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Page 4 of 14 Ansari et al. J Cancer Metastasis Treat 2019;5:20 I http://dx.doi.org/10.20517/2394-4722.2018.68

viability of the cells was measured at 48 and 72 h post‐treatment using the CellTiter‐Glo® Luminescent Cell
Viability Assay kit. Compounds that showed at least two‐fold greater toxicity against BBM1 cells compared
to astrocytes cells were further analyzed for their concentration-dependent effects on BBM1 cells. For
concentration-dependent toxicity, cells were treated with 0, 31.2, 62.5, 125, 250, 500, 1000 nmol/L final concentration
of the compounds (n = 8). The viability was measured at 72 h post-treatment. The data normalization
compared to control and IC determination was done using GraphPad Prism 7.
50
Real-time PCR and Western blot analysis
Total RNA from astrocytes and BBM1 and BBM2 cells was extracted using Trizol (Invitrogen) and treated
with RNase-free DNase (Qiagen), according to the manufacturer’s instructions. To analyze gene expression,
cDNA was synthesized using an iScript reverse transcription kit (Bio-Rad). Real-time PCR quantification
was conducted using gene-specific primers and SYBR Select Master Mix (Applied Biosystems) using a CFX
real-time PCR system (Applied Biosystems). Control PCR reactions were conducted using glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) and/or actin-specific primers. PCR data was analyzed using the CFX
manager (Bio-Rad).

For Western blot analysis, total cell lysates were prepared in protein lysis buffer (50 mmol/L Tris-HCl, pH 7.5;
100 mmol/L NaCl; 1% Triton X-100; 1 mmol/L EDTA; 1 mmol/L EGTA, 50 mmol/L β-glycerophosphoran,
1 mmol/L dithiothreitol, 1 mmol/L phenylmethanesulfonyl fluoride; 2 mmol/L sodium orthovanadate, 10 μg/mL
aprotinin; 10 μg/mL leupeptin; and 10 μg/mL pepstatin A) by incubating cells for 20 min at 4 °C, followed
by centrifugation (15000 × g, 15 min, 4 °C). Protein extracts were analyzed by using antibodies specific
to ERBB2; phosphorylated ERBB2 (pERBB2); Protein kinase B (AKT); phosphorylated AKT (pAKT)
were obtained from Millipore; Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit, Alpha
(PIK3CA), phosphorylated PIK3CA (pPIK3CA); SRC Proto-Oncogene (SRC); phosphorylated SRC (pSRC);
MYC Proto-Oncogene (MYC); Mammalian Target of Rapamycin (mTOR); phosphorylated mTOR (pmTOR);
procaspase3 and cleaved caspase 3 were obtained from Cell Signaling. Actin and α-tubulin antibodies were
also obtained from Cell Signaling.

Apoptosis analysis
Apoptosis induction in treated and control cells was measured using Annexin V-FITC staining followed by
FACS analysis. In brief, cells were treated with the compounds 24 to 48 h prior to fixing in 4% PFA. Control
cells were treated with vehicle alone. The cells were immunostained with Annexin V-FITC (Invitrogen)
followed by PI (Life Technology) and analyzed using BD FACSAria Flow Cytometer (BD Biosciences). The
experimental control cells were stained with IgG with or without PI.

Statistical analysis
Verification of RNA-seq data was done with real-time PCR in at least three biological replicates to confirm
reproducibility and repeated at least twice. Results are expressed as the means ± SEM. Unless otherwise
stated, the two groups comparisons analyses were performed by using Mann-Whitney U tests in GraphPad
Prism® 7.0 (GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 was considered to be statistically significant.

RESULTS
Breast cancer brain metastases display a unique brain-like transcription profile
Primary HER2(ERBB2)+ breast cancer tissue (PT) and HER2+ breast to brain metastatic tissue (MT)
collected from the consented patients, along with normal breast (nBreast) and normal brain (nBrain) tissues,
were subjected to RNA-seq analysis. Hierarchical clustering of the normalized expression data of 20,257
transcripts revealed that, whereas PT shows transcriptome similarity with nBreast tissue, MT exhibits a
brain-like transcription profile [Figure 1A and B]. IPA of the 367 MT-specific revealed ERBB2 signaling as a