AMG 900 and Breast Cancer Kalous, Conklin et al.

SUPPLEMENTARY MATERIAL

Supplementary Methods

Cell lines, cell culture, and reagents

The cell line panel included 41 breast cancer cell lines and 3 immortalized breast epithelial cell lines representing the known molecular subgroups of breast cancer and was described in detail previously [1-3]. The panel included MDA-MB-415, MDA-MB-134, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC-1569, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3, MDA-MB-361, UACC-893, UACC-812, UACC-732, T-47D, MDA-MB-453, MDA-MB-468, CAMA-1, MDA-MB-157, MCF-7, MDA-MB-435, ZR-75-1, BT-20, MDA-MB-231, BT-549, DU-4475, HCC-1395, HCC-2218, 184A1, 184B5 and MCF-10A that were purchased from American Type Culture Collection (Rockville, MD). The cell lines EFM-192A, KPL-1, EFM-19, COLO-824 and CAL-51 were obtained from the German Tissue Repository DSMZ (Braunschweig, Germany). Both cell line banks perform the cell lines authentication by short tandem repeat analysis. The cell lines SUM-190 and SUM-225 were obtained from the University of Michigan (Ann Arbor, MI). Upon receipt, all cell lines were assessed for Mycoplasma contamination using a multiplex PCR method [4] and mitochondrial DNA from the cells was sequenced to confirm their correct identity [5]. Cell lines were then expanded and these procedures were repeated for all cell lines prior to cryopreservation. All cell lines were passaged for less than 6 months before use in this study.

AMG 900 was obtained from Amgen Inc. (Thousand Oaks, CA) and diluted in DMSO.

Proliferation assays

Cells were seeded in duplicate at 5 x 103 to 5 x 104 cells per well in 24-well plates. A day after plating, cells were treated with a dose range of AMG 900 starting at 1 mM and decreasing by six 10-fold dilutions. t = 0 control wells were counted at the time of treatment. The remaining control and treated wells were counted after 5 days of treatment. Trypsinized cells were placed in an Isotone solution and immediately counted using a Coulter Z1 particle counter (Beckman Coulter Inc, Fullerton, CA). Suspension cultures were counted using a Coulter Vi-Cell counter (Beckman Coulter Inc, Fullerton, CA).

The average number of cell population doublings (or generations) from baseline and generational percent inhibition were calculated as previously described [1]. Non-linear curve fitting to the dose-response data described above was performed as described [1]. IC50 values were interpolated from the resulting curves. Using our model of growth inhibition, IC50 can be interpreted as the drug concentration needed to prevent 50% of cell population doublings. Lethality was defined as the average post-treatment cell counts significantly lower than the average t = 0 control counts. Percentage lethality was calculated using the following formula:

All experiments were performed at least twice.

Microarray analysis of cell lines

Agilent microarray analyses were developed for each cell line. RNA extraction was performed as described [2]. Microarrays were then performed on the Agilent Human Whole Genome 44Kx4 chip. Characterization of individual breast cancer cell lines by comparison to a breast cell line mixed reference pool was conducted as described [2]. Microarray slides were read using an Agilent Scanner, and the Agilent Feature Extraction software version 9.5 was used to calculate gene expression values. Extracted data was imported into Rosetta Resolver 7.2 to create expression profiles for each individual breast cell line experiment. These microarray data are available with GEO accession number GSE44552.

DNA isolation and oligonucleotide array Comparative Genomic Hybridization (aCGH) analysis

Extraction of genomic DNA was performed from frozen cell pellets using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Agilent 105K oligo aCGH was performed as described [6].

Quantitative analysis of cell cycle and apoptosis by flow cytometry

The effects of AMG 900 on cell cycle were assessed using Nim-DAPI staining (NPE Systems, Pembroke Pines, FL) as described previously [1]. The cells were plated evenly in control and experimental wells, allowed to grow to log phase, and then treated with 10 nM AMG 900 for a specified time period. For apoptosis, cells were plated evenly in control and experimental wells, allowed to grow to log phase, and then treated with 10 nM AMG 900 for a specified time period and analyzed using Annexin V-FITC as described [1].

