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Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.
Rationally designed targeted therapies could represent the future of personalized medicine for cancer patients. Trastuzumab, one of the most successful examples of these therapies, is a humanized antibody targeting human epidermal growth factor receptor-2 (HER2 or ERBB2). It shows considerable clinical efficacy and extends the overall survival of certain patients with ERBB2-overexpressing breast cancer1,2. However, the overall response rate to trastuzumab-containing therapies remains modest: ~26% when used as a single therapy and 40–60% when used in combination with systemic chemotherapy1,3,4. Many patients do not respond to initial trastuzumab treatment (de novo resistance), and many trastuzumab-responsive patients develop resistance after continuous treatment (acquired resistance)5,6. Although multiple trastuzumab resistance mechanisms have been identified in preclinical studies, no effective regimen has been developed to overcome trastuzumab resistance in patients. More important, inherent tumor heterogeneity and alternative survival pathways developed during drug treatment pose additional challenges to the clinical management of patients with trastuzumab-resistant tumors of different genesis.
Two major categories of trastuzumab resistance mechanisms have been proposed: de novo resistance due to genetic alterations of receptor tyrosine kinases (RTKs) and their downstream signaling targets; and acquired resistance primarily due to the acquisition of alternative RTK signaling activation that compensate for ERBB2 inhibition after trastuzumab treatment. The most prevalent de novo resistance mechanisms include constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway owing to phosphatase and tensin homolog (PTEN) deficiency7 or PIK3CA gene mutations8, and the accumulation of truncated ERBB2 receptors (p95HER2), which lack an extracellular trastuzumab-binding domain9. The overexpression of other RTKs, such as epidermal growth factor receptor (EGFR) family receptors10–12, insulin-like growth factor-1 receptor (IGF-1R)13,14 and hepatocyte growth factor15, also contribute to both de novo and acquired trastuzumab resistance. Heterodimerization between RTKs may redundantly trigger cell proliferation signals and confer resistance when ERBB2 is inhibited by trastuzumab16,17.
One could design targeted therapy corresponding to a specific resistance mechanism. However, it is possible that multiple resistance mechanisms may coexist in patients with late-stage, heterogeneous, metastatic breast cancer. It would be more effective and clinically practical to identify and target the key nodes common to several (if not all) of the aforementioned resistance mechanisms.
Here we report a rationally designed therapy targeting the key node in multiple signaling pathways driving trastuzumab resistance, which might broadly benefit trastuzumab-resistant patients. We identify the nonreceptor tyrosine kinase SRC as a common node, which is hyperactivated in various trastuzumab resistance models. We also show a direct dephosphorylation of SRC by PTEN’s protein phosphatase activity, which is involved in de novo trastzumab resistance, conferred by PTEN deficiency. SRC activation confers trastuzumab resistance in breast cancer cells and is correlated with both lower response and poorer survival in patients who received trastuzumab-based therapy. Notably, targeting this key node with a SRC inhibitor universally sensitized cells bearing distinct resistant mechanisms to trastuzumab treatment in vitro and in vivo. Our data indicate that combinatorial treatment with SRC inhibitor plus trastuzumab could be a clinically useful strategy for overcoming trastuzumab resistance conferred by multiple mechanisms.
First, we modeled the development of acquired resistance in patients, by treating ERBB2-overexpressing breast cancer cells (BT474, AU565, and SKBR3) with increasing doses of trastuzumab for >6 months to select trastuzumab-resistant sublines (TtzmR). Compared with parental lines, TtzmR cells were significantly (P = 0.003, 0.001 and 0.001) more resistant to trastuzumab treatment in vitro (Fig. 1a and Supplementary Fig. 1). Orthotopic xenograft tumors of BT474. TtzmR cells were significantly (P = 0.001) less responsive to trastuzumab treatment (Fig. 1b). Prolonged treatment with trastuzumab leads to alterations (‘reprogramming’) of various RTKs17. Indeed, we found a substantial downregulation of ERBB2 and a prominent upregulation of EGFR (in all three cell lines), HER3 (in AU565) and IGF-1R (in AU565) in the TtzmR sublines compared with parental cell lines (Fig. 1c,d), suggesting that increased EGFR, HER3 and IGF-1R signaling may be acquired resistance mechanisms. Consistently, EGFR phosphorylation (at Tyr1068) was increased in TtzmR lines, whereas ERBB2 receptor was downregulated (Fig. 1e). Notably, TtzmR lines showed increased SRC phosphorylation at Tyr416 suggesting SRC activation, which was further confirmed by SRC kinase assay (Supplementary Fig. 2). The unchanged PTEN protein and AKT phosphorylation in TtzmR lines (Fig. 1e) indicate that the acquired trastuzumab resistance was not due to selection of a pre-existing sub-population with PI3K pathway alterations.
