Supplemental Figures from Rictor/mTORC2 Drives Progression and Therapeutic Resistance of HER2-Amplified Breast Cancers

Supplemental Figure S1. Kaplan meier analysis and oncoprint mapping of Luminal A/B (A) and Basal-like (B) TCGA-curated breast cancers, showing the tumors in each set with upregulation of Akt1/2/3 mRNA (red boxes) or phospho-S473 (black arrows), and the survival of these patients (red Kaplan-Meier line) versus other patients (blue Kaplan-Meier line). Log-rank test shows insignificance. Supplemental Figure S2. Rictor expression in human breast cancer. Supplemental Figure S3. Oncoprint of TCGA-curated invasive breast cancers (TCGA, Cell 2015, N = 971) assessed for RICTOR genomic alterations. Analysis shows 8% of cases (76/971) with gene amplification, overexpression, missense mutation, or deep deletion. Supplemental Figure S4. Rictor mRNA expression was measured by qRT-PCR of total RNA from mammary glands harvested from virgin female 12-week old mice. Supplemental Figure S5. Genetic ablation of Rictor decreases growth and survival of HER2-positive breast cancer cells. Supplemental Figure S6. A. Cells were cultured 6 hours with FITC-labeled Annexin V, then imaged. B. MCF10-HER2 cells (parental, Rictor, SFN, and Rictor ZFN + exogenous expression of myc-tagged Rictor) were cultured 6 h with An-nexin V-FITC. C. The percentage of cells in panel B that were labeled with Annexin V-FITC was quantitated. Averages (midlines) ± S.D. were assessed for significance using Student€™s two-tailed unpaired T-test. Supplemental Figure S7. Rictor-mediated Akt activity is necessary and sufficient for breast cancer cell survival. Supplemental Figure S8. Cells were treated with lapatinib for the time course indicated. Total cell RNA was used for qRT-PCR to measure relative RPTOR mRNA expression. Supplemental Figure S9. Cells were cultured 7 days in 10% serum plus lapatinib (1 μM) or PP242 (1 μM) or the combination of lapatinib + PP242. Cells were stained with crystal violet. Crystal violet fluorescence was scanned using the odyssey. Representative images are shown. Supplemental Figure S10. Loss of Rictor/mTORC2 sensitizes parental and resistant cells to lapatinib in vitro Supplemental Figure S11. Cells were treated 6h with PP242 (1μM) ± lapatinib (1μM) and assessed by luminescent caspase-3/7 assay.