miR-20b is up-regulated in brain metastases from primary breast cancers

Abstract

Brain metastases are frequent in patients with advanced breast cancer and are associated with poor prognosis. However, unique molecular biomarkers have not yet been established. We hypothesized that microRNA-20b (miR-20b) plays a role in breast cancer brain metastasis. Our study cohort comprised of eleven breast cancer patients with brain metastasis and nine control patients (age, stage, and follow-up matched) with breast cancer without brain metastasis. Cases were reviewed microscopically to select tumor blocks with >50% tumor cells, RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks and expression of miR-20b analyzed using qRT-PCR. We further tested the effect of miR-20b overexpression on colony formation and invasion in vitro using MCF-7 and MDA-MB-231 cells. In the patient-derived samples, miR-20b expression was significantly higher in brain metastases of breast cancer patients, compared to primary breast tumors as well as the patients without brain metastasis. miR-20b also significantly induced the colony formation and invasiveness of breast cancer cells. Further, miR-20b levels were observed to be high in brain-metastasizing cells, compared to bone-metastasizing cells. Together, our findings suggest a novel role of miR-20b in breast cancer brain metastasis that warrants further investigation for its potential to be developed as prognostic and/or therapeutic target.

Keywords: brain metastasis; breast cancer; miR-20b.

Figure 1. Patient with solitary breast cancer brain metastasis involving the posterior aspect of the left middle frontal gyrus

Post-contrast T1-weighted axial (A), coronal (C), and sagittal (D) images and fluid-attenuated inversion recovery (FLAIR) axial image (B) showing a 2.5 × 3.3 × 3.1 cm mass (white arrows) with extensive peritumoral vasogenic edema (yellow arrows). Of note, this patient had the highest expression of miR-20b in the resected tumor specimen.

Figure 2

(A) MCF-7 and MDA-MB-231 breast cancer cells were transfected with non-specific pre-miRNA controls (NS) or pre-miR-20b (miR-20b). Relative expression of miR-20b was determined by quantitative RT-PCR. RNU48 was used as internal miRNA control against which the data was normalized. (B) miR-20b-transfected MCF-7 and MDA-MB-231 cells exhibited significantly increased anchorage-independent colony formation in soft agar, and invasiveness, compared to control cells (non-specific pre-miR-transfected cells). *p < 0.05, **p < 0.01.

Figure 3. Relative expression of miR-20b in patient samples

miRNAs extracted from FFPE samples of breast cancer patients with or without breast cancer brain, and analyzed for expression of miR-20b. RNU48 was used as miRNA control against which the data was normalized. *p < 0.05.

Figure 4. Relative expression of miR-20b in individual patient samples

miRNAs extracted from FFPE samples of individual breast cancer patients with or without breast cancer brain metastasis were analyzed for expression of miR-20b. RNU48 was used as miRNA control against which the data was normalized.

Figure 5. Relative expression of miR-20b in brain vs. bone-seeking breast cancer cells

Expression of miR-20b was determined by quantitative RT-PCR. RNU48 was used as internal miRNA control against which the data was normalized. BR, Brain-metastasizing MDA-MB-231 cells; BO, Bone-metastasizing MDA-MB-231 cells. *p < 0.01.