10810

WASF3

WAS protein family, member 3;WASF3;WAS protein family, member 3

WASP family Verprolin-homologous protein 3|protein WAVE-3|verprolin homology domain-containing protein 3|wiskott-Aldrich syndrome protein family member 3

13q12

protein-coding

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease comprised of at least five genetically distinct subtypes, which together form the second leading cause of cancer death in women in the United States. Within BC subtypes, those classified as Triple Negative BCs (TNBCs) exhibit dismal survival rates due to their propensity to develop distant metastases. We have identified the WAVE3 protein, which is a critical regulator of actin cytoskeleton dynamics that are required for the motility and invasion of cancer cells through its activation of the Arp2/3 complex, as a key regulator of the different steps of the invasion-metastasis cascade in BC, especially in the more aggressive TNBCs. Our published studies have also shown that elevated expression levels of WAVE3 in the TNBC cell lines directly contribute to their increased invasion and metastasis potentials both in vitro and in vivo in murine models of BC metastasis. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we utilized both immunohistochemistry (IHC) of primary human BC tumors as well as quantitative real-time RT-PCR of WAVE3 in the peripheral blood of BC patients to clearly establish that WAVE3 is a predictive marker of overall BC patients' survival. High levels of WAVE3 were predictive for reduced distant recurrence-free survival as well as for decreased disease-specific mortality. Our analysis of WAVE3 expression levels in the peripheral blood of BC patients showed that WAVE3 is highly expressed in the blood of patients who developed metastatic breast cancer compared to those who did not. WAVE3 expression was also highly upregulated in the blood of BC patients with the more aggressive TNBC subtype. CONCLUSIONS: Together, these findings establish WAVE3 as a novel marker for increased risk of breast-cancer-specific mortality and for the metastatic potential of the TNBCs, and also identify WAVE3 as an attractive therapeutic target for the treatment of metastatic BC.

Breast cancer is the second leading cause of cancer death in women in the United States. metastasis accounts for the death of ~90 % of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that play essential roles in regulating cell morphology, actin polymerization, cytoskeleton remodeling, cell motility, and invasion. Accordingly, we demonstrated previously that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human breast cancers. Herein, we show that transforming growth factor-beta (TGF-beta) selectively and robustly induced the expression of WAVE3 in metastatic breast cancer cells, but not in their nonmetastatic counterparts. Moreover, the induction of WAVE3 expression in human and mouse triple-negative breast cancer cells (TNBCs) by TGF-beta likely reflects its coupling to microRNA expression via a Smad2- and beta3 integrin-dependent mechanism. We further demonstrate the requirement for WAVE3 expression in mediating the initiation of epithelial-mesenchymal transition (EMT) programs stimulated by TGF-beta. Indeed, stable depletion of WAVE3 expression in human TNBC cells prevented TGF-beta from inducing EMT programs and from stimulating the proliferation, migration, and the formation of lamellipodia in metastatic TNBC cells. Lastly, we observed WAVE3 deficiency to abrogate the outgrowth of TNBC cell organoids in 3-dimensional organotypic cultures as well as to decrease the growth and metastasis of 4T1 tumors produced in syngeneic Balb/C mice. Indeed, WAVE3 deficiency significantly reduced the presence of sarcomatoid morphologies indicative of EMT phenotypes in pulmonary TNBC tumors as compared to those detected in their parental counterparts. Collectively, these findings indicate the necessity for WAVE3 expression and activity during EMT programs stimulated by TGF-beta; they also suggest that measures capable of inactivating WAVE3 may play a role in alleviating metastasis stimulated by TGF-beta.

MicroRNAs are small non-coding RNAs that are directly involved in the regulation of gene expression by either translational repression or degradation of target mRNAs. Because of the high level of conservation of the target motifs, known as seed sequences, within the 3'-untranslated regions, a single microRNA can regulate numerous target genes simultaneously, making this class of RNAs a powerful regulator of gene expression. The miR200 family of microRNAs has recently been shown to regulate the process of epithelial to mesenchymal transition during tumor progression and metastasis. Here, we report that the expression of WAVE3, an actin cytoskeleton remodeling and metastasis promoter protein, is regulated by miR200 microRNAs. We show a clear inverse correlation between expression levels of WAVE3 and miR200 microRNAs in invasive versus non-invasive cancer cells. miR200 directly targets the 3'-untranslated regions of the WAVE3 mRNA and inhibits its expression. The miR200-mediated down-regulation of WAVE3 results in a significant reduction in the invasive phenotype of cancer cells, which is specific to the loss of WAVE3 expression. Re-expression of a miR200-resistant WAVE3 reverses miR200-mediated inhibition of cancer cell invasion. Loss of WAVE3 expression downstream of miR200 also results in a dramatic change in cell morphology resembling that of a mesenchymal to epithelial transition. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties and morphology of cancer cells associated with their metastatic potential.

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade.

Inactivation of HSP90 and HSP70 leads to loss of invasion in a variety of cancer cell types, presumably as a result of destabilization of, as yet, undefined clients of these molecular chaperones that influence this phenotype. The WASF3 gene has been shown to be up-regulated in high-grade tumors and its down-regulation leads to loss of invasion and metastasis. WASF3 phosphorylation by ABL kinase is essential for its ability to regulate invasion. Mass spectroscopy analysis now shows that HSP90 is present in the WASF3 immunocomplex from prostate cancer cells. Inactivation of HSP90 in these and other cell types does not affect WASF3 stability but prevents its phosphoactivation as a result of destabilization of ABL. HSP70 was also found in the WASF3 immunocomplex and inactivation of HSP70 results in destabilization of WASF3 through proteasome degradation. Knockdown of WASF3, HSP90, and HSP70 individually, all lead to loss of invasion but as knockdown of WASF3 in the presence of robust expression of HSP90/70 has the same effect, it seems that the influence these chaperone proteins have on invasion is mediated, at least in part, by their control over the critical invasion promoting capacity of the WASF3 protein. Overexpression of HSP70 in WASF3 null cells does not enhance invasion. These observations suggest that targeting HSP90/70 may have efficacy in reducing cancer cell invasion.