Supplementary Figures S1-S5 from AKT-Induced Tamoxifen Resistance Is Overturned by RRM2 Inhibition

Abstract

<p>PDF file - 917 KB, Legends for Supplementary Figures: Supplementary Fig. S1: AKT1 and AKT3 are highly expressed in patient breast cancer tumors and AKT3 expression correlates with recurrence and ER negative status. Box and whisker plot of AKT isoforms expression in (A) normal versus breast carcinoma specimens, (B) tumors of different grade, and tumors with respect to (C) recurrence and (D) estrogen receptor status using Oncomine TCGA datasets. Supplementary Fig. S2: Tamoxifen induces cell proliferation in breast cancer cells with high levels of Akt. Venn diagrams show overlaps of differentially expressed genes (A) C, E and T treated parental MCF-7 versus Akt3 cells (MC:AC, ME:AE, and MT:AT). (B) C versus E treated parental MCF-7 (MC:ME) and T treated parental MCF-7 versus Akt overexpressing MCF-7 cells (MT:AT). Ingenuity pathway analysis was used to generate the network of differentially expressed genes (C) upon estrogen versus control treatment in parental MCF-7 cells and (D) upon tamoxifen treatment in parental MCF-7 versus Akt3 overexpressing cells. Genes shown in red are up- regulated, genes shown in green are down-regulated and central nodes are estradiol (C) and NFkB (D). Supplementary Fig. S3: Differential gene expression variations in breast cancer cells RT-qPCR confirmation analysis of (A) up-regulated and (B) down-regulated genes was examined in cells upon Akt overexpression. Fold change is shown relative to control treated parental MCF-7 cells. Supplementary Fig. S4: Altered gene expression in normal versus breast carcinoma specimens using the Oncomine database. Altered expression of (A) up-regulated and (B) down-regulated genes is shown. Fold changes (FC) of breast carcinoma were calculated relative to normal breast tissue and comparisons between groups were made by two-sample t-tests. Supplementary Fig. S5: (A) Knockdown efficiencies of siRRM2#1 and siRRM2#2 as compared to non-targeting control (NT C). (B) Western blots showing efficient knockdown of RRM2 protein in six breast cancer cell line. (C) Cellular proliferation between siRNA treatments were compared relative to NT C. (D) Knockdown efficiencies of siRRM2 remained high over seven days as validated by RT-qPCR. (E) Images were captured by phase contrast microscopy using a 4? objective at 20 h after removal of inserts for siRRM2 transfected and non-targeting control. siRRM2 significantly inhibited motility in Akt1 and Akt3 overexpressing MCF7 cells and MDA-MB-231 cells indicated by arrows.</p>