Next, we investigated the role of alternative RTK pathways in SRC activation by ectopic overexpression of EGFR or IGF-1R in the parental trastuzumab-sensitive BT474 cells. Overexpression of either RTK led to increased phosphorylation of SRC Tyr416 (Fig. 1f) and a trastuzumab-resistant phenotype (Fig. 1g). Knockdown of EGFR in the BT474.TtzmR cells inhibited phosphorylation of SRC Tyr416 (Fig. 1h). shRNA-mediated knockdown of either EGFR (Fig. 1i) or SRC (Fig. 1j) considerably resensitized the TtzmR cells to trastuzumab treatment. Collectively, these data indicate that SRC activation is a critical event downstream of alternative RTK signaling pathways contributing to acquired trastuzumab resistance.
PTEN deficiency confers de novo trastuzumab resistance7. Knocking down PTEN with either antisense oligonucleotides (PTEN.as) or shRNA (PTEN-shRNA) conferred considerable resistance to trastuzumab compared with control cells (Fig. 2a and Supplementary Fig. 3). Notably, although PTEN knockdown had no discernible effect on EGFR, it led to an increase in phosphorylation of SRC Tyr416, indicating that PTEN loss also led to SRC activation (Fig. 2b and Supplementary Fig. 4). Moreover, PI3K inhibitors (LY294002 or wortmannin) did not inhibit SRC activation in PTEN-knockdown cells (Fig. 2c and Supplementary Fig. 5), indicating that SRC activation is not due to PTEN loss–mediated AKT activation.
As PTEN is a dual lipid and protein phosphatase18, we next explored whether SRC Tyr416 is a direct substrate for PTEN protein phosphatase activity and if increased SRC phosphorylation at Tyr416 is a direct result of PTEN loss. In PTEN-deficient MDA-MB-468 cells (Fig. 2d) or PTEN-knockdown BT474 cells (Supplementary Fig. 6), we reconstituted either wild-type PTEN or two PTEN mutants identified from patients’ tumors (a C124S mutant, deficient in lipid and protein phosphatase activities; a G129E mutant, deficient in lipid phosphatase activity only19). The wild-type PTEN and the G129E mutant (but not the C124S mutant) substantially reduced phosphorylation of SRC Tyr416 without affecting Tyr527. Moreover, coimmuno-precipitation (Fig. 2e) and GST pull-down assays (Supplementary Fig. 7) demonstrated a direct interaction between PTEN and SRC proteins. Furthermore, in a cell-free PTEN phosphatase assay, GST-PTEN G129E did not dephosphorylate Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) as wild-type PTEN did (Supplementary Fig. 8). However, the G129E mutant readily released phosphates from the SRC peptide phosphorylated at Tyr416 without strong effect on the SRC peptide phosphorylated at Tyr527 (Fig. 2f). The data indicate that PTEN directly and specifically dephosphorylates SRC at Tyr416 by its protein phosphatase activity. Notably, knockdown of SRC in PTEN-deficient BT474 parental cells significantly (P = 0.013 and P = 0.008 compared with shPTEN) restored the sensitivity of these cells to trastuzumab treatment (Fig. 2g,h). Therefore, loss of PTEN protein phosphatase activity led to SRC activation that contributed to the de novo trastuzumab resistance in PTEN-deficient cells.
As SRC is commonly activated in EGFR-overexpressing, IGFR-overexpressing and PTEN-deficient cells, SRC activation seems to be a common event downstream of various signaling pathways leading to trastuzumab resistance. To determine whether SRC activation is sufficient to confer trastuzumab resistance, we stably expressed a constitutively active SRC Y527F mutant and a dominant-negative SRC K295R mutant20, in both BT474 and AU565 cells. Indeed, cells expressing SRC Y527F showed increased SRC activation and hyperphosphorylation of SRC Tyr416 and the SRC downstream target paxillin at Tyr118 (Fig. 3a and Supplementary Fig. 9). A notably increased phosphorylation of SRC Tyr527 (an inhibitory site for SRC activity) and reduced phosphorylation of paxillin Tyr118 indicated reduced SRC activity in SRC K295R–transfected cells (Fig. 3a). Compared with controls, SRC Y527F cells were highly resistant to trastuzumab-mediated growth inhibition whereas SRC K295R cells were more sensitive (Fig. 3b). Similarly, overexpression of wild-type SRC also conferred SRC activation and trastuzumab resistance (Supplementary Figs. 10 and 11).
Additionally, SRC Y527F cells responded poorly to trastuzumab in a three-dimensional (3D) tumor spheroid assay (Fig. 3c). Furthermore, trastuzumab treatment did not inhibit the growth of BT474.SRC Y527F mammary fat pad tumors (Fig. 3d). Collectively, these data indicate that SRC activation is sufficient to confer trastuzumab resistance.
To examine whether SRC activation is involved in clinical trastuzumab resistance, we retrospectively evaluated the relationship between SRC activation and patient response to trastuzumab-based therapies. We assessed SRC activation in primary breast tumors from 57 patients, who were subsequently treated with trastuzumab-based therapies (Supplementary Table 1), by immunohistochemistry staining using a validated antibody for SRC phosphorylated at Tyr416 (pSRC-Y416; Supplementary Fig. 12).
In general, the phosphorylation of SRC Tyr416 is positively correlated (Pearson r = 0.297, P = 0.025) with total SRC protein abundance (data not shown). Notably, patients with high amounts of phosphorylated SRC (pSRC) in tumors showed a lower clinical response rate and a higher progressive disease rate after trastuzumab treatment than patients having low pSRC tumors (Fisher’s exact test, P = 0.011, Fig. 3e). The overall survival of patients with high pSRC tumors (median 34.2 months) was significantly (log-rank test, P = 0.044, Fig. 3f) lower than that of patients with low pSRC tumors (median 57.9 months) after trastuzumab-based therapy (Fig. 3f). These data suggest that SRC activation is correlated with clinical trastuzumab resistance.
To study the mechanism of SRC activation–mediated trastuzumab resistance in TtzmR cells, we modulated SRC activity using either the small-molecule inhibitor saracatinib or SRC shRNA in TtzmR cells. In TtzmR cells, compared with parental cells, the amount of total EGFR and receptor dimerization increased under EGF stimulation (Supplementary Fig. 13). SRC knockdown inhibited EGF-induced EGFR dimerization (Fig. 4a) and suppressed EGFR phosphorylation at Tyr845, a known SRC-dependent EGFR phosphorylation site21, and at Tyr1068, an autophosphorylation site, under both basal and EGF-stimulated conditions (Fig. 4b). Saracatinib treatment also inhibited EGFR phosphorylation in TtzmR cells (Fig. 4c). These data indicate an essential role of SRC in mediating EGFR signaling and the existence of a positive feedback loop between EGFR and SRC22. Notably, trastuzumab treatment transiently induced ERBB2 receptor dimerization and phosphorylation, which led to a further increase of SRC activation in TtzmR cells (Supplementary Figs. 14 and 15). Inhibition of SRC activity by either saracatinib or knocking down SRC (shRNA.SRC2) diminished trastuzumab-induced transient ERBB2 phosphorylation (Fig. 4d,e). Additionally, SRC inhibition potentiated trastuzumab-mediated inhibition of phosphorylation of HER3, a critical dimerization partner of ERBB2 (refs. 17,23,24; Fig. 4d,e), although SRC inhibition had no discernable effect on ERBB2-HER3 heterodimerization (Supplementary Fig. 16). These data indicate that SRC activation induces trastuzumab resistance by facilitating EGFR, ERBB2 and HER3 activation in TtzmR cells.
