The International Journal of Biochemistry & Cell Biology
The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell–matrix adhesion
Introduction
The gene SASH1 (SAM- and SH3-domain containing 1) is a candidate tumor suppressor gene in breast cancer located at chromosome 6q24.3 (Zeller et al., 2003). Loss of heterozygosity of this region occurred in 30% of primary breast cancers and was associated with poor survival and increase in tumor size. Correspondingly, SASH1 expression was reduced in the majority of breast tumors (Zeller et al., 2003). A multi-tissue tumor suppressor gene is suspected at chromosome 6q24, since this region is lost in many malignancies (Alcock et al., 2003, Barghorn et al., 2001, Bell et al., 1997, Lemeta et al., 2006, Zhang et al., 1997). We showed recently that SASH1 expression on mRNA level, as well as on protein level, was strongly reduced in primary colon cancer and liver metastases (Zhang et al., 1997, Rimkus et al., 2006). Low expression of SASH1 was significantly associated with metastasis formation and reduced survival, and constituted an independent negative predictive factor for prognosis.
SASH1 is a member of the evolutionarily conserved SLY-family (Beer et al., 2001) that comprises SASH1, SLY1 (SH3-domain protein containing expressed in lymphocytes), and SLY2 (or: SAMSN1/HACS1/NASH1) (Claudio et al., 2001, Uchida et al., 2001, Zhu et al., 2004). All three proteins share a central conserved region with a bipartite nuclear localization signal (NLS), and two protein–protein interaction motifs, an SH3-domain (Src homology domain 3), and a SAM-domain (sterile alpha motif). SH3 domains were first described in the non-receptor tyrosine kinase Src (Koch et al., 1991), they bind to proline-rich protein motifs (Claudio et al., 2001, Pawson, 1995), whereas SAM domains have the capacity to form homo- or heterophilic protein–protein complexes (Kim et al., 2001, Kim et al., 2002). Both domains are frequently found in signal adapter proteins or scaffolding factors. In contrast to SLY1 and SLY2, SASH1 has long C-terminal and N-terminal sequence extensions and a second SAM-domain at the C-terminus of the protein. SLY1 is involved in regulation of the adaptive immune system (Beer et al., 2001, Beer et al., 2005). SLY2 was reported to be differentially expressed in malignant haematopoietic cells, but also in colorectal tumors (Claudio et al., 2001, Uchida et al., 2001, Williams et al., 2003). The expression of SLY1 and SLY2 is mainly restricted to hematopoietic cells, whereas SASH1 is widely expressed in normal human tissue, with the exception of lymphocytes and dendritic cells (Rimkus et al., 2006, Zeller et al., 2003). However, the biological function of SASH1 and its involvement in malignant transformation remain largely unknown. We have employed cell biological and biochemical methods to begin to unravel the biological role of SASH1. We report here a dual localization of SASH1, in the nucleus, as well as in the cytoplasm, with an enrichment in cortical, highly dynamic and F-actin rich membrane protrusions. The central region of SASH1, which is conserved among the SLY-family, was necessary and sufficient for the recruitment to lamellipodia. Moreover, we establish the oncoprotein cortactin as the first known binding partner of SASH1. Lastly, our data indicate that SASH1 is critically involved in cell migration and cell–matrix adhesion, processes that are important for invasion and metastasis of tumor cells.
Section snippets
Plasmid construction
Expression plasmid pBUDCE4.1-SASH1 (Invitrogen, San Diego, CA, USA) was a gift from Dr. Constanze Zeller (Max Delbrück Centrum, Berlin). Deletion constructs were generated by using an endogenous EcoRV site (1749 bp). The plasmid was digested with EcoRV and NotI, and treated with DNA polymerase I (Klenow fragment). Re-ligation lead to the deletion of the 5′-domain (SASH1-ΔNter). An oligonucleotide linker was introduced 3′ of the SASH1 ORF between the BglII and the BstBI sites to create a XhoI and
SASH1 localizes to the nucleus and membrane-proximal areas
A previously described antiserum (pAb #1540), raised against N-terminal epitopes conserved between human and mouse SASH1 (Rimkus et al., 2006), was used to analyze the intracellular distribution of endogenous SASH1. As described earlier, SASH1 is frequently down-regulated in more advanced tumor stages, but still expressed in early stages. Therefore, we have analyzed tissue cryosections from human stage II colon carcinoma with the antiserum #1540, and have detected SASH1 expression mainly in
Discussion
SASH1 is a candidate tumor suppressor from the SLY-family of signal adapter proteins. Decreased SASH1 expression has been observed in breast and colon cancer, it is correlated to reduced survival, aggressive tumor growth and metastasis formation (Rimkus et al., 2006, Zeller et al., 2003). However, the physiological interaction partners of SASH1, and its function in tumor biology remained elusive. SASH1 contains several protein–protein interaction motifs: one SH3-domain, and two SAM-domains, as
Conclusions
SASH1 localizes to the nucleus and to the cytosol, and is enriched at the cell periphery. Down-regulation of SASH1 expression leads to decreased cell adhesion to the extracellular matrix, whereas increased SASH1 expression stimulates actin polymerization, cell–matrix adhesion and inhibits cell migration. Thus, our findings offer a first insight into the biological role of SASH1 in cancer biology. Reduced expression of SASH1, which is clinically correlated to aggressive tumor growth and
Acknowledgements
The authors thank Anja Conrad, Widya Johannes, and Carmen Marthen for excellent technical assistance. The work was supported by Deutsche Forschungsgemeinschaft (SFB 456). We thank Dr. Constanze Zeller for the V5-SASH1 expression plasmid, and Dr. Michael Sixt for the help with timelapse-video microscopy.
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