Astrocyte-induced Reelin expression drives proliferation of Her2+ breast cancer metastases

Abstract

Breast cancer metastasis to the brain develops after a clinical latency of years to even decades, suggesting that colonization of the brain is the most challenging step of the metastatic cascade. However, the underlying mechanisms used by breast cancer cells to successfully colonize the brain's microenvironment remain elusive. Reelin is an archetypal extracellular glycoprotein that regulates migration, proliferation, and lamination of neurons. It is epigenetically silenced in various cancers, and its expression in multiple myelomas is linked to poor patient survival. We found that Reelin expression was low in primary breast cancer tissue. However, its expression was significantly higher in Her2⁺ breast cancers metastasizing to the brain. In particular, Reelin was highly expressed in the tumor periphery adjacent to surrounding astrocytes. This augmented Reelin expression was seen in Her2⁺ metastases, but not in triple negative (TN) primary tumors or in TN breast to brain metastasis cells co-cultured with astrocytes. Furthermore, the elevated expression was sustained in Her2⁺ cells grown in the presence of the DNA methyltransferase inhibitor 5-azacytidine, indicating epigenetic regulation of Reelin expression. The relative growth and rate of spheroids formation derived from Her2⁺ primary and BBM cells co-cultured with astrocytes were higher than those of TN primary and BBM cells, and knockdown of both Reelin and Her2 suppressed the astrocyte-induced growth and spheroid forming ability of Her2⁺ cells. Collectively, our results indicate that within the neural niche, astrocytes epigenetically regulate Reelin expression and its interaction with Her2 leading to increased proliferation and survival fitness.

Keywords: Astrocytes; Brain metastasis; Breast cancer; ERBB2; Epigenetics; Her2; Neural microenvironment; Reelin; Triple negative breast cancer.

Figure 1.. Expression of Reelin in primary breast cancer and BBM cells is subtype and microenvironment specific.

(a-c) Immunohistochemical analysis of Reelin expression in Her2⁺ and TN primary breast cancer tissue. Quantification of Her2 and Reelin is shown on right (n = 12, p ≤ 0.01, bars indicate SEM). (d-f) Immunohistochemical analysis of Reelin expression in Her2⁺ and TN BBM patient specimens. Quantification of Her2 and Reelin is shown on right (n = 3, p ≤ 0.01, bars indicate SEM). (g) Immunohistochemical analysis of spatial distribution of Reelin within Her2⁺ BBM tumor and within tumor periphery. Tumor periphery and peritumor regions are separated by blue and red dashed lines. Triangle in lower panel indicates expression gradient of Reelin from tumor core to tumor periphery (B = brain, T = tumor, PT= peritumor regions). (h) Immunohistochemical analysis of astrocyte in tumor periphery and peritumor regions of BBM tumor. Tumor periphery is separated by blue dashed lines. Red arrow indicate presence of infiltrative astrocyte within BBM tumor (B = brain, T = tumor)

Figure 2.. Expression of Reelin is induced by astrocytes.

Primary breast cancer (BT474 and MDA-MB231) and BBM (BBM1 and BBM3) cells were cultured in astrocyte CM or BBM2 CM for 24–72h in serum-free media, after which cells were harvested for RNA and protein extraction. (A-B) Real-time PCR quantification of Reelin and Her2 expression in BBM1, BT474, BBM3, and MDA-MB-231 cells were plotted (RQ = relative quantification compared to Actin, n = 3, **p<0.05, ***p<0.01, bars indicate SEM). (C) Western blot analysis of Reelin, pHer2, and Her2 in BBM1, BT474, BBM3, and MDA-MB-231 cells co-cultured with astrocytes for 48 h. Tubulin was used as a loading control. (D) Exosomes (EVs) were extracted from astrocytes and BBM2 (Her2+) cells grown in serum-free media and used to treat BBM1, BT-474, BBM3, and MDA-MB-231 cells for 48 h. Real-time PCR quantification of Reelin expression is plotted (n = 6, ***p<0.01, bars indicate SEM).

Figure 3.. Astrocyte CM induced growth of tumor cell spheroids.

BBM1, BT-474, BBM3, and MDA-MB-231 cells were grown in serum-free astrocyte CM in low attachment flasks for 120 h. The cells were analyzed by light microscopy at 24 h intervals for the appearance of spheroids. (A) Relative sizes of spheroids (n = 12, Bars indicate SEM). (B) Spheroids derived from BBM1 and BT474 cells. (C-D) BT474 cells were cultured in serum-free media in a low attachment flask for 72 h. Spheroids were transferred to serum-free astrocyte CM for an additional 24 h before harvesting. Co-immunofluorescently stained spheroids using Her2 and Ki67 (C) and Her2 and Reelin (D) specific antibodies. Nuclear counterstaining was done with DAPI.

Figure 4.. Functions of astrocytic factors on Reelin transcription.

(A) Real-time PCR quantification of Reelin expression in Her2⁺ (BBM1) cells co-cultured with astrocytes (Astro cc) in the presence of 5-azacytidine for 24 h in serum-free media, followed by an additional 48 h of growth with or without 5-azacytidine (n = 3, **p<0.05, ***p<0.01, RQ = relative quantification compared to Actin). Bars indicate SEM. (B) Reelin promoter region showing cis-elements for transcription factors. (C) Set1 histone methyltransferase and GATA1 transcription factor were knocked down in BBM1 cells by shRNA and cocultured with astrocytes for 24 h. Real-time PCR quantification of Reelin expression (n = 3, ***p<0.01, bars indicate SEM).

Figure 5:. Effect of Reelin knockdown on astrocyte CM-induced Reelin expression and cell proliferation.

Her2⁺ (BBM1 and BT-474) and TN (BBM3 and MDA-MB-231) cells were transfected with shRNA1 and shRNA2 specific to Reelin. Control cells were transfected with a lentiviral plasmid expressing scramble control (Scr). Cells were purified by FACS and Reelin knockdown was confirmed by RT-PCR and Western blot analysis. (A) Real-time PCR quantification of Reelin expression (n = 3, ***p<0.01, bars indicate SEM). (B) The cells were grown in CM or in normal DMEM (control) in 24-well plates (20,000 cells/per well) for up to 120 h. Numbers of viable cells were measured using MTT assay (n = 12, ***p<0.01, bars indicate SEM).

Figure 6.. Reelin knockdown affects spheroid formation by BBM cells.

BBM1, BT-474, BBM3, and MDA-MB-231 cells were transfected with Reelin shRNA-expressing lentiviral vectors. Control cells were transfected with scramble shRNA-expressing vectors (Scr). Cells were grown in serum-free media in low attachment flask for 120 h. (A) immunofluorescence staining of Reelin in BBM1 spheroids. Nuclear counterstaining was done using DAPI. (B) Relative numbers of cells in spheroids compared to the total cell in the flask at different time points (n = 12, ***p<0.01, **p<0.05 bars indicate SEM).