Furthermore, we examined the effects of saracatinib and trastuzumab treatment on the signaling events downstream of RTKs. Compared with a complete inhibition of AKT phosphorylation by trastuzumab in parental cells (Supplementary Fig. 17), TtzmR cells were largely resistant to trastuzumab-mediated AKT inhibition (Fig. 4f), perhaps owing to alternative RTK activation. Concurrent treatment with saracatinib inhibited AKT activation in TtzmR cells (Fig. 4f). Moreover, this combination also inhibited AKT phosphorylation, along with SRC phosphorylation, in PTEN-deficient de novo trastuzumab-resistant cells (Fig. 4g), SRC.Y527F cells (Fig. 4h) and EGFR-overexpressing cells (Supplementary Fig. 18). Together, these data indicate that RTK activation and their downstream signaling events (AKT activation) involved in trastuzumab resistance can be effectively blocked by inhibiting SRC.
As our data indicate that SRC sits on the signaling hub mediating multiple types of trastuzumab resistance of different mechanisms (both acquired and de novo), we hypothesized that targeting this key node could be a more effective strategy to overcome trastuzumab resistance conferred by multiple mechanisms. To target SRC, we used saracatinib, an orally available small-molecule SRC inhibitor tolerated well in phase 1 and 2 clinical trials25. Saracatinib treatment universally sensitized trastuzumab-resistant cells to trastuzumab treatment in all models tested, including acquired resistance, PTEN deficiency, EGFR overexpression, IGF-1R overexpression (Fig. 5a) and SRC constitutive activation (Fig. 5b). Combination treatment also suppressed 3D tumor spheroid formation in both BT474 acquired resistant cells (Fig. 5c) and PTEN-deficient cells (Supplementary Fig. 19). We also observed similar inhibitory effects in clonogenic assays (Supplementary Fig. 20).
Next, we examined whether the AKT inhibitor triciribine could have a similar sensitization effect to saracatinib. In all models we tested, saracatinib was more effective in sensitizing resistant cells to trastuzumab compared with triciribine (Fig. 5d and Supplementary Fig. 21). As treatment with a combination of trastuzumab and saracatinib effectively suppressed AKT signaling (Fig. 4f,g) and induced cell blebbing in 3D drug response assays (Supplementary Fig. 19), we examined whether SRC inhibition could sensitize trastuzumab-resistant cells to treatment partially through induction of apoptosis. Indeed, combinatorial treatment considerably increased the number of TUNEL-positive cells in PTEN knockdown cells (Fig. 5e) and increased sub-G1 populations in PTEN-deficient and SRC Y527F cells (Fig. 5f). These consistent data from multiple trastuzumab resistance models demonstrate that inhibition of SRC enhances trastuzumab-mediated growth inhibition by promoting apoptosis.
To test whether targeting SRC could be an effective strategy to overcome trastuzumab resistance in vivo, we first knocked down SRC in trastuzumab-resistant, PTEN-deficient mammary fat pad tumors by intratumoral injection of viral particles carrying SRC shRNA. Trastuzumab treatment led to a significant (P = 0.0003) shrinkage of tumors injected with SRC shRNA but not control shRNA (Fig. 6a). In the acquired resistance model, trastuzumab plus saracatinib greatly inhibited BT474.TtzmR tumors, whereas saracatinib alone at 25 mg per kg body weight daily (a dosage tolerated well by humans25,26) was not effective. This tumor inhibition by trastuzumab plus saracatinib was more substantial than trastuzumab plus triciribine (Fig. 6b). Notably, in the PTEN-deficiency model, combinatorial treatment eliminated tumors and most tumors were barely detectable 21 d after treatment (Fig. 6c). Phosphorylation of AKT in PTEN.shRNA xenograft tumors was also decreased by combinatorial treatment but not by single treatment alone (Fig. 6d). Notably, although combinatorial treatment showed a statistically insignificant trend of decreased Ki67 staining (data not shown) compared with controls, there was a significant (P = 0.039) increase of TUNEL-positive staining in these tumors, suggesting an induction of apoptosis by the combinatorial treatment (Fig. 6e). Collectively, these data indicate that SRC inhibition by saracatinib sensitizes trastuzumab-resistant tumors to trastuzumab in vivo, at least partially through induction of apoptosis.