Western blots and protein quantification

In order to determine the p21, Aurora A, and Aurora B baseline protein expression, cells in log-phase growth were harvested and lysates taken as described [1]. In order to determine the effect of AMG 900 on the expression of p53 and p21, cells in log-phase growth were treated with 10 nM or 100 nM AMG 900 and were harvested at specified times ranging from 10 min to 48 hours. The expressions of total p53 and p21 were detected by anti-p53 and anti-p21 antibody (Cell Signaling, Beverly, MA). Aurora A and B were detected by anti-Aurora A (Cell Signaling) and anti-Aurora B (Abcam, Cambridge, MA) antibodies. Densitometry was performed using the ImageJ 1.45s (NIH) software.

Statistical analysis

Response ratio (RR) and corresponding p-values for each predictor were determined by Cochran-Mantel-Haenszel Chi-square analysis using the PROC FREQ function in SAS for Windows version 9.2 (SAS Institute, Inc., Cary, NC). Pearson correlation coefficients and their corresponding p values were calculated using the PROC CORR function in SAS.

All graphs and tables were created in Microsoft Excel.

REFERENCES:

1. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, Ginther C, Atefi M, Chen I, Fowst C, Los G, Slamon DJ (2009) PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res 11 (5):R77. doi:10.1186/bcr2419

2. Finn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N, Slamon DJ (2007) Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/"triple-negative" breast cancer cell lines growing in vitro. Breast Cancer Res Treat 105 (3):319-326. doi:10.1007/s10549-006-9463-x

3. Kalous O, Conklin D, Desai AJ, O'Brien NA, Ginther C, Anderson L, Cohen DJ, Britten CD, Taylor I, Christensen JG, Slamon DJ, Finn RS (2012) Dacomitinib (PF-00299804), an irreversible Pan-HER inhibitor, inhibits proliferation of HER2-amplified breast cancer cell lines resistant to trastuzumab and lapatinib. Mol Cancer Ther 11 (9):1978-1987. doi:10.1158/1535-7163.MCT-11-0730

4. Uphoff CC, Drexler HG (2002) Detection of mycoplasma in leukemia-lymphoma cell lines using polymerase chain reaction. Leukemia 16 (2):289-293. doi:10.1038/sj.leu.2402365

5. Ginther C, Issel-Tarver L, King MC (1992) Identifying individuals by sequencing mitochondrial DNA from teeth. Nat Genet 2 (2):135-138. doi:10.1038/ng1092-135

6. Konecny GE, Winterhoff B, Kolarova T, Qi J, Manivong K, Dering J, Yang G, Chalukya M, Wang HJ, Anderson L, Kalli KR, Finn RS, Ginther C, Jones S, Velculescu VE, Riehle D, Cliby WA, Randolph S, Koehler M, Hartmann LC, Slamon DJ (2011) Expression of p16 and retinoblastoma determines response to CDK4/6 inhibition in ovarian cancer. Clin Cancer Res 17 (6):1591-1602. doi:10.1158/1078-0432.CCR-10-2307

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure S1: Aurora A and Aurora B baseline protein levels. Aurora A and Aurora B baseline protein levels were measured by Western blots on the cell line panel as described in Supplementary Methods. Cell lines were grouped based on their sensitivity to AMG 900 as described in Table 1; (a) highly sensitive, (b) less sensitive cell lines. α-tubulin was used as a loading control; +*, common control (HepG2); densitometry data available in Online Resource Supplementary Table S2.

SUPPLEMENTARY TABLES

Supplementary Table S1: Molecular subtypes of breast cancer, ER status, and HER2 amplification do not predict for response to AMG 900

Supplementary Table S2: Aurora A and Aurora B baseline protein levels quantified by densitometry

Supplementary Table S3: Cell lines and their p53 status

Supplementary Table S4: p21 baseline protein levels quantified by densitometry

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