Download Supplementary Figure Legends and Methods

Supplementary Figure Legends:
Supplementary Table S1: (A) Oncomine database analysis of RRM2 expression between ERpositive and ER-negative breast cancer patients (25-29). (B) GEO database analysis of RRM2
expression between relapse free survival and no relapse free survival in breast cancer who received
the tamoxifen as an adjuvant therapy for 5 years (30-34).
Supplementary Fig. S1: Transient expression of RRM2 modulates tamoxifen sensitivity in MCF-7
and T-47D breast cancer cell lines. (A) Western blot for RRM2 overexpression in vector control
(VC) and RRM2 (R2) transfected breast cancer cell lines 48 hours after transient transfection. (B)
Effect of RRM2 transient transfection on growth curves upon tamoxifen treatment in MCF-7 breast
cancer cells. (C) Effect of RRM2 transient transfection on growth curves upon tamoxifen treatment
in T-47D breast cancer cells. (D) Tamoxifen IC50 and resistance ratio values upon RRM2 transient
transfection in MCF-7 and T-47D breast cancer cell lines.
Supplementary Fig. S2: Stable overexpression of RRM2 reduces tamoxifen sensitivity in MCF-7
breast cancer cells. (A) Western blot from MCF-7 vector (MCF-7 VC), and RRM2 overexpressing
MCF-7 cells (clone MCF-7/R2.1, clone MCF-7/R2.3 and pool population MCF-7/R2.pool). Colony
formation assay shown as (B) representative images and (C) quantification upon tamoxifen and
Didox treatment in RRM2 overexpressing breast cancer cells. (D) MCF-7 VC, (E) MCF-7/R2.1, and
(F) MCF-7/R2.3 cell lines were treated with vehicle control (C), 0.1µM estrogen (E), 1µM tamoxifen
(T), 30µM of Didox (D) and a combination of 1µM of T and 30µM of D (T+D) for 7 days. Error bars
represent mean ± SEM of three individual experiments done in triplicate. Comparisons between
groups were made by two-sample t-tests (p < 0.05). ● indicate significant differences between
control treated versus estrogen treated MCF-7 cells. ** indicate significant differences between
control treated versus tamoxifen treated MCF-7 cells. * indicate significant differences between
control treated versus T+D treated MCF-7 cells.
Supplementary Fig. S3: Didox resensitizes RRM2 overexpressing and TamR cells to tamoxifen
induced cell death. The CellTox™ Green Dye assay (Promega) was used to measure cell death
according to manufacturer’s protocol. Tamoxifen induces cell death in vector control (MCF-7 VC),
parental MCF-7, and parental T-47D cells but RRM2 overexpressing and TamR cells become
insensitive to tamoxifen induced cell death. Didox resensitizes RRM2 overexpressing and TamR
cells to tamoxifen induced cell death. The combination of tamoxifen and Didox (T+D) significantly
induced cell death as measured after 4 days by CellTox Green assay in RRM2 overexpressing MCF7 breast cancer cell lines and acquired tamoxifen resistant breast cancer cell lines. Error bars
represent mean ± SEM of three individual experiments done in triplicate.
Supplementary Fig. S4: Didox cooperates with tamoxifen to reduce cell proliferation. (A) Doseresponse curves for tamoxifen (T), Didox (D) and their combination (T+D) in MCF-7, RRM2
overexpressing MCF-7 cells (MCF-7/R2.1), and MCF-7 TamR cells. (B) Combination index of
MCF-7, MCF-7/R2.1, and MCF-7TamR cells (p<0.05).
Supplementary Fig. S5: Cell viability was measured with varying concentrations of (A) tamoxifen
and (B) Didox to determine that the tamoxifen IC50 is 4.35 M in MCF-7 TamR cells (lowered
sensitivity), while it is 1.25 M for MCF-7 cells. The CellTox™ Green Dye assay was multiplexed
with the CellTiter-Glo® Cell Viability Assay (Promega) to measure both cell toxicity and viability
after treatment with (C) tamoxifen or (D) Didox in MCF-7, MCF/R2.1 and MCF- TamR cells for 4
days.
Supplementary Fig. S6: Effect of Didox treatment on cell motility on MCF-7 RRM2
overexpressing breast cancer cells. Images were captured by phase contrast microscopy using a 4×
objective at 20 h after removal of inserts with and without Didox treatment in RRM2 overexpressing
MCF-7 breast cancer cells. Didox treatment significantly inhibited motility as indicated by arrows.
Supplementary Methods:
Cell treatments
For experiments, cells were washed with Hank's Balanced Salt Solution (HBSS) and kept in
phenol-red-free DMEM-F12 media supplemented with 5% charcoal-stripped-serum (CSS) for 48
hours.
Cells were then washed with HBSS and treated with 0.1 µM ethanol as vehicle control
(C), 0.1µM E, 1µM T, 30µM of Didox (D) or a combination of 1µM T + 30µM of Didox (T+D) in
phenol-red-free, serum-free DMEM-F12 for 24 hours. For cell treatments, Didox was dissolved in
0.9% sterile saline solution, then filtered through a 0.45 μm syringe filter and stored at 4°C in the
dark for a maximum of 1 week.
Gene expression data retrieval and processing
We retrieved the gene expression data from five different NCBI GEO database, namely
GSE12093, GSE6532, GSE9195, GSE9893 and GSE1456 (http://www.ncbi.nlm.nih.gov/geo) (3034). All datasets contained data from ER-positive patients who received tamoxifen as mono-or
adjuvant therapy. Patient tumors were classified as tamoxifen-resistant if there was annotation (in the
GSM description) that metastasis had occurred; otherwise, they were classified as tamoxifensensitive. Correlations and statistical analyses were carried out and all p-values were calculated by
means of a two-sided t-test and p-values <0.05 were considered significant.
Patient Survival Analysis
An online database was used to assess the relevance of RRM2 expression to relapse free
survival (RFS), overall survival (OS), and distant metastasis free survival (DMFS) in ER positive
tamoxifen treated breast cancer patients (35). Briefly, RRM2 gene was entered into the database
(http://kmplot.com/breast/) to obtain Kaplan-Meier survival plots where the number-at-risk is
indicated below the main plot. Hazard ratios (at 95% confidence intervals) and p-values were
calculated and displayed on the webpage. Hazard ratio is defined as the chance of an event occurring
in the treatment arm divided by the chance of the event occurring in the control arm of a study.
Hazard ratio more than 2 is indicative of the significant effect of expression of genes of interest in a
study.
RNA extraction and quantification
Total RNA was isolated using RNeasy according to the manufacturer’s protocol
(Qiagen). All RNA samples were examined for their concentration, purity, and integrity. The
absorbance ratio at 260/280 of the RNA samples was in the range from 1.86 to 2.06 (mean
1.98±0.04) indicating pure and protein-free RNA. The RNA was run on ethidium bromide (Sigma)
stained 1 % agarose gels (Invitrogen), and Kodak 1D Image Analysis Software (CareStream Health)
was used to determine the intensity and the ratio of the 28S to 18S ribosomal RNA bands (> 1.75).