Increased expression levels of WAVE3 are associated with the progression and metastasis of triple negative breast cancer

Abstract

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.

Figure 1. WAVE3 expression levels positively correlate with the aggressiveness of breast cancer cell lines.

(A) Western bolt analysis of protein lysates isolated from the indicated BC cell lines using WAVE3 antibody. Actin was used as a loading control. (B) Representative micrographs of Matrigel invasion of the indicated BC cell lines and quantification of the Matrigel invasion (C). The results are shown as the mean±s. d. of at least 3 independent assays.

Figure 2. Loss of WAVE3 attenuates the aggressiveness of the BC cells of Triple-negative subtype.

(A) Quantitative Real-time RT-PCR of WAVE3 in the indicated BC cell lines after transient transfection with either a control siRNA or WAVE3-specific siRNA. (B) Western blot analysis of protein lysates following the same treatment as in (A). Actin was used a loading control and WAVE2 was used a negative control. (C and D) Matrigel invasion assay after the indicated treatments. Representative micrographs are shown in (C), and the quantitation is shown in (D). The results are shown as the mean±s. d. of at least 3 independent assays.

Figure 3. WAVE3 protein staining levels are increased in the high grade BC.

Representative WAVE3 IHC micrographs with matching hematoxylin & eosin of BC tumors. (A & B) mSBR grade 1 with negative staining and (C &D) mSBR grade 3 with diffusely strongly positive (score 300) staining. WAVE3 staining is shown as brown color in panels B and D. The final WAVE3 score is determined as product of the staining intensity (0, 1, 2 or 3) times the percent of tumor cells showing WAVE3 staining (0% to 100%). Therefore the final WAVE3 score can vary from 0: no cells are being satined for WAVE3, to 300, where 100% of tumor cells show strong WAVE3 staining. Insert figure in panel D shows cytoplasmic expression of WAVE3 (100x).

Figure 4. WAVE3 protein staining levels are positively correlated with reduced distant recurrence free survival and with decreased disease specific survival.

Kaplan-Meier analysis of (A) Overall survival, (B) Distant recurrence free survival and (C) Disease specific survival. The WAVE3 score, represented as the mean of WAVE3 staining level, was dichotomized as positive or negative to determine the relative contribution of the WAVE3 score to each of the three disease outcome parameters, respectively. The midpoint of the difference between the 75% CI for the WAVE3 score of patients with overall survival, no distant recurrence or breast cancer related mortality and the 25% CI was (212) was used.

Figure 5. WAVE3 mRNA is highly expressed in the peripheral blood of patients with metastatic breast cancer.

(A) Semi-quantitative RT-PCR from total RNA extracted from a mix of 5 million cells of EBV-lin (a PBMC) and MDA-MB-231 (a highly metastatic breast cancer cell line), at the indicated ratios. MDA-MB-231 cells and EBV-Lin cells were used alone as a positive and negative controls, respectively. WAVE3 mRNA could be amplified from the positive control cells (MDA-MB-231), but not form from the white blood cells (EBV-Lin). WAVE3 mRNA could also be amplified from as few as 1 cancer cell in a million blood cells. GAPDH was used as an internal control for the integrity of the RNA and as equal loading control. (B) Semi-quantitative RT-PCR and (C) quantitative real-time RT-PCR analyses of WAVE3 mRNA expression levels in the blood of 10 patients with metastatic BC and 10 healthy controls. MCF10A was used as a control. The graphs were plotted in a logarithmic scale with the average fold change to MCF10A ± s. d. is shown under the respective bar. GAPDH was used an internal normalization control. The results are shown as the mean±s. d. of at least 3 independent assays.

Figure 6. WAVE3 mRNA expression levels in the blood of BC patients correlate positively with the aggressiveness of the primary tumor.

WAVE3 expression levels were analyzed by quantitative real-time RT-PCR and plotted against the nuclear grade (A) and the SBR grade (B) of the primary tumor. WAVE3 RT-PCR values were normalized to GAPDH and plotted as the fold change to MCF10A. The results are shown as the mean±s. d. of at least 3 independent assays.

Figure 7. WAVE3 mRNA expression levels in the blood of BC patients correlate positively with the aggressive TNBC subtype.

WAVE3 expression levels were analyzed by quantitative real-time RT-PCR and plotted against the ER (A) and PR (B) hormone receptor status of the primary tumor. In (C) WAVE3 expression levels were compared between the patients with TNBC and other subtypes. WAVE3 RT-PCR values were normalized to GAPDH and plotted as the fold change to MCF10A. The results are shown as the mean±s. d. of at least 3 independent assays.