Cytoplasmic BRMS1 expression in malignant melanoma is associated with increased disease-free survival

Abstract

Background/aims: Breast cancer metastasis suppressor 1 (BRMS1) blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis.

Methods: Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines.

Results: A significantly higher percentage of nevi (87%), compared to primary melanomas (20%) and metastases (48%), expressed BRMS1 in the nucelus (p < 0.0001). Strong nuclear staining intensity was observed in 67% of nevi, and in 9% and 24% of the primary and metastatic melanomas, respectively (p < 0.0001). Comparable cytoplasmic expression was observed (nevi; 87%, primaries; 86%, metastases; 72%). However, a decline in cytoplasmic staining intensity was observed in metastases compared to nevi and primary tumors (26%, 47%, and 58%, respectively, p < 0.0001). Score index (percentage immunopositive celles multiplied with staining intensity) revealed that high cytoplasmic score index (≥ 4) was associated with thinner tumors (p = 0.04), lack of ulceration (p = 0.02) and increased disease-free survival (p = 0.036). When intensity and percentage BRMS1 positive cells were analyzed separately, intensity remained associated with tumor thickness (p = 0.024) and ulceration (p = 0.004) but was inversely associated with expression of proliferation markers (cyclin D3 (p = 0.008), cyclin A (p = 0.007), and p21Waf1/Cip1 (p = 0.009)). Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013) and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033). Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016) and decreased relapse-free period (p = 0.043). Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011), a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro.

Conclusion: Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion.Please see related article: http://www.biomedcentral.com/1741-7015/10/19.

Figure 1

Immunohistochemical staining of BRMS1 in benign nevi showing cytoplasmic and nuclear (A,) and nuclear only expression (B), primary melanomas with cytoplasmic expression (C, D) and metastatic melanomas demonstrating cytoplasmic and nuclear (E) and cytoplasmic only immunoreactivity (F). In the two melanoma cell lines (G, H) BRMS1 is localized mainly in the nucleus, although some cytoplasmic expression is also present.

Figure 2

Kaplan-Meier curve demonstrating relationship between BRMS1 cytoplasmic score index (A) and between percentage of cells expressing nuclear BRMS1 (B) and disease-free survival.

Figure 3

Effect of BRMS1 on migration and invasion of melanoma cells. BRMS1 expression was transiently down-regulated using siRNA in the metastatic melanoma cell lines WM239 and FEMX-1 (A) and analyzed for migration and invasion ability in a transwell chamber assay (B). Bars represent mean ratio of cells ± SE of cells in the lower compartment compared to total number of cells in both compartments from at least three independent biological experiments. WM239: invasion; p = 0.047, migration; p = 0.013, FEMX-1; invasion; p = 0.016, migration; p = 0.43.