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

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

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

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

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