Recent studies have highlighted a scenario in which, upon prolonged treatment by a highly specific targeted therapy (such as trastuzumab), tumor cells reprogram themselves to develop alternative compensatory pathways to sustain cell proliferation, ultimately leading to drug resistance17,27. Additionally, pre-existing genetic alterations of the RTK-PI3K–AKT pathway (such as PTEN loss and IGF-1R overexpression) make tumor cells inherently more resistant to trastuzumab7,8,13. Owing to the heterogeneous nature of tumors, different resistance mechanisms may coexist in the same patient. Thus, targeting one molecule or one resistance mechanism is usually ineffective27. Targeting multiple altered signaling pathways that confer resistance is warranted. However, targeting multiple pathways by simply combining several US Food and Drug Administration–approved drugs against each pathway may not always be feasible and may cause severe side effects in some patients28. To develop more effective regimens to overcome drug resistance, we need to further dissect the signaling events in resistant tumors. Identification of the ‘common signaling nodes’ involved in multiple resistance mechanisms may help researchers to rationally design new combinatorial therapies.
SRC interacts with multiple RTKs through its SH2 domain and facilitates RTK-mediated signaling20,29–34. Small-molecule inhibitors targeting multiple kinases including SRC (such as PP2 and Dasatinib) potentiate the initial efficacy of anti-RTK therapies in naive tumor cells35–37. A recent study also suggested that SRC activation may contribute to transient trastuzumab resistance induced by TGF-β treatment36. Here we demonstrate that, in both acquired and de novo trastuzumab-resistant tumors, SRC has a universal role in mediating multiple resistance pathways including alternative activation of RTK pathways and PTEN deficiency. This places SRC at a common signaling node for trastuzumab resistance (Fig. 6f). Thus, we propose that instead of targeting each of the RTK or PI3K-AKT pathway alterations individually, researchers may effectively overcome trastuzumab resistance resulting from multiple mechanisms by inhibiting this common key node. Our data support this new concept. Targeting SRC universally sensitized trastuzumab-resistant cells to trastuzumab treatment and significantly suppressed tumor growth in vivo in multiple preclinical resistance models (Fig. 6f). This new combination strategy warrants further clinical investigation in patients with trastuzumab-resistant tumors having different de novo or acquired resistance mechanisms. Furthermore, similar strategies may also be applied to overcoming resistance to other RTK-targeting drugs.
Previously, we tested whether we could overcome de novo trastuzumab resistance conferred by PTEN deficiency by combining trastuzumab with PI3K-AKT pathway inhibitors38. In our side-by-side comparison of trastuzumab plus SRC inhibitor versus trastuzumab plus AKT inhibitor in vitro and in vivo, we observed that combination with the SRC inhibitor saracatinib was more effective in overcoming trastuzumab resistance in both TtzmR and PTEN deficiency models38. Notably, from a clinical perspective, toxicity is a critical consideration in prescribing further treatment for trastuzumab-resistant patients who have already been heavily treated. The extensively documented side effects of the clinically applied AKT inhibitor triciribine39 may preclude the combination of this drug with trastuzumab in trastuzumab-resistant patients, although this combination showed some efficacy in our TtzmR xenograft model. In contrast, the SRC inhibitor saracatinib is tolerated well in phase 1 and 2 clinical trials25. Thus, combination with SRC-targeting agents is a clinically more applicable treatment for trastuzumab-resistant patients.
In addition to the AKT pathway40, SRC also modulates many downstream targets, including focal adhesion kinase41, signal transducer and activator of transcription-3 (ref. 42), mitogen-activated protein kinases43,44, the protein kinase C family45 and c-MYC46. These signaling pathways have diverse roles in regulating tumor cell survival47–49 and metastasis50,51. Combinatorial therapy with trastuzumab and saracatinib could have a broad impact on tumor progression. Particularly, this combination treatment inhibited migration of trastuzumab-resistant cells (Supplementary Fig. 22), suggesting a potential benefit of this regimen in preventing further metastatic recurrence for patients with metastatic breast cancer.
In conclusion, our studies identified SRC activation as a key convergence point of multiple trastuzumab resistance mechanisms. The combinatorial regimen of SRC inhibitor plus trastuzumab is effective in overcoming various de novo and acquired resistance mechanisms. Our findings may directly impact the clinical management of patients with ERBB2-overexpressing breast cancer.
We obtained AU565, SKBR3 and MDA-MB-468 cells from American Type Culture Collection. We obtained the tumorigenic BT474.m1 subline (BT474) as previously described7. We purchased pLKO.1-based shRNAs from Sigma-Aldrich and obtained the SRC mutants (Y527F and K295R) and GST-PTEN constructs from Addgene. We established acquired trastuzumab-resistant cells as described52. We carried out 3D tumor spheroid culture as described53. We subcloned human EGFR cDNA into pcDNA6 vector.
We carried out cell proliferation assay using the CellTiter 96 AQueous cell proliferation assay kit (Promega). We calculated the percentage of inhibition of cell proliferation as [1 – (treated cells / untreated cells)] × 100. We carried out cell cycle analysis and APO-BrdU TUNEL assay (Invitrogen) on the basis of the manufacturer’s protocols.
To detect receptor dimerization, we cross-linked membrane RTKs by resuspending the cells in 5 mM Sulfo-EGS prepared in PBS on ice for 2 h followed by adding 10 mM Tris (final concentration). For coimmunoprecipitation experiments, we incubated the total cell lysate with first antibody overnight at 4 °C and immunoprecipitated the immunocomplex with protein G–agarose. For detection of ERBB2 and HER3 interaction, we covalently immobilized antibody to ERBB2 on UltraLink Biosupport resin (Thermo Fisher Scientific) before carrying out immunoprecipitation.
We reconstituted the synthetic SRC peptides phosphorylated at Tyr416 and Tyr527 (SRC-Y416 (RRLIEDNE-pY-TARG) and SRC-Y527 (TSTEPQ-pY-QPGENL); AnaSpec) in phosphate-free water. We carried out the phosphatase assay by incubating 250 ng of purified GST-PTEN protein with 100 µM phosphorylated SRC peptide in 25 µl phosphatase assay buffer (25 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 10 mM DTT) for 30 min at 37 °C. We measured the released free phosphate from substrate using Malachite Green reagent (Echelon Biosciences).
We collected primary breast cancer specimens from women at MD Anderson Cancer Center from 1996 to 2005 under a protocol approved by the MD Anderson Cancer Center Institutional Review Board. Informed consent was obtained from all subjects. We included patients who received first-line trastuzumab-based therapies (without prior chemotherapy) in our retrospective analysis. We evaluated the clinical response to trastuzumab-based therapy every 8–12 weeks using response evaluation criteria in solid tumors with modifications. We defined clinical benefit as complete response, partial response or stable disease longer than 6 months. We defined overall survival time after trastuzumab treatment as the time from initiation of trastuzumab treatment to death.
We used the following primary antibodies for immunohistochemistry staining: PTEN (138G6, Cell Signaling), phospho-SRC-Y416 (2101, Cell Signaling), phospho-AKT-S473 (736E11, 1:150, Cell Signaling) and Ki67 (MIB-1, 1:75, Dako). Retrieval butters were: 0.01 M citrate retrieval buffer, pH 6.0, for Ki67 staining; 1 mM EDTA, pH 8.0, for PTEN, phospho-SRC-Y416 and phospho-AKT-S473 staining. We scored staining by H-score according to the staining intensity and percentage of positive cells within the whole tissue section. We considered cases with phospho-SRC-Y416 H-score > 20 (>10%, ++; 20%, +) to have high phospho-SRC-Y416 staining. We classified phospho-AKT-S473 staining intensity into four groups (0, 1, 2 and 3+) by H-score. Phospho-AKT staining with H-score ≥ 250 is defined as 3+. We quantified PTEN expression using the immunoreactive score as described7.
All mouse experiments were carried out in accordance with approved protocols from the Institutional Animal Care and Use Committee of MD Anderson Cancer Center. We implanted 6-week-old female severe combined immunodeficiency mice with 0.72 mg 60-d release 17β-estradiol pellets (Innovative Research). Then we established mammary fat pad tumors by injection of 8 × 106 BT474.m1 cells in 100 µl of PBS-Matrigel mixture (1:1 ratio) orthotopically into the mammary fat pads. When xenografts reached about 100–150 mm3, we started to treat the mice with trastuzumab (10 mg per kg−1) or vehicle control (IgG) intraperitoneally once per week. We treated mice with saracatinib daily at a dose of 25 mg per kg body weight in vehicle (0.5% wt/vol hydroxypropyl-methylcellulose with 0.1% vol/vol Tween 20) via oral gavage. We treated mice with triciribine by intraperitoneal daily at dose of 0.5 mg per kg body weight. For intratumoral lentivirus injection, we concentrated lentiviral particles containing pLKO.con.shRNA or pLKO.SRC shRNA using PEG-it virus precipitation solution (System Biosciences) to reach ~2.6 × 107 transduction units (TU) ml−1. We carried out intratumoral injection of 50 µl of concentrated virus every 3 d. We calculated tumor volume as length × width2/2. For tissue TUNEL staining, the apoptotic index was expressed as a percentage of TUNEL-positive cells per section. We excluded necrotic areas in the tumors from evaluation.
We analyzed quantitative results either by one-way analysis of variance (multiple groups) or t test (two groups). For patient samples, we calculated correlation by Fisher’s exact test. We carried out survival analysis using Kaplan-Meier log-rank test. We considered a difference with P< 0.05 (two-sided) statistically significant.
Detailed methodology is described in Supplementary Methods.
We thank AstraZeneca for providing us with saracatinib and W. Zhang in the Department of Pathology, MD Anderson Cancer Center for providing us with the IGF-1R expression vector. We also thank C. Neal, J. Lu, S. Rehman and F.J. Lowery for insightful comments on this manuscript. This work was supported by US National Institutes of Health grants P30-CA 16672 (MD Anderson Cancer Center), RO1-CA112567 (D.Y.), PO1-CA099031 project 4 (D.Y.), MD Anderson Cancer Center Breast SPORE P50 CA116199 (G.N.H.) project 4 (D.Y.), US Department of Defense Center of Excellence subproject W81XWH-06-2-0033 (D.Y.), Susan G. Komen Breast Cancer Foundation promise grant KG091020 (D.Y.), Cancer Prevention Research Institute of Texas grant RP100726 (D.Y.), the MD Anderson Cancer Center Breast SPORE Career Development Award (S.Z.) and the Susan G. Komen Breast Cancer Foundation postdoctoral fellowship (S.Z.). D.Y. is the Hubert L. and Olive Stringer Distinguished Chair in Basic Science at MD Anderson Cancer Center.
Note: Supplementary information is available on the Nature Medicine website.
AUTHOR CONTRIBUTIONSS.Z., W.-C.H. and D.Y. designed experiments and analyzed data; S.Z., W.-C.H., H.G., P.L., S.-B.P., S.W.B., Y.X., L.-M.T. and Z.D. carried out experiments; S.Z., H.G. and S.-H.L. did statistical analysis of clinical data; A.A.S. collected tumor samples and evaluated immunohistochemistry staining with H.G.; G.N.H. and F.J.E. collected clinical patient information and analyzed patient response data; S.Z., S.W.B. and D.Y. wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.