer samples indicating that SCAI expression might be linked to the etiology of human cancer. To establish a functional link between SCAI and tumorigenesis we performed affinity columns to identify SCAI-interacting proteins. Our data show that SCAI interacts with the tumor suppressing SWI/SNF chromatin remodeling complex to promote changes in gene expression and the invasive capacities of human tumor cells. Moreover our data implicate a functional hierarchy between SCAI and BRM, since SCAI function is abrogated in the absence of BRM expression

LASP1 is a novel BCR-ABL substrate and a phosphorylation-dependent binding partner of CRKL in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by a genomic translocation generating a permanently active BCR-ABL oncogene with a complex pattern of atypically tyrosine-phosphorylated proteins that drive the malignant phenotype of CML. Recently, the LIM and SH3 domain protein 1 (LASP1) was identified as a component of a six gene signature that is strongly predictive for disease progression and relapse in CML patients. However, the underlying mechanisms why LASP1 expression correlates with dismal outcome remained unresolved. Here, we identified LASP1 as a novel and overexpressed direct substrate of BCR-ABL in CML. We demonstrate that LASP1 is specifically phosphorylated by BCR-ABL at tyrosine-171 in CML patients, which is abolished by tyrosine kinase inhibitor therapy. Further studies revealed that LASP1 phosphorylation results in an association with CRKL - another specific BCR-ABL substrate and bona fide biomarker for BCR-ABL activity. pLASP1-Y171 binds to non-phosphorylated CRKL at its SH2 domain. Accordingly, the BCR-ABL-mediated pathophysiological hyper-phosphorylation of LASP1 in CML disrupts normal regulation of CRKL and LASP1, which likely has implications on downstream BCR-ABL signaling. Collectively, our results suggest that LASP1 phosphorylation might serve as an additional candidate biomarker for assessment of BCR-ABL activity and provide a first step toward a molecular understanding of LASP1 function in CML

BRM, a core component of the SWI/SNF complex, associates with SCAI.

<p>(A) Coomassie stained gel of GST and GST-SCAI (aa 35–280) associated proteins using a mouse brain high salt extract as a source of proteins. (B) Subunit composition of the human SWI/SNF complex (modified from Roberts and Orkin, 2004). (C) SCAI coimmunoprecipitates with the ATPase BRM. HEK 293 cells were transfected with Myc-tagged BRM and full length SCAI, SCAI aa 460–606 (ΔN) and SCAI aa 1–212 (n-t) and subjected to immunoprecipitation using Flag-beads. Immunoprecipitates were analyzed by immunoblot using the indicated antibodies. (D) The N-terminus of BRM is required for SCAI interaction. HEK 293 cells were transfected with Flag/GFP-tagged SCAI and indicated HA-tagged BRM deletion mutants and subjected to immunoprecipitation using Flag-beads. Immunoprecipitates were analyzed by immunoblot using the indicated antibodies.</p

SCAI requires SWI/SNF to modulate SRF-dependent reporter gene activity.

<p>SW13 cells were transfected with the SRF reporter 3DA.Luc, pRLTK (Renilla luciferase) and indicated expression plasmids. Reporter gene activity was assessed 48 h after transfection. Statistical analysis of three independent experiments (+/− s.d.) is shown in (A). (B) HEK 293 cells were transfected with the SRF reporter 3DA.Luc, pRLTK and indicated expression plasmids. Reporter gene activity was measured 16 h after transfection. Statistical analysis of three independent experiments (+/− s.d.) is shown. (C) HEK 293 cells were transfected with siRNA specific to hBRM. 48 h later cells were transfected with the SRF reporter 3DA.Luc, pRLTK and an expression plasmid for SCAI wt and the reporter gene activity was measured 16 h later. Statistical analysis of three independent experiments (+/− s.d.) is shown. Representative immunoblots assessing the expression of SCAI/BRM constructs as well as endogenous BRM and HDAC2 as loading control is shown below the bar charts for each experiment. Please note that the Flag-antibody recognizes an unspecific band above 72 kDa in SW13 cells.</p

SCAI expression in human tumor samples.

<p>(A) Analysis of SCAI expression in human tissue using rat mAB 1H2 (Brandt et al 2009). After stripping, membranes were reprobed with anti-GAPDH mAb serving as a loading control. (B) Breast tissues were deparaffinized and probed with a rat anti-SCAI mAb and a secondary HRP-labeled goat anti-rat antibody. Staining was performed with DAB. To depict cells of epithelial origin, a consecutive tissue section was probed with a pan-specific cytokeratin antibody. (C) Western Blot analysis of SCAI, and RhoC protein expression in breast cancer specimen (n = 36) and normal tissue (N). MAPK served as a loading control. Specimen number and stage of disease according to the AJCC classification are given on top of the figure. (D and E) Relative levels of SCAI and RhoC protein expression in relation to stage of disease. Signal intensities were calculated after densitometric analysis of Western blots shown in (C) normalized to the MAPK signal. Expression levels of SCAI and RhoC in normal breast tissue (N) are given by the green dashed lines. No significant association between tumor stage and levels of protein expression were observed for SCAI whereas levels of RhoC expression were strongly correlated to the stage of disease with higher levels of RhoC in advanced breast cancer.</p

Silencing of the SWI/SNF core subunit BRM phenocopies SCAI mediated effects on invasive cell migration.

<p>(A) MDA-MB-435 cells were transfected with indicated siRNAs. After 48 h cells were analyzed for their invasive properties using a 3D matrix (matrigel). Representative images show confocal sections of invaded cells stained for F-actin (red) and DAPI (blue) at 20 µm distance to the transwell membrane. Three-dimensional reconstruction shows a side view of experiments with the location of invaded cells with respect to the transwell membrane (dashed line). A quantification of three independent experiments (+/−s.d.) is shown in (B) for MDA-MB-435 cells and in (C) for MDA-MB-231 cells. (D) MDA-MB-435 cells were processed for immunoblot analysis after 48 h of siRNA treatment and the abundance of BRM protein was assessed using the indicated antibodies.</p