nWASP and WAVE2 in breast cancer cell invasion

A study has observed three-dimensional invasion of MDA-MB-231 cells that infiltrate through membrane pores in response to platelet-derived growth factor signals in a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent manner, shown by using PI3K inhibitors.

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Immunofluorescence staining of these invading cells showed that WAVE2 and nWASP were colocalized with intensive F-actin accumulations at the site of infiltration. Further, diminution of WAVE2 and nWASP using siRNA techniques demonstrated a significant reduction in cell invasion, and accumulation of F-actin was also depleted. 

These findings suggest that nWASP and WAVE2 have a role in the three-dimension invasion of MDA-MB-231 through small gaps in response to platelet-derived growth factor through reorgani­zation of actin filaments within the cell.83

WAVE3 and breast cancer

Sossey-Alaoui et al identified WAVE3 as a candidate tumor suppressor gene on analysis of a patient with ganglioneuro­blastoma in whom a chromosomal translocation causing a truncation of WAVE3 was seen.

It was proposed that WAVE3 is important in cell differentiation and motility, considering it is part of the WASP/WAVE family that is involved in reor­ganization of the actin cytoskeleton, and that loss of WAVE3 activity seemed to be related to tumor formation in some forms of neuroblastoma.84

Since this initial proposal, further work by Sossey-Alaoui et al has focused on elucidating the role of WAVE3 in cell motility and cancer metastasis.

It has been indicated that WAVE3 expression correlates with breast cancer progression, as immunohistochemical staining has shown significantly higher levels of WAVE3 in grade III tumors compared with low or no WAVE3 pro­tein levels in normal breast or grade I tumors from human patients.85

A separate study has also identified WAVE3 activ­ity as an indicator for some characteristics of breast cancer. WAVE3 expression levels were found to correlate with the invasiveness of MDA-MB-231 and BT-549 breast cancer cell lines compared with the less aggressive MCF7, T47D, and SKBr3 cell lines, shown in a Matrigel invasion assay. Immunohistochemistry and quantitative RT-PCR were also used to detect WAVE3 activity in tumor and blood samples, respectively, taken from breast cancer patients.

The findings demonstrated that WAVE3 levels correlate with poor patient outcome in terms of incidence of metastatic disease, aggres­siveness of subtype, and patient survival.86

Transient suppression of WAVE3 using siRNA techniques has been shown to impair the motility and invasiveness of MDA-MB-231 human breast cancer cells on Matrigel migra­tion and gap closure assays.87 The same researchers also created a stable knockdown of WAVE3 protein expression in MDA-MB-231 cells using shRNA techniques.

Suppres­sion of WAVE3 activity was found to reduce the invasive ability of these cells by approximately fivefold on a Matrigel invasion assay. Further, knockdown of WAVE3 appeared to have an inhibitory effect on the metastatic potential of MDA-MB-231 cells.

This was shown by the significantly reduced number of lung surface metastases seen after injection of the knockdown cells into the tail veins of SCID mice when compared with control cells.

Finally, stable WAVE3 knockdown breast cancer cells were used in an orthotopic xenograft model where they were implanted into the mam­mary fat pads of female SCID mice. WAVE3-deficient cells were significantly less tumorigenic, with the tumor incidence reduced by 60%–80% compared with controls, and tumors that did develop grew more slowly and were less angiogenic.85

A more recent study has implicated WAVE3 in transform­ing growth factor-β-mediated epithelial to mesenchymal transition and metastasis of breast cancer cells. The highly metastatic murine triple-negative breast cancer cell model, 4T1, showed reduced levels of invasion in a Boyden chamber Matrigel assay when WAVE3 was inactivated through RNA silencing methods.

Further, WAVE3 deficiency reduced the outgrowth of 4T1 organoids in three-dimensional cultures measured after 8 days and also reduced the growth and metastasis of triple-negative 4T1 breast cancer cells engrafted into mammary glands or injected in the tails, respectively, of BALB/c mice.88

These studies further highlight the role of WAVE3 in the tumorigenic behavior of breast cancer cells and suggest that measures capable of inactivating WAVE3 may be able to act as a therapy targeted to breast cancer metastasis.

Interestingly, a study carried out by another group has disputed the finding by Sossey-Alaoui that WAVE3 is involved in the invasion of breast cancer cells in a Matrigel assay.90

Using RNA interference methods, Spence et al demonstrated that depletion of WAVE3 in MDA-MB-231 cells results in slower migration and larger and less dynamic lamellipodia when observed on a two-dimensional matrix.89

However, when invasion into Matrigel was observed in a three-dimensional environment, no difference was seen between WAVE3-deficient cells and controls, so the group suggested that WAVE3 does not have a critical role in driving invasiveness of MDA-MB-231 cells in three-dimensional invasion assays.

However, their results did show a preference of WAVE3-deficient cells to invade in clusters of cells, which was specu­lated to indicate a loss of plasticity in WAVE3-deficient cells, thereby making them less able to change direction and break away from such clusters.

No difference was found between WAVE3-deficient cells in MV3 and A375 metastatic mela­noma cells.89 This study disagrees with the results of a similar study by Sossey-Alaoui et al, who reported that WAVE3 is important for breast cancer cell invasion through a Matrigel matrix.85

It has been suggested that the variation in outcomes observed in these similar studies could be due to differences in experimental procedures or perhaps the unknown use of a different cell line other than the intended MDA-MB-231 cells.89,90

Nonetheless, the involvement of WAVE3 in invasion and regulation of motility, and its metastatic potential has been demonstrated in numerous studies using several different cancer cell lines.68,70,85–88,91

It is clear that WAVE3 is a critical player in cancer metastasis and a biomarker for progression of breast cancer.

More insights into the role of WAVE3 in the development of cancer are detailed in a comprehensive review by Sossey-Alaoui, who has been involved in many of the advances in our understanding of the role of WAVE3.90