Supplementary Fig. 1: ATM is phosphorylated in HER2 breast cancer cell lines. (A) ATM is phosphorylated in SKBR3 cells depending on ATM and HER2 activity. Upper panel: Representative histograms for FACS analysis of ps1981 ATM expression (ATM-p). Incubation with AlexaFluor488 -conjugated alone (488, white histograms) served as negative control. M.F.I., Mean Fluorescence Intensity; KU55933, 10µM for 48 hours,; Trastuzumab, Tzb, 20 µg/ml for 48 hours; CTR, untreated cells. Lower panel: ATM phosphorylation on Ser1981 (ATM-p) was detected by western blot upon immunoprecipitation with anti-atm antibody. (B) MCF7 treated cells with Adriamycin (Adr, 1 µm for 3 hours) have been used as control of ATM activation. Upper panel: representative histograms showing FACS analysis of phosho-ser-1981 ATM (ATM-p) expression. For each cell line, incubation with AlexaFluor488 -conjugated alone (488, white histograms) served as negative controls. MCF7 cells have been treated with Adriamycin (Adr, 1 µm for 3 hours) alone or in combination with KU55933 (10µM, 30 min of pretreatment) and have been used as control for ATM activation to set up flow cytometry (FACS) analysis with ps1981 ATM antibody. Lower panel: ATM phosphorylation on Ser1981 (ATM-p) was detected by western blot upon immunoprecipitation with anti-atm antibody.
Supplementary Fig. 2: ATM promotes HER2 dependent transformation. (A-D) Anchorage-independent growth of HER2 + breast cancer cell lines (BT474, in A), HER2+ ovarian cancer cell line (SKOV3, in B) and HER2- breast cancer cell lines (T47D and MDA-MB-231, respectively in C and D) infected with retrovirus expressing specific RNA interference for ATM (shatm #1) or a control sequence (shr5, labeled as -). Colonies are counted after 10 days and represented as mean±s.d. (n=6 for each condition). The panels on the right display representative western blots.
Supplementary Fig. 3: ATM sustains cell growth independently of HER2 expression.growth curve assay for MCF7(A), MCF7-HER2 (B) SKBR3 (C), MDA-MB-231 (D), T47D (E), cell lines infected with lentiviral construct expressing ATM shrna interference sequence or control shrna interference sequence. 10 5 Cells were seeded in complete medium and grown for 4 days. Cell numbers have been counted at the indicated times. Data are shown as mean +SD of three independent experiments each of one performed in triplicate for each condition.
Supplementary Fig. 4: ATM sustains HER2 dependent tumorigenicity and cell growth of MCF10A cells (A-B) Western blot for detection of ATM protein, NeuT protein and Ras protein on total protein extract of MCF10A-pBabe, MCF10A-NeuT and MCF10A-RasV12 stable cell lines infected with lentivirus expressing control vector (-) or ATM RNA interference vector (+).
Supplementary Fig. 5: ATM interference enhances MCF7 tumorigenicity in vivo. MCF7shR5, MCF7shATM, cells were subcutaneously injected into nude mice and tumor growth was monitored. (A) Quantification of tumor size during five weeks of xenograft growth. Data are shown as mean +SD. The experiment has been performed on 6 animals for each group. (B) Pictures representative of tumors after dissection.
Supplementary Fig. 6: KU-55933 systemic administration in vivo does not exert major toxicity
and efficiently targets ATM activity. (A) KU-55933 in vivo administration by intraperitoneal injection does not affect cell proliferation in vivo suggesting that this compound does not exert strong toxicity. MITO-LUC transgenic mice 46 harbor the luciferase gene under control of nuclear factor Y- dependent cyclin B2, allowing imaging of areas where proliferation is actively occuring, Mice were injected with 10 mg/kg KU-55933 (upper panels) or with DMSO as control (lower panels) every two days for 2 weeks. Bioluminescence imaging of a representative animal has been performed as previously described 46 and displays areas of active proliferation compared to pretreated animals (Pre). (B) KU-55933 administration in vivo significantly dampens ATM activation in response to Ionizing Radiation (IR). Mice were injected intraperitoneally with 10 mg/kg KU-55933 and 24 hours later they were irradiated as indicated. Immonoblots were performed on protein extracts derived from thymus collected at indicated times upon IR.
Supplementary Fig. 7: ATM modulates HER2-HSP90 interaction and coimmunoprecipitates with HER2 and HSP90 in vitro. (A) Cellular extracts from SKBR3 cell lines were prepared for immunoprecipitation with anti-her2 antibody and subjected to western blotting with anti-hsp90. Cells were infected with retrovirus expressing shatm RNA interference or shgfp as control or treated with ATM kinase inhibitor KU55933 (10µM for 24 hours). HSP90 inhibitor, 17-AAG (1 µm for 2 hours), was used as a positive control. Dissociation of HSP90 from HER2 was revealed after extensive immunoprecipitation of HER2 protein. Immunoblotting to detect HER2 expression in supernantants upon immunoprecipitation confirmed that HER2 was efficiently immunoprecipitated (left upper panel) as the supernatant was immunodepleted for HER2 (right upper panel). The amount of HSP90 interacting protein upon
treatments was independent of the decrease in HER2 protein levels. Western blots on total extracts were carried out loading the same amount of total proteins for each sample in order to assess the effect of the different treatments on HER2 protein levels. Tubulin has been used as loading control (lower panel). (B) ATM phosphorylation on Ser1981 (ATM-p) was detected by immunoblotting on protein extracts from HEK293T cells transiently transfected as indicated and incubated in the presence or not of ATM kinase inhibitor KU-55933 (24 hours). (C) HER2 has been immunoprecipitated by the indicated protein extracts and interacting proteins revealed by immunoblotting with the indicated antibodies.
Supplementary Fig. 8: Representative pictures of immunohistochemical analysis with ps1981atm (ATM-p) monoclonal antibody in HER2+ and HER2- human primary tumors samples (scale bars 30 m)
Supplementary Fig. 9: Full scans for Figure 1
Supplementary Fig. 10: Full scans for Figure 3 B, C, D
Supplementary Fig. 11: Full scans for Figure 3 F, G, H
Supplementary Fig. 12: Full scans for Figure 4A, B
Supplementary Fig. 13: Full scans for Figure 4 C
Supplementary Fig. 14: Full scans for Figure 4 D
Supplementary Fig. 15: Full scans for Figure 5 D
Supplementary Fig. 16: Full scans for Figure Supplementary 4A, B
Supplementary Fig. 17: Full scans for Supplementary Figure 7 B, C
Supplementary Table 1. Distribution of ATM-p positivity in breast carcinomas developed in multiple breast glands of NeuT mice treated or untreated with KU-55933 ATM-p positivity KU55933 Treated DMSO control Mouse 1 Mouse 2 Mouse 3 Total 1/4* (25%) 1/5* (20%) 3/4* (75%) 5/13* (39%) Mouse 1 Mouse 2 9/10* (90%) 6/6* (100%) 15/16* (94%) * n. of positive breast carcinomas developed in multiple breast glands; χ 2 : 0.003
Supplementary Table 2 Distribution of HER2 positivity in breast carcinomas developed in multiple breast glands of KU-55933 treated mice KU55933 Treated Untreated control HER2 Reduced Positivity Mouse 1t Mouse 2t Mouse 3t Total 4/4* (100%) 5/5* (100%) 3/4* (75%) 12/13* (92%) Mouse 1u Mouse 2u 0* 0* 0/16* *n. of reduced HER2 positivity breast carcinomas developed in multiple breast glands; F-Fisher: <0.0001
Supplementary Table 3 Clinico-pathological characteristics of 167 breast carcinomas Total number of pts 167 Age, median year (range) 56 (24-94) Tumor size T1 96 (57.5%) T2 57 (34.1%) T3/4 14 (8.4%) Grading G1 12 (7.2%) G2 82 (49.1%) G3 73 (43.7%) Lymph Node Negative 90 (53.9%) Positive 77 (46.1%) Estrogen Negative 76 (45.5%) Positive 91 (54.5%) Progesterone Negative 84 (50.3%) Positive 83 (49.7%) HER2 status Negative 80 (47.9%) Positive 87 (52.1%) Ki67 Low 65 (39.6%) High 99 (60.4%) ATM-p Negative 68 (40.7%) Positive 99 (59.3%) HER2 negative: 0/1+/2+NA; HER2 positive: 2+A/3+; Ki67 low: 15; high: >15
Supplementary Table 4 Univariate Analysis Factors Univariate analysis Patients n. 167 HR (95% CI) P- value Tumor size T2-T4 vs T1 1.20 (0.65-2.20) 0.554 Lymph node status Positive vs Negative 2.23 (1.18-4.19) 0.013 Grading G2 vs G1 1.29 (0.30-5.54) 0.736 G3 vs G1 1.60 (0.37-6.84) 0.528 Estrogen Positive vs Negative 0.33 (0.17-0.63) 0.001 Progesterone Positive vs Negative 0.52 (0.27-0.97) 0.041 HER2 Positive vs Negative 4.98 (2.20-11.28) <0.001 Ki67 High vs Low 4.01 (1.68-9.58) 0.002 ATM-p score Positive vs Negative 3.72 (1.72-8.08) 0.001 ER/PgR: negative: <10; positive 10; HER2 negative: 0/1+/2+Non Amplified; HER2 positive: 2+Amplified/3+; KI67 low: 15; high: >15 The Cox univariate model evidenced that Lymph node status (HR 2.23, C.I. 95%: 1.18-4.19, p=0.013), ER (HR 0.33, C.I. 95%: 0.17 0.63, p =0.001), PGR (HR 0.52, C.I. 95%: 0.27-0.97, p= 0.041), HER2 (HR 4.98, C.I. 95%: 2.20-11.28, p<0.001), Ki67 proliferation index (HR 4.01, C.I. 95%: 1.68-9.58, p = 0.002), and ATM-p positivity (HR 3.72, C.I. 95%: 1.72-8.08, p= 0.001) significantly impact on DFS.
SUPPLEMENTARY METHODS DNA constructs shatm construct #1, position 912: 5 -GACTTTGGCTGTCAACTTTCG-3 shatm #3 position, 8538, 5 - GGAGCGCACCATCTTCTTC-3 control shgfp: 5 - GGAGCGCACCATCTTCTTC-3 sir5: 5 -GGGATATCCC TCTAGATTA-3 Patients A retrospective series of 167 patients diagnosed with invasive breast cancer (BC) were selected from the surgical pathology files of the Regina Elena National Cancer Institute, (Rome, Italy) in order to obtain two different series: one HER2-positive (87 pts) and one HER2-negative (80 pts). The two series tients included 154 (92%) invasive ductal carcinomas, 11 invasive lobular carcinomas (7%), and 2 medullary carcinomas (1%). Ninety-six BC (57.5%) were T1, 57 (34.1%) were T2 and 14 (8.4%) were T3-T4. Twelve (7.2%) BC were graded as well differentiated (G1), 82 (49.1%) and 73 (43.7%) as moderately (G2) and poorly differentiated (G3) carcinomas, respectively. Ninety (53.9%) patients were node negative and 77 (46.1%) node positive. Estrogen (ER), Progesterone Receptors (PgR) and Ki-67 were positive in 91 (54.5%), 83 (49.7%) and 99 (60.4%) tumors respectively Furthermore, 90 (53.9%) patients were node positive and 77 (46.1%) node negative. Estrogen (ER) and Progesterone Receptors (PgR) were positive
in 91 (54.5%) BC and HER2 was positive in 87 (52.1%) cases. Ninety-nine cases presented a high proliferation index by Ki-67 (60.4%). ATM-p was positive, with variable degree of intensity (see Immunohistochemistry paragraph), in 99 (59.3%) cases (Supplementary Table S2). Immunohistochemistry and scoring criteria ER and PgR were analyzed by using the monoclonal antibodies (MoAbs) 6F11, 1A6 (Menarini, Florence, Italy) respectively. HER2 and Ki-67 were assessed by using the polyclonal antibody A0485 (PoAb, Dako, Milan, Italy) and the MoAb MIB-1 (Dako), respectively. ATM-p monoclonal antibody expression was analyzed using the MoAb 7C10D8, (Rockland, tebu-bio, Italy). The following scoring criteria have been adopted. ATM-p was considered as positive when at least 10% of the neoplastic cells showed a nuclear/cytoplasmic immunoreactivity. HER2 was scored as 0 and 1+ as negative, 2+ equivocal (to be confirmed by ISH), and 3+ positive. ER and PgR were considered positive when >10% of the neoplastic cells showed distinct nuclear immunoreactivity. Based on the median value of our series, Ki-67 was regarded as high if >15% of the cell nuclei were immunostained. Evaluation of the IHC results, blinded to all patient data, was performed independently by two investigators (MM, ADB). Statistical Analysis For Immunohistochemistry experiments on primary tumors sample descriptive statistics were used to summarize pertinent study information. The associations were tested by the Pearson Chi-Square test or Fisher Exact test when appropriate. P values <0.05 denotes statistically significant associations. The DFS curves were estimated by the Kaplan- Meier method. DFS was calculated from the date of tumor diagnosis to the date of first recurrence, including local relapses or distant metastases. Patients without recurrence were censored at the time of the last follow-up. We used the Log-rank test to compare the
survival curves. The Hazard Ratio (HR) and the 95% confidence interval (95% CI) were evaluated for each variable using the Cox univariate model. A multivariate Cox proportional hazard model was also developed using stepwise regression (forward selection) with predictive variables that were significant in the univariate analyses. The enter limit and remove limit were p = 0.10 and p = 0.15, respectively. Statistical analyses were carried out using SPSS software (SPSS version 20.0, SPSS Inc., Chicago, Illinois, USA).