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AKT antagonist AZD5363 influences estrogen receptor function in endocrine resistant

breast cancer and synergises with fulvestrant (ICI182780) in vivo

Ricardo Ribas1*, Sunil Pancholi1*, Stephanie K. Guest1*, Elisabetta Marangoni2, Qiong Gao1,

Aurélie Thuleau2, Nikiana Simigdala1, Urszula Polanska3, Hayley Campbell3, Aradhana Rani1,

Gianmaria Liccardi1, Stephen Johnston4, Barry R. Davies3, Mitch Dowsett1,4, Lesley-Ann

Martin1§

* These authors contributed equally

1Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, United

Kingdom; 2Translational Research Dept., Institut Curie, Paris, France 3Oncology Innovative

Medicines, AstraZeneca, Alderley Park, Macclesfield, United Kingdom; 4 Royal Marsden

Hospital, London, United Kingdom

Running title: Effect of AZD5363 in endocrine resistant breast cancer

Financial support: This study was supported by the Mary-Jean Mitchell Green

Foundation, Breakthrough Breast Cancer funding to M. Dowsett and L.A. Martin and The

AstraZeneca Global Alliance to L.A. Martin, M. Dowsett and S. Johnston. We also

acknowledge NHS funding to the Royal Marsden Hospital’s NIHR Biomedical Research

Centre received by M. Dowsett and S. Johnston

§ Corresponding author:

Dr Lesley-Ann Martin, The Breakthrough Breast Cancer Research Centre, Institute of Cancer

Research, 237 Fulham Road, London, SW3 6JB, UK

Email. [email protected], Tel: +44(0) 207 153 5329, Fax: +44(0) 207 153 5340

on June 15, 2020. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 26, 2015; DOI: 10.1158/1535-7163.MCT-15-0143

http://mct.aacrjournals.org/

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Conflict of Interest: RR, SP, SKG, EM, QG, AT, NS, HC, AR, GL, SJ, MT and LAM declare

no conflict of interest. UP is an employee of AstraZeneca and BRD is an employee and

shareholder in AstraZeneca.




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Abstract

Phosphoinositide-3-kinase/protein-kinaseB/mammalian target of rapamycin (PI3K/AKT/mTOR)

signalling plays an important role in breast cancer (BC). Its interaction with estrogen receptor

(ER) signalling becomes more complex and inter-dependent with acquired endocrine resistance.

Targeting mTOR combined with endocrine therapy has shown clinical utility, however, a

negative feedback-loop exists downstream of PI3K/AKT/mTOR. Direct blockade of AKT

together with endocrine therapy may improve BC treatment. AZD5363, a novel pan-AKT kinase

catalytic inhibitor, was examined in a panel of ER+ BC cell lines (MCF7, HCC1428, T47D,

ZR75.1) adapted to long-term-estrogen-deprivation (LTED) or tamoxifen (TamR). AZD5363

caused a dose-dependent decrease in proliferation in all cell lines tested (GI50<500nM) except

HCC1428 and HCC1428-LTED. T47D-LTED and ZR75-LTED were the most sensitive of the

lines (GI50~100nM). AZD5363 re-sensitised TamR cells to tamoxifen and acted synergistically

with fulvestrant. AZD5363 decreased p-AKT/mTOR targets leading to a reduction in ERα-

mediated transcription in a context specific manner and concomitant decrease in recruitment of

ER and CREB-binding protein (CBP) to estrogen-response-elements located on the TFF1, PGR

and GREB1 promoters. Furthermore, AZD5363 reduced expression of cell-cycle-regulatory

proteins. Global gene expression highlighted ERBB2-ERBB3, ERK5 and IGF1 signaling

pathways driven by MYC as potential feedback-loops. Combined treatment with AZD5363 and

fulvestrant showed synergy in an ER+ patient derived xenograft and delayed tumour progression

post-cessation of therapy. These data support the combination of AZD5363 with fulvestrant as a

potential therapy for BC that is sensitive or resistant to E-deprivation or tamoxifen and that

activated AKT is a determinant of response, supporting the need for clinical evaluation.




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Introduction

Around 80% of breast cancers (BCs) express estrogen receptor alpha (ER) and depend on

estrogen (E) to grow. Strategies to target ER activity include depriving the hormone dependent

tumour cells of E by the use of aromatase inhibitors (AI) (1) or the use of anti-estrogens such as

tamoxifen or fulvestrant, both of which compete with E for the ER. Despite the efficacy of these

agents, many tumours exhibit de novo or develop acquired mechanisms of resistance.

De-regulated signalling through the PI3K/AKT/mTOR pathway is a feature of most types of

cancer cells (2) and has been linked to endocrine resistant BC (3-5). Activation of PI3K/AKT

occurs via gain-of-function mutations in the catalytic domain of PI3K (PIK3CA) and the

regulatory subunit p85α (PIK3R1), amplification of PIK3CA, PIK3CB and PDK1; or by reduced

expression of phosphatase-and-tensin-homolog-deleted on chromosome ten (PTEN), an

endogenous inