Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation
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Estrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation.
Inhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547.
Treatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/β and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation.
These data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.
KeywordsAKT ER+ breast cancer endocrine resistance IGF-IR InsR
estrogen receptor α-positive
fetal bovine serum
fibroblast growth factor receptor
forkhead box class O
insulin-like growth factor-I receptor
improved minimum essential medium
long-term estrogen deprivation
PI3K catalytic subunit p110α
- PIK3R1 PI3K
regulatory subunit p85α
PI3K catalytic subunit p110β
quantitative polymerase chain reaction
receptor tyrosine kinases
small interfering RNA
tyrosine kinase inhibitor.
AKT is a serine/threonine kinase downstream of phosphatidylinositol-3 kinase (PI3K) that plays a critical role in cellular survival, proliferation, metabolism and resistance to apoptosis . Upon activation by growth factor receptor tyrosine kinases (RTKs) and G-protein-coupled receptors, PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 then recruits pleckstrin homology (PH) domain-containing proteins such as PDK1, SGK and AKT to the plasma membrane, where AKT is phosphorylated at T308 by PDK-1 and, subsequently, at S473 by TORC2, becoming fully activated [1, 2].
The PI3K/AKT signaling pathway is the most frequently mutated pathway in breast cancer [2, 3, 4]. PI3K is activated via several mechanisms, including gain-of-function mutations in the PI3K catalytic subunit p110α (PIK3CA) and regulatory subunit p85α (PIK3R1), amplification of wild type PIK3CA, p110β (PIK3CB) and PDK1, loss/inactivation of the PIP3 phosphatases PTEN and INPP4B, mutation and/or amplification of AKT1-3 and amplification of RTKs, such as HER2, IGF-IR, MET, FGFR1 and EGFR [3, 5]. These cumulative data have suggested AKT as a rational molecular target for breast cancer therapy.
About 80% of breast cancers express estrogen receptor α (ER) and/or progesterone receptor (PR), biomarkers indicative of hormone dependence . Therapies against ER+ breast cancers inhibit ER function either by antagonizing ligand binding to ER (tamoxifen), downregulating ER (fulvestrant) or blocking estrogen biosynthesis (aromatase inhibitors (AIs)). However, many tumors exhibit de novo or acquired resistance to endocrine therapies. Overexpression of the ErbB2/HER2 protooncogene has been shown to promote clinical resistance to antiestrogen therapy [7, 8]. However, <10% of ER+ breast cancers overexpress HER2, suggesting that, for the majority of ER+ breast cancers, mechanisms of escape from endocrine therapy remain to be discovered.
The PI3K pathway has been causally associated with resistance to endocrine therapy [9, 10, 11, 12, 13, 14]. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on PI3K/AKT signaling . Herein we show that inhibition of AKT using the catalytic inhibitor AZD5363, currently in phase I clinical trials, suppressed hormone-independent ER+ breast cancer growth. However, upregulation of IGF-IR/InsR and their ligands compensated for AKT inhibition and limited the effect of AZD5363. Addition of an IGF-IR/InsR tyrosine kinase inhibitor (TKI) enhanced the action of AZD5363 against MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation, suggesting a novel and testable therapeutic combination for patients with ER+ breast cancer.
Cell lines (ATCC, Manassas, VA, USA) were maintained in improved minimum essential medium (IMEM)/10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA) and authenticated by short tandem repeat profiling using Sanger sequencing (sequenced in March 2011). Long-term estrogen deprived (LTED) cells were generated and maintained in phenol red-free IMEM with 10% dextran/charcoal-treated FBS (DCC-FBS) .
Immunoblot analysis and RTK arrays
Lysates from cells treated with AZD5363 , IGF-I, IGF-II, IGFBP-3 (R&D Systems, Minneapolis, MN, USA), AEW541  or BKM120  (Selleck Chemicals, Houston, TX, USA) were subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblot analysis  using antibodies against P-AKTS473, P-AKTT308, AKT, P-PRAS40, P-GSK-3α/β, P-S6S240/244, S6, P-IGF-IRβY1131/P-InsRβY1146, P-HER3Y1197, P-HER2Y1248, P-SrcY416, P-FRS2-αY436, EGFR (Cell Signaling, Danvers, MA, USA), InsRβ, IGF-IRβ, ERα (F-10), HER3, HER4, FGFR2 (Santa Cruz Biotechnology, Dallas, TX, USA), HER2 (NeoMarkers, Fremont, CA, USA), PR (Dako, Carpinteria, CA, USA), IRS-1 (EMD Millipore, Billerica, MA, USA), and actin (Sigma-Aldrich, St. Louis, MO, USA). Densitometric analysis was performed using ImageJ. Phospho-RTK arrays were performed using the Human Phospho-RTK Array Kit according to the manufacturer's protocol (R&D Systems).
Cells seeded in triplicate in 12-well plates (2.5 × 104 cells/well for MCF-7/LTED and 4 × 104 cells/well for other lines) were treated in 10% DCC-FBS ± AZD5363, selumetinib (AZD6244, ARRY-142886)  (Selleck Chemicals), fulvestrant (ICI 182780, R&D Systems), 17β-estradiol (E2) or AZD9362 (AstraZeneca, Cambridge, MA, USA). AZD9362 is a reversible, ATP-competitive small molecule inhibitor of IGF-IR and insulin receptor. In isolated enzyme assays, it inhibits the IGF-IR enzyme with an IC50 of 14 nM. In cellular assays, the compound prevents autophosphorylation of IGF-IR in fibroblasts from IGF-IR knockout mice stably transfected with human IGF-IR with an IC50 of 48 nM; it inhibits autophosphorylation of human InsR in CHO-T cells with an IC50 of 186 nM. AZD9362, dosed at 25 mg/kg qd, also inhibits phosphorylation of IGF-IR by >50% for at least six hours and induces >70% inhibition of tumor volume in NIH3T3 fibroblasts stably transfected with IGF-IR. Media and inhibitors for proliferation assays were replenished every three days; after five to ten days, adherent cells were trypsinized and counted using a Coulter Counter or fixed/stained with crystal violet . For siRNA experiments, cells were transfected in 100-mm dishes using HiPerfect Transfection Reagent according to the manufacturer's protocol (Qiagen, Germantown, MD, USA). The next day, cells were re-seeded in 10% DCC-FBS for immunoblot analyses as described previously  or cell proliferation assays and counted five to ten days later. siRNAs targeting IGF-IR, InsR, HER3, or non-silencing control were obtained from Qiagen.
Cells grown in 10% DCC-FBS ± AZD5363 were harvested and their RNA extracted using the RNeasy Mini Kit (Qiagen). Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 µg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SABiosciences (Qiagen). For siRNA experiments, cells were transfected with siRNA targeting forkhead box class O (FoxO3) (Life Technologies), ER or non-silencing control (Qiagen) using Dharmafect 1 according to the manufacturer's protocol (Thermo Fisher Scientific, Pittsburgh, PA, USA). Two days later cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours followed by RNA isolation and RT qPCR.
MCF-7/LTED cells plated in 35-mm dishes with No. 1.5 coverglass coated with Poly-d-lysine (MatTek, Ashland, MA, USA) were transfected with 2.5 µg of an AKT-PH-GFP plasmid (provided by Dr. Gordon Mills, MD Anderson Cancer Center, Houston, TX, USA) using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen, Life Technologies, Grand Island, NY, USA). On day four, cells were treated with 10% DCC-FBS ± AZD5363, AEW541 or BKM120 for four hours. Cells were viewed on an LSM 510Meta confocal microscope at 40x magnification at the Vanderbilt University Cell Imaging Shared Resource.
Mouse xenograft experiments
Animal experiments were approved by the Vanderbilt Institutional Animal Care and Use Committee. Female ovariectomized athymic mice were implanted s.c. with a 14-day-release E2 pellet (0.17 mg; Innovative Research of America, Sarasota, Florida, USA). The next day, 107 MCF-7 cells suspended in IMEM and mixed with matrigel (BD Biosciences, San Jose, California, USA) at 1:1 ratio were injected s.c. into the right flank of each mouse. After >2 weeks, mice bearing tumors ≥150 mm3 were randomized to treatment with vehicle (25% (2-hydroxypropyl)-β-cyclodextrin), AZD5363 (150 or 100 mg/kg bid p.o.), fulvestrant (5 mg/week i.p.), AZD9362 (25 mg/kg/day p.o.) or AZD4547  (12.5 mg/kg/day p.o.). Combining 150 mg/kg/day AZD5363 with AZD9362 and AZD4547 resulted in excessive toxicity, so a lower dose of AZD5363 (100 mg/kg/day p.o.) was used in this experiment. Tumor diameters were measured twice weekly and volume in mm3 calculated as volume = width2 x length/2. Tumors were harvested one or four hours after the last dose of AZD5363 or 24 hours after the last dose of fulvestrant and flash-frozen in liquid nitrogen or fixed in 10% formalin prior to paraffin-embedding. Frozen tumors were homogenized using the TissueLyser II (Qiagen). Tumor lysates were prepared, subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblot analysis .
In cell proliferation assays, significant differences were determined by one-way analysis of variance (ANOVA) (one cell line) or two-way ANOVA with Bonferroni post-hoc tests corrected for multiple comparisons. Unpaired t-tests were used to determine significant differences in crystal violet assays and real-time qPCR assays. Two-way ANOVA with Bonferroni post-hoc tests corrected for multiple comparisons was used to determine significance in real-time qPCR assays comparing multiple cell lines. In tumor growth assays, significant differences were determined by unpaired t-tests. Significant differences in immunohistochemistry (IHC) histoscores were determined by unpaired t-tests. P <0.05 was considered significant.
[See Additional file 1 for Supplementary materials and methods.]
Inhibition of AKT suppresses hormone-independent breast cancer cell growth
Combined inhibition of AKT and ER suppresses growth of MCF-7 xenografts
We next assessed the effects of the AKT inhibitor ± fulvestrant on tumor growth in vivo. ER+/PIK3CA mutant MCF-7 xenografts were established in ovariectomized athymic female mice supplemented with a 14-day-release E2 pellet. The E2 pellets expired after 14 days. Four weeks after tumor cell inoculation, mice with tumors measuring ≥150 mm3 were randomized to treatment with vehicle, AZD5363, fulvestrant or the combination. AZD5363 and fulvestrant significantly inhibited tumor growth compared to vehicle-treated controls (Figure 2C). Treatment with both AZD5363 and fulvestrant suppressed xenograft growth >90%; this effect was statistically better than either drug alone (Figure 2C). AZD5363-treated tumors exhibited lower levels of P-PRAS40 and P-S6 but higher levels of P-AKT compared to control tumors (Figure 2D). Treatment with fulvestrant alone or with both drugs downregulated ER and IGF-IR protein levels. Tumors treated with fulvestrant for six weeks exhibited higher levels of T308 P-AKT (Figure 2D). Finally, addition of AZD5363 enhanced fulvestrant-induced inhibition of tumor cell proliferation as measured by Ki67 IHC [see Additional file 2, Figure S4]. These data suggest that simultaneous inhibition of AKT and ER is more effective than inhibition of each molecular target alone against MCF-7 xenografts in vivo. They also imply that AKT and ER inhibitors induce an adaptive response that limits their efficacy as single agents; that is, cells may compensate by signaling with the alternative (spared) pathway when only one pathway is inhibited.
Inhibition of AKT was also effective against other models of endocrine resistance. HBCx-3 ER+ luminal B breast cancer xenografts were established in nude mice after resection from a post-menopausal woman with no previous treatment . These xenografts were negative for PTEN and HER2 protein by IHC [see Additional file 2, Figure S5A]. Although these xenografts were resistant to tamoxifen and fulvestrant, treatment with AZD5363 suppressed tumor growth [see Additional file 2, Figure S5B-C]. Further, AZD5363 treatment increased ER protein levels in the HBCx-3 xenografts [see Additional file 2, Figure S5D], suggesting that active AKT represses ER expression both in vitro (Figure 2A; Additional file 2, Figure S3) and in vivo.
Inhibition of AKT results in upregulation of RTKs in vitro and in vivo
We next assessed the effects of AZD5363 on a wider panel of RTKs. Following inhibition of AKT in MCF-7/LTED, ZR75-1/LTED and MDA-361/LTED cells, phospho-RTK array analysis revealed increased phosphorylation of multiple RTKs, including InsR, IGF-IR, HER3, EGFR, HER2, HER4, Dtk, VEGFR1 and FGFR2-4 (Figure 3C; Additional file 2, Figure S7). To validate these findings in vivo, we treated ovariectomized mice bearing MCF-7 xenografts with AZD5363 for one or three days. Inhibition of AKT upregulated the tumor levels of P-InsR/IGF-IR, InsR, P-HER3, HER3, P-HER2, HER2, the FGFR substrate P-FRS2 and FGFR2 proteins (Figure 3D). Further, treatment with AZD5363 for one to three days also increased tumor levels of InsR, IGF-IR and FGFR 1-4 mRNAs (Figure 3E).
Inhibition of IGF-IR/InsR or PI3K abrogates AZD5363-induced AKT membrane localization and phosphorylation
Inhibition of AKT results in FoxO-dependent upregulation of IGF-IR/InsR ligands
Estrogen is known to modulate IGF-I signaling in breast cancer, and ER induces IGF-IR and IGF-II expression . The IGF-IR and InsR gene promoters also contain binding sites for the FoxO transcription factors, including FoxO3a, which is inhibited when phosphorylated by AKT . FoxO proteins can bind directly to insulin-responsive sequences (IRSs), such as those found in the IGFBP-1 promoter, or IRS-like DNA-sequences . Blockade of AKT inhibits FoxO3a phosphorylation, resulting in translocation of FoxO3a to the nucleus, where it regulates gene transcription. Further, FoxO3a has been shown to interact functionally with ER [34, 35, 36, 37, 38], prompting us to speculate that IGF-IR, IGF-I, and IGF-II are regulated by both ER and FoxO. Since AZD5363 induces FoxO3a nuclear translocation in ER+/PIK3CA mutant breast cancer cells  and ER mRNA in LTED cells (Figure 2A), we examined whether knockdown of ER and/or FoxO3a affects AZD5363-induced transcription of IGF-IR, InsR, and IGF ligands. siRNA-mediated knockdown was confirmed by RT qPCR (Figure 5C). Downregulation of FoxO3a or ER, either alone or in combination, abrogated AZD5363-mediated induction of IGF-IR, IGF-I, IGF-II and ER mRNA (Figure 5C-D). Knockdown of FoxO3a, but not ER, inhibited the induction of InsR mRNA following treatment with AZD5363 (Figure 5D). This result was expected, since InsR is not ER-regulated. These results suggest that the AZD5363-induced upregulation of IGF-IR, IGF-I, and IGF-II is dependent on ER and FoxO3a, whereas upregulation of InsR is dependent on FoxO3a.
Pharmacological inhibition of IGF-IR/InsR enhances the anti-tumor effect of AZD5363 in vivo
PI3K/AKT/mTOR pathway activation has been implicated in endocrine resistance in breast cancer [9, 10, 12, 13, 14]. High AKT expression in breast tumors has also been associated with a poor response to antiestrogen therapy [39, 40]. In support of this notion, we show herein that the catalytic AKT inhibitor AZD5363 inhibited the growth of ER+ human breast cancer cells with acquired resistance to estrogen deprivation and prevented the emergence of hormone-independent cells. Inhibition of AKT suppressed growth of MCF-7 xenografts in ovariectomized mice and in a patient-derived breast cancer resistant to tamoxifen and fulvestrant. Combined inhibition of ER and AKT was more effective than each intervention alone. AKT inhibition resulted in feedback upregulation and activation of RTKs in vitro and in vivo, including IGF-IR, InsR, HER3 and FGFRs. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and AKT phosphorylation following treatment with AZD5363. Inhibition of AKT resulted in upregulation of ER- and FoxO-dependent IGF-IR, IGF-I, and IGF-II. Treatment with IGFBP-3 blocked the AZD5363-induced phosphorylation of IGF-IR/InsR and AKT, suggesting that the induced ligands activated IGF-IR/InsR. Finally, inhibition of IGF-IR/InsR enhanced the antitumor effect of the AKT inhibitor both in vitro and in vivo.
Inhibition of AKT with AZD5363 resulted in upregulation and activation of several RTKs. Others have seen upregulation of RTKs upon inhibition of the PI3K/AKT/mTOR pathway, including HER3 [22, 23, 24]. We show that this feedback reactivation also occurs in antiestrogen-resistant breast cancer cells and xenografts using a catalytic inhibitor of AKT. AZD5363 treatment resulted in prominent upregulation of IGF-IR/InsR expression and activity both in vitro and in vivo (Figure 3). In turn, InsR/IGF-IR stimulated membrane localization and phosphorylation of AKT in T308 likely as a result of increased production of PIP3. Indeed, inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and P-AKT following treatment with AZD5363 (Figure 4). While the increase in InsR/IGF-IR levels can be explained by increased FoxO-dependent mRNA transcription (Figure 5D; [22, 23]), it is less clear why receptor phosphorylation would increase following inhibition of AKT. However, we observed that upon inhibition of AKT, IGF-I and IGF-II mRNA were increased whereas IGFBP-3 mRNA levels were reduced (Figure 5A-B), thus revealing a previously unreported autocrine loop. Treatment with IGFBP-3 blocked AZD5363-induced phosphorylation of IGF-IR/InsR and AKT (Figure 6), suggesting that increased IGF-IR/InsR ligand production and activation of IGF-IR/InsR activates PI3K upstream AKT.
Inhibition of the PI3K/AKT pathway using AZD5363 or BKM120 induced ERα expression (Figure 2A; Additional file 2, Figure S3). In agreement with our data, Guo and colleagues reported that constitutively active AKT reduces ERα expression, whereas AKT inhibition increases ERα levels . Knockdown of FoxO3a reduced ERα mRNA and limited the AZD5363-mediated induction of ERα (Figure 5C), suggesting that its compensatory upregulation may be dependent on FoxO3a. In support of this, Guo and colleagues reported that expression of a dominant negative FoxO3a decreased ERα levels in MCF-7 cells . Further, FoxO3a has been shown to transactivate ERα[35, 38]. In contrast, others have shown that FoxO3a negatively regulates ER transcriptional activity [34, 36, 37]. These differing reports may be due to the use of different cellular systems and the presence or absence of estrogen. Importantly, we also identified a novel role for FoxO3a in regulating AZD5363-induced ER, IGF-I and IGF-II transcription. Further, AZD5363-induced upregulation of IGF-IR, IGF-I and IGF-II mRNA was dually regulated by FoxO3a and ER (Figure 5D). We propose that inhibition of AKT induces FoxO3a nuclear translocation  and transcriptional activation (Figure 5C), leading to increased ER, InsR, IGF-IR, IGF-I and IGF-II expression. ER also regulates IGF-IR, IGF-I and IGF-II transcription, ultimately leading to enhanced phosphorylation of IGF-IR/InsR and AKT.
Compensation for AKT inhibition through InsR/IGF-IR signaling has therapeutic implications in cancer. Although treatment with AZD5363 upregulated HER3 mRNA and protein levels (Figures 3A-B), knockdown of HER3 did not sensitize to AZD5363 treatment in MCF-7 cells (Figure 7A). Consistent with this result, treatment with the EGFR/HER2 dual kinase inhibitor lapatinib, which blocks HER3 phosphorylation in MCF-7 cells, does not suppress P-AKT in MCF-7 cells . These data suggest that HER3 does not appreciably activate PI3K in these cells. In contrast, RNAi-mediated knockdown or pharmaceutical inhibition of IGF-IR/InsR sensitized breast cancer cells to the AKT inhibitor (Figures 7A,C). We have previously identified IGF-IR/InsR signaling as a mechanism of escape from hormone dependence in ER+ breast cancer . In keeping with this, inhibition of IGF-IR/InsR with AZD9362 suppressed MCF-7 xenograft growth in ovariectomized mice devoid of estrogen supplementation (Figure 7D). Importantly, treatment with AZD9362 also enhanced the anti-tumor effects of the AKT inhibitor against MCF-7 xenografts (Figure 7D), suggesting that combined inhibition of IGF-IR/InsR and AKT should be more effective than either agent alone in treating ER+ breast cancers that adapt to estrogen deprivation. We also showed that long-term treatment with the pan-PI3K inhibitor BKM120 increased IRS-1 levels in T47D cells [see Additional file 2, Figure S3], providing an additional rationale for combining PI3K/AKT and IGF-IR/InsR antagonists. Addition of the FGFR inhibitor AZD4547 also increased the anti-tumor effects of AZD5363 in vivo, albeit modestly (Figure 7D). FGFR1 amplification has been shown to drive endocrine therapy resistance, and patients with ER+ positive tumors that overexpress FGFR1 exhibit a shorter relapse-free survival after adjuvant tamoxifen . Thus, combined inhibition of AKT with FGFR in the setting of antiestrogen resistance warrants further investigation.
Upregulation of IGF-IR/InsR and their ligands compensates for AKT inhibition in breast cancer cells with acquired resistance to estrogen deprivation, implying that AKT inhibitors may have limited clinical activity in endocrine-resistant breast cancers when used as single agents. Inhibition of the IGF-IR/InsR signaling pathway enhanced the action of AZD5363 against estrogen-deprived breast cancers, suggesting that combined treatment with an AKT inhibitor and a dual IGF-IR/InsR TKI merits evaluation as a potential treatment for endocrine-resistant breast cancer.
We thank Violeta Sánchez (Vanderbilt University) for performing the MCF-7 xenograft IHC. We gratefully acknowledge Pascal Leuraud, Jean-Gabriel Judde and Myriam Lassalle (Xentech, Evry, France) for carrying out the HBC-x3 anti-tumor study. AZD5363 was discovered by AstraZeneca subsequent to a collaboration with Astex Therapeutics (and its collaboration with the Institute of Cancer Research and Cancer Research Technology Limited) and is currently in phase I clinical trials. This work was supported by Postdoctoral Fellowship Grant #PF-10-184-01-TBE from the American Cancer Society (EMF), Breast Cancer Specialized Program of Research Excellence (SPORE) grant P50 CA98131, Vanderbilt-Ingram Cancer Center Support grant P30 CA68485, a Breast Cancer Research Foundation grant (CLA); ACS Clinical Research Professorship Grant CRP-07-234; the Lee Jeans Translational Breast Cancer Research Program; Stand Up to Cancer Dream Team Translational Research Grant, a Program of the Entertainment Industry Foundation (SU2C-AACR-DT0209) and Susan G. Komen for the Cure Foundation grant SAC100013 (CLA).
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