Supplemental Figure Legends. Figure S1. ERBB3 mrna levels are elevated in Luminal A breast cancers harboring ERBB3 ErbB3 gene copy number gain. Supplemental Figure S1. ERBB3 mrna levels are elevated in Luminal A breast cancers harboring ERBB3 gene copy number gain. ERBB3 mrna levels measured in 235 TCGA-curated Luminal A breast cancers (1) were assessed. ERBB3 mrna was highest in those tumors with ERBB3 gene copy number gains, and lowest relative levels of expression in those tumors with ERBB3 copy number loss. Figure S2. ErbB3 overexpression in BT-474, HCC1428, and MDA-MB-361 cells. Luminal breast cancer cells were transduced with retrovirus expressing ErbB3-IRES-RFP and selected with puromycin. Puromycin-resistant clones were assessed by western analysis to confirm overexpression of ErbB3. Figure S3. ErbB3 targeting with the antibody U3-1287 does not decrease growth of luminal breast cancer lines grown in monolayer. MCF7, T47D, and MDA-MB-361 cells were cultured 10 days in 10% serum with U3-1287 (5 µg/ml) or control IgG. Crystal violet stained cells were imaged on a flatbed scanner and quantitated using Image J. Figure S4. U3-1287 targeting of ErbB3 in luminal breast cancer xenografts. MDA-MB-361 and T47D xenograft-bearing mice were randomized to treatment groups to receive U3-1287 (10 mg/kg, twice weekly) or control IgG after tumors reached >150 mm 3. Mice were treated for 14-28 days. Tumors were harvested 24 hours after the final treatment with U3-1287 or IgG. Figure S5. Decreased ErbB3 expression and signaling in response to U3-1287 in vivo. T47D whole tumor lysates were assessed by western analysis for expression of ErbB3 and P-Akt (S473). Figure S6. U3-1287 does not induce expression of tumor stem cell markers in MCF7 cells. MCF7 cells were treated with U3-1287 or control IgG for 48 h. Total RNA was assessed by qrt- PCR for relative levels of transcripts encoding Twist, Slug, E-cadherin, and Snail. Average ± S.D. relative transcript levels are shown (N = 3) in relation to GAPDH levels (using the ΔΔCT method), and the average value of IgG-treated samples for each transcript were given a value of 1. Figure S7. U3-1287 down-regulates basal and fulvestrant-induced levels of ErbB3. MCF7 1
and MDA-MB-361 cells were cultured 24 hours in 10% serum in the presence of fulvestrant (1 µm) or DMSO, with either U3-1287 (5 µg/ml) or isotype-matched IgG. Whole cell lysates were assessed by western analysis using antibodies indicated at left. Figure S8. U3-1287 downregulates fulvestrant-induced ErbB4 tyrosine phosphorylation without affecting ErbB4 levels. MCF7 cells cultured in 10% serum in the presence of fulvestrant (1 µm) or DMSO with U3-1287 (5 µg/ml) or control IgG were used for western analysis of whole cell lysate, using antibodies indicated at left. Figure S9. The ErbB3/ErbB4 ligand NRG decreases response to fulvestrant. MCF7 cells were cultured 10 days in 10% serum in the presence of fulvestrant (1 µm) and NRG (2 ng/ml). Crystal violet stained cells were imaged on a flatbed scanner and quantitated using Image J. Values shown represent the average total colony area per well ± S.D., N = 3. *P < 5. **P < 1. Student s T-test. Figure S10. ErbB3 targeting with U3-1287 improves response to fulvestrant. MCF7, T47D and MDA-MB-361 cells were cultured in 10% serum for 10 days in the presence of fulvestrant (1 µm), NRG-1 (2 ng/ml), and U3-1287 (5 µg/ml) or isotype-matched human IgG. Crystal violet stained cells were imaged on a flatbed scanner and quantitated using Image J. Values shown represent the average total colony area per well ± S.D., N = 3. **P < 1 Student s T-test. Figure S11. ErbB3 targeting improves luminal breast tumor cell response to fulvestrant. MCF7, T47D, CAMA-1, HCC1428, and MDA-MB-175 VII cells were suspended in Matrigel and cultured for 14 days in the presence of fulvestrant (1 µm) or DMSO, and U3-1287 (5 µg/ml) or isotype-matched human IgG. Colonies were photodocumented. Representative images are shown. Figure S12. ErbB3 targeting with U3-1287 improves fulvestrant-mediated inhibition of luminal breast tumor growth in vivo. MCF7 xenograft bearing mice were treated once weekly with fulvestrant in the presence of twice weekly U3-1287 or IgG. Tumor weight (± S.D) was measured on treatment day 42. P values calculated using Student s T-test. N = 6-10 per treatment group. Figure S13. ErbB3 targeting with U3-1287 improves fulvestrant-mediated inhibition of 2
luminal breast tumor growth in vivo. MDA-MB-361 xenograft bearing mice were treated once weekly with fulvestrant in the presence of twice weekly U3-1287 or IgG. Tumor weight (± S.D) was measured on treatment day 21. P values calculated using Student s T-test. N = 6-10 per treatment group. Figure S14. Tumor response to the combination of U3-1287 and fulvestrant. T47D xenograft bearing mice (A) and MDA-MB-361 xenograft breaing mice (B) were randomized into treatment arms to receive U3-1287 or IgG (each at 10 mg/kg, twice weekly) in the presence or absence of fulvestrant (once weekly). Representative images of hematoxylin and eosin-stained tumor sections are shown. Figure S15. Fulvestrant increases P-S6 in MCF7 xenografts. Immunohistochemical detection of P-S6 in MCF7 xenografts treated 8 days with fulvestrant (1µM). Figure S16. Inhibition of PDK1, but not SGK, block fulvestrant-induced mtor activation. MCF7 cells were cultured for 24 h in the presence of fulvestrant (1µM) or DMSO, and for the final 2 hours of culture with the addition of a PDK1 inhibitor or an SGK inhibitor. Whole cell lysates were assessed by western analysis using the antibodies indicated at the right. Figure S17. Increased cell death in MCF7 cells cultured in the presence of U3-1287 and fulvestrant. MCF7 cells were treated 24 hours with fulvestrant (1 µm) in the presence of either U3-1287 (5 µg/ml) or RAD001 (0.2 µg/ml). Annexin V-FITC was added during the final 2 hours of culture to detect dying cells. Representative images are shown. Figure S18. Fulvestrant-mediated upregulation of P-Akt is blocked by ErbB3 inhibition, but enhanced by mtorc1 inhibition. MCF7 and MDA-MB-361 cells were cultured 24 hours in 10% serum with fulvestrant (1 µm), U3-1297 (5 µg/ml), or RAD001 (0.2 µm). Whole cell lysates were assessed by western analysis for antibodies indicated at left. Figure S19. Fulvestrant and U3-1287 cooperate to alter gene expression in MCF7 cells. A. MCF7 tumors treated in vivo for 8 days with fulvestrant, U3-1287, or both were assessed for gene expression changes (as compared to untreated tumors) by screening total tumor RNA with the SABiosciences RT2-Profiler qpcr Array (Breast Cancer and ER signaling). Significantly altered 3
genes in each group are shown as waterfall plots in upper panels. Genes whose expression is altered in a unique was in the combination as compared to the individual treatments are shown in lower panels. N = 3 per group, analyzed in duplicate. Values are shown relative to values measured in IgG-treated tumors, which was given a value of 1. Genes whose expression values were statistically altered are shown. References Cited 1. 2012. Comprehensive molecular portraits of human breast tumours. Nature 490:61-70. 4
A
Supplemental Figure S2. ErbB3 overexpression in BT-474, HCC1428, and MDA-MB-361 cells. Luminal breast cancer cells were transduced with retrovirus expressing ErbB3-IRES-RFP and selected with puromycin. Puromycin-resistant clones were assessed by western analysis to confirm overexpression of ErbB3.
U3-1287 IgG MCF7 MDA-MB- 361 T47D Supplemental Figure S3. ErbB3 targeting with the antibody U3-1287 does not decrease growth of luminal breast cancer lines grown in monolayer. MCF7, T47D, and MDA-MB-361 cells were cultured 10 days in 10% serum with U3-1287 (5 µg/ml) or control IgG.
IgG U3-1287 MDA-MB-361 xenografts IgG U3-1287 T47D xenografts Supplemental Figure S4. U3-1287 targeting of ErbB3 in luminal breast cancer xenografts. MDA- MB-361 and T47D xenograft-bearing mice were randomized to treatment groups to receive U3-1287 (10 mg/kg, twice weekly) or control IgG after tumors reached >150 mm 3. Mice were treated for 14-28 days. Tumors were harvested 24 hours after the final treatment with U3-1287 or IgG.
IgG U3-1287 ErbB3 P-Akt Akt Supplemental Figure S5. U3-1287 decreased ErbB3 signaling in vivo. T47D whole tumor lysates were assessed by western analysis for expression of ErbB3 and P-Akt (S473).
Average fold change normalized to GAPDH expression Average fold change normalized to GAPDH expression Average E-cadherin expression normalized to GAPDH Average Snail expression normalized to GAPDH Twist 1.5 n.s. 2.0 Slug n.s. E-cadherin 1.5 n.s. 2.0 Snail n.s. 1.0 1.5 1.0 1.5 1.0 1.0 0.5 0.5 0.5 0.5 IgG Control U3-1287 IgG Control U3-1287 IgG Control U3-1287 IgG Control U3-1287 Supplemental Figure S6. U3-1287 does not induce expression of tumor stem cell markers in MCF7 cells. MCF7 cells were treated with U3-1287 or control IgG for 48 h. Total RNA was assessed by qrt-pcr for relative levels of transcripts encoding Twist, Slug, E-cadherin, and Snail. Average ± S.D. relative transcript levels are shown (N = 3) in relation to GAPDH levels (using the CT method), and the average value of IgG-treated samples for each transcript were given a value of 1.
U3-1827 Fulv. ErbB3 ER - + - + - + - + - - + + - - + + MCF7 MDA-MB-361 Supplemental Figure S7. U3-1287 down-regulates basal and fulvestrant-induced levels of ErbB3. MCF7 and MDA-MB-361 cells were cultured 24 hours in 10% serum in the presence of fulvestrant (1 µm) or DMSO, with either U3-1287 (5 µg/ml) or isotype-matched IgG. Whole cell lysates were assessed by western analysis using antibodies indicated at left.
U3-1827 - + - + Fulv. - - + + P- ErbB3 P-ErbB4 ErbB4 MCF7 Supplemental Figure S8. U3-1287 downregulates fulvestrant-induced ErbB4 tyrosine phosphorylation without affecting ErbB4 levels. MCF7 cells cultured in 10% serum in the presence of fulvestrant (1 µm) or DMSO with U3-1287 (5 µg/ml) or control IgG were used for western analysis of whole cell lysate, using antibodies indicated at left.
NRG-1 control Average total colony area DMSO Fulv. 150000 ** ** * 100000 50000 0 DMSO Fulv. NRG-1 NRG-1 + fulv. Supplemental Figure S9. The ErbB3/ErbB4 ligand NRG decreases response to fulvestrant. MCF7 cells were cultured 10 days in 10% serum in the presence of fulvestrant (1 µm) and NRG (2 ng/ml). Crystal violet stained cells were imaged on a flatbed scanner and quantitated using Image J. Values shown represent the average total colony area per well ± S.D., N = 3. *P < 5. **P < 1. Student s T-test.
Fold change in cell number U3-1287+ NRG-1 IgG + NRG-1 U3-1287 IgG Average colony area A DMSO Fulv. DMSO Fulv. DMSO Fulv. B 120000 MCF7 100000 80000 ** 60000 40000 20000 ** 0 IgG + - + - + - U3-1287 - + - + - + Fulv. - - + + + + IgG U3-1287 IgG + fulv. U3-1287 + fulv. NRG-1 + fulvestrant U3-1287 + NRG-1 + fulv. NRG-1 - - - - + + MCF7 T47D MDA-MB-361 C MCF7 5 **** 4 3 2 1 0 DMSO U3 Fulv U3/Fulv Supplemental Figure S10. ErbB3 targeting with U3-1287 improves response to fulvestrant. A. MCF7, T47D and MDA-MB-361 cells were cultured in 10% serum for 10 days in the presence of fulvestrant (1 µm), NRG-1 (2 ng/ml), and U3-1287 (5 µg/ml) or isotype-matched human IgG. Crystal violet stained cells were imaged on a flatbed scanner and (B) quantitated using Image J. Values shown represent the average total colony area per well ± S.D., N = 3. **P < 1 Student s T-test. C. MCF7 cells were cultured in serum-containing media in the presence of fulvestrant (1µM), U3-1287 (5 µg/ml), or both. Cells were counted at 48 hour intervals. Values shown represent the average (± S.D.) fold change in cell number over 48 hours within each treatment group. N = 3 per group. ****P < 001, One-way ANOVA.
MDA-MB- 175 VII HCC1428 CAMA-1 T47D MCF7 A DMSO IgG DMSO U3-1287 Fulv. IgG Fulv. U3-1287 Supplemental Figure S11. ErbB3 targeting improves luminal breast tumor cell response to fulvestrant. MCF7, T47D, CAMA-1, HCC1428, and MDA-MB-175 VII cells were suspended in Matrigel and cultured for 14 days in the presence of fulvestrant (1 µm) or DMSO, and U3-1287 (5 µg/ml) or isotype-matched human IgG. Colonies were photodocumented. Representative images are shown.
MCF7 tumor weight (g) 1.5 P < 001 1.0 P < 1 0.5 - - + + Fulv. - + - + U3-1287 IgG U3-1287 IgG + Fulv. U3-1287 + Fulv. Supplemental Figure S12 ErbB3 targeting with U3-1287 improves fulvestrantmediated inhibition of luminal breast tumor growth in vivo. MCF7 xenograft bearing mice were treated once weekly with fulvestrant in the presence of twice weekly U3-1287 or IgG. Tumor weight was measured on treatment day 42. P values calculated using Student s T-test. N = 6-10 per treatment group.
MDA-MB-361 tumor weight 1.2 n.s. 0.8 *** 0.4 - - + + Fulv. - + - + U3-1287 IgG U3-1287 IgG + Fulv. U3-1287 + Fulv. Supplemental Figure S13. ErbB3 targeting with U3-1287 improves fulvestrantmediated inhibition of luminal breast tumor growth in vivo. MDA-MB-361 xenograft bearing mice were treated once weekly with fulvestrant in the presence of twice weekly U3-1287 or IgG. Tumor weight was measured on treatment day 21. P values calculated using Student s T-test. N = 6-10 per treatment group.
H&E H&E A IgG U3-1287 IgG + Fulv. U3-1287 + Fulv. T47D tumors harvested after 7 days of treatment B IgG U3-1287 IgG + Fulv. U3-1287 + Fulv. MDA-MB-361 tumors harvested after 14 days of treatment Supplemental Figure S14. Tumor response to the combination of U3-1287 and fulvestrant. T47D xenograft bearing mice (A) and MDA-MB-361 xenograft breaing mice (B) were randomized into treatment arms to receive U3-1287 or IgG (each at 10 mg/kg, twice weekly) in the presence or absence of fulvestrant (once weekly). Representative images of hematoxylin and eosin-stained tumor sections are shown.
Fulvestrant Control P-S6 IHC Supplemental Figure S15. Fulvestrant increases P-S6 in MCF7 xenografts. Immunohistochemical detection of P-S6 in MCF7 xenografts treated 8 days with fulvestrant (1µM).
Fulv. - + - + - + - + PDKi - - + + - - + + SGKi - - - - + + + + ErbB3 ER P-Akt (T308) Akt P-S6 S6 Supplemental Figure S16. Inhibition of PDK1, but not SGK, block fulvestrant-induced mtor activation. MCF7 cells were cultured for 24 h in the presence of fulvestrant (1µM) or DMSO, and for the final 2 hours of culture with the addition of a PDK1 inhibitor or an SGK inhibitor. Whole cell lysates were assessed by western analysis using the antibodies indicated at the right.
DMSO Fulvestrant U3-1287 IgG Rad001 MCF7 Supplemental Figure S17. Increased cell death in MCF7 cells cultured in the presence of U3-1287 and fulvestrant. MCF7 cells were treated 24 hours with fulvestrant (1 µm) in the presence of either U3-1287 (5 µg/ml) or RAD001 (0.2 µg/ml). Annexin V-FITC was added during the final 2 hours of culture to detect dying cells. Representative images are shown.
MCF7 Fulvest. - - - + + + U3-1287 - + - - + - Rad001 - - + - - + MDA-MB-361 - - - + + + - + - - + - - - + - - + ER ErbB3 P-Akt Supplemental Figure S18. Fulvestrant-mediated upregulation of P-Akt is blocked by ErbB3 inhibition, but enhanced by mtorc1 inhibition. MCF7 and MDA-MB-361 cells were cultured 24 hours in 10% serum with fulvestrant (1 µm), U3-1297 (5 µg/ml), or RAD001 (0.2 µm). Whole cell lysates were assessed by western analysis for antibodies indicated at left.
Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change Log2 Fold Change PGR FLRT SLC7A5 DLC1 VEGFA JUN CTSD MAP2K7 STC2 HSPB1 KLF5 CLDN7 TFF1 IGFBP2 NME1 TGFA BCL2L2 AR BAD CCNA2 TOP2A GSN MKI67 SCGB1D2 NFYB STC2 SCGB1D2 CTSB SCB2A1 TGFA CCNA2 MKI67 TOP2A BAD FOSL1 TP53 NME1 FAS AR CCND1 IGFBP2 SERPINA3 THBS1 PGR SPRR1B VEGFA SCGB2A1 MUC1 CTSD ERBB2 BCL2L2 ESR1 TGFA BAG IL6ST TNFAIP2 GSN THBS2 FOSL1 AR BAD DLC1 FAS THBS1 HMGB1 TOP2A CCNA2 MKI67 NGFR SERPINA3 CCNA1 GABRP PGR SCGB1D2 Log2 Fold Change Log2 Fold Change Log2 Fold Change 2.0 U3-1287 Fulvestrant Fulvestrant/U3-1287 1 1.5 1 1.0 0 0 0.5-1 -1-0.5-2 2-1.0-2 -3 NGFR TNFAIP2 SCGB1D2 2 1.0 1 1 0-1 0.5-0.5-1.0 0-1 -2-2 fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-1.5fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-3 fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888 TOP2A CCNA2 MKI67-0.5-0.5-0.5-1.0-1.0-1.0-1.5-1.5-1.5-2.0fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-2.0fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-2.0 fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888 BAG1 BAD GSN 1.0 1.0 1.0 0.5 0.5 0.5-0.5 fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-0.5 fulv U31287 fulv + AMG fulvestrant AMG888 fulvestrant + AMG888-0.5 fulvestrant AMG888 fulvestrant AMG888 fulv U31287 fulv + Supplemental Figure S19. Fulvestrant and U3-1287 cooperate to alter gene expression in MCF7 cells. A. MCF7 tumors treated in vivo for 8 days with fulvestrant, U3-1287, or both were assessed for gene expression changes (as compared to untreated tumors) by screening total tumor RNA with the SABiosciences RT2-Profiler qpcr Array (Breast Cancer and ER signaling). Significantly altered genes in each group are shown as waterfall plots in upper panels. Genes whose expression is altered in a unique was in the combination as compared to the individual treatments are shown in lower panels. N = 3 per group, analyzed in duplicate. Values are shown relative to values measured in IgG-treated tumors, which was given a value of 1. Genes whose expression values were statistically altered are shown.
Supplemental Methods. RNA was extracted from flash-frozen MCF7 tumors using the RNeasy kit (Qiagen). Total RNA was quantified and 2 ug RNA was used to synthesize cdna (High Capacity cdna Reverse Transcription kit - Applied Biosystems). Quantitative RT PCR was performed on a BioRad icycler iq5 machine using the following primer sequences: Human snai2 F: 5 -GGGGAGAAGCCTTTTTCTTG-3 Human snai2 R: 5 -TCCTCATGTTTGTGCAGGAG-3 Human snai F: 5 -CCTCCCTGTCAGATGAGGAC-3 Human snai R: 5 -CCAGGCTGAGGTATTCCTG-3 Human twist F: 5 -GGAGTCCGCAGTCTTACGAG-3 Human twist R: 5 -TCTGGAGGACCTGGTAGAGG-3 Human cdh1 F: 5 -TGGGCCAGGAAATCACATCCTACA-3 Human cdh1 R: 5 -TTGGCAGTGTCTCTCCAAATCCGA-3
MCF7 T47D MDA-MB-361 BT474 CAMA-1 ZR75-1 HCC1428 ERα + + + + + + + HER2 overexpression - - + + - - - PTEN loss of function - - - - + + - PIK3CA mutant E545K H1047R E545K *K117N - - - Supplemental Table S1. Cell lines purchased from ATCC were used for analysis within 10 passages of receipt from ATCC. Expression of ERα, HER2 levels, and mutation status of PTEN and PIK3CA genes are indicated for each cell line. Specific PIK3CA mutations are shown. Although E545K and H1047R mutations are known to be activating mutations in p110α, the oncogenic effect of K117N mutation in p110α remains unclear.