WAVE3, an actin-polymerization gene, is truncated and inactivated as a result of a constitutional t(1;13)(q21;q12) chromosome translocation in a patient with ganglioneuroblastoma

Journal of Biological Chemistry

Ranalli

et al. 2005

Self Cite

165

WAVE3 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. The precise role of WAVE3 in cell migration and its regulation, however, have not been elucidated. Here we show that endogenous WAVE3 was found to be concentrated in the lamellipodia at the leading edge of migrating MDA-MB-231 cells. Platelet-derived growth factor (PDGF) treatment induced lamellipodia formation as well as two-dimensional migration of cells in the wound-closure assay and chemotactic migration toward PDGF in three-dimensional migration chambers. Knockdown of WAVE3 expression by RNA interference prevented the PDGF-induced lamellipodia formation and cell migration. Treatment of cells with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), also abrogated the PDGF-induced lamellipodia formation and cell migration, suggesting that PI3K may be required for WAVE3 activity. WAVE3 and the PI3K regulatory subunit, p85, were found to interact in a yeast two-hybrid screen, which was confirmed through coimmunoprecipitation. The WAVE3-p85 interaction was mediated by the N-terminal region of WAVE3 and the C-terminal SH2 domain of p85. These results imply that the WAVE3-mediated migration in MDA-MB-231 cells via lamellipodia formation is activated downstream of PI3K and induced by PDGF. The findings of the WAVE3-p85 partnership also suggest a potential regulatory role for p85 in WAVE3-dependent actin-cytoskeleton reorganization and cell migration.

Section: Resultsmentioning

confidence: 79%

mentioning

confidence: 91%

WAVE3-mediated Cell Migration and Lamellipodia Formation Are Regulated Downstream of Phosphatidylinositol 3-Kinase

Journal of Biological Chemistry

Ranalli

et al. 2005

Self Cite

165

“…Primers for WAVE3 were as previously reported. 13 The antibodies used in this study were as follows: rabbit anti-human WAVE3/Scar antibodies from either Upstate Biotechnology (Charlottesville, VA) or from New England Peptide, Inc. (Gardner, MA); mouse anti-human PI3-kinase p85 obtained from BD Biosciences (San Diego, CA), and rabbit anti-human vascular endothelial growth factor (VEGF) from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies were obtained from Amersham Biosciences (GE Healthcare, Buckinghamshire, UK).…”

Section: Methodsmentioning

confidence: 99%

Down-Regulation of WAVE3, a Metastasis Promoter Gene, Inhibits Invasion and Metastasis of Breast Cancer Cells

The American Journal of Pathology

Safina

et al. 2007

Self Cite

108

187

The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogenactivated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominantnegative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis. The processes of cellular migration and invasion are critical for the ability of tumor cells to metastasize locally and to distant sites.1 These events are controlled by a complex interaction of genetic pathways that can be specific to different cell types and facilitated through disruption of phenotypes that restrict the motility of the cell type involved.2 These phenotypic changes occur on a background of genetic events that allow the cell to invade surrounding tissue as well as access the vasculature to facilitate relocation to distant organs. This latter process can only be achieved if the metastasizing cell can survive in the blood stream and re-establish in the new organ site. Although the matrix metalloproteinases are perhaps the best studied proteins facilitating tumor cell invasion, the factors regulating the actin cytoskeleton dynamics have recently emerged as critical contributors to the metastatic phenotype. 2-6Cell motility and invasion require highly coordinated regulation of actin dynamics within the cell. 7,8 The proteins that influence this process have been implicated in metastasis. [3][4][5][6]9,10 We have recently shown that WAVE3, a member of a WASP protein family controlling actin polymerization, has a profound effect on motility and invasion of breast cancer cells. The proteins of this family regulate actin polymerization through the recruitment of the Arp2/3 protein complex via a verprolin-cofilin-acidic domain at the C terminus. It is thought that the formation of the multimeric complex comprising the verprolin-cofilin-acidic domain, the actin monomer, and the Arp2/3 complex is a critical step in actin polymerization. 11,12 We have found that suppression of WAVE3 by small interfering RNA (siRNA) in breast cancer MDA-MB-231 cells dramatically reduces lamelli...

“…We analysed the 14 coding exons from a total of 58 breast tumours and nine cell lines using WAVEt DHPLC as described previously (Sossey-Alaoui et al, 2002b). We detected three different missense substitutions in four primary breast tumours (Table 1).…”

Section: Clca2 Gene Is Not Commonly Mutated In Breast Cancermentioning

confidence: 99%

“…The WAVE instrument (Transgenomic, NE, USA) was utilized to perform denaturing high-performance liquid chromatography (DHPLC) and search for mutations in CLCA2 as described elsewhere (Sossey-Alaoui et al, 2002b). Oligonucleotide primers specific to each exon of CLCA2 (Table 2) were used for the mutational screening of primary breast tumours and cell lines.…”

Section: Mutation Analysis Of Primary Tumours and Cell Linesmentioning

confidence: 99%

CLCA2 tumour suppressor gene in 1p31 is epigenetically regulated in breast cancer

Cowell

2004

Oncogene

Self Cite

The calcium-activated chloride channel gene family is clustered in the 1p31 region, which is frequently deleted in sporadic breast cancer. Recent studies have indicated the association of the second member of this gene family (CLCA2) with the development of breast cancer and metastasis. We have now shown the absence of expression of CLCA2 in several breast cancer tumours and cell lines, which confirms the results from other reports. When overexpressed in CLCA2-negative cell lines, their tumorigenicity and metastasis capability were significantly reduced, suggesting a tumour suppressor role for CLCA2 in breast cancer. The mechanisms behind the silencing of CLCA2 in breast cancer, however, have not been elucidated to date. Although we were able to identify CLCA2 mutations in breast cancers, somatic mutations are not the major cause of CLCA2 gene silencing. On the other hand, treatment of breast cancer CLCA2-negative cell lines with demethylating agents was able to restore CLCA2 expression, suggesting an epigenetic inactivation of this gene. Bisulphite-sequencing of the promoterassociated CpG island of the CLCA2 gene in breast tumours demonstrated that the absence of expression in these tumours was caused by hypermethylation of the promoter CpG island. In contrast, in breast cancer cell lines, tumours, and control cell lines that express CLCA2, a much lower level, and often absence, of methylation of the promoter were demonstrated. These findings demonstrate that CLCA2 is frequently inactivated in breast cancer by promoter region hypermethylation, which makes it an excellent candidate for the 1p31 breast cancer tumour suppressor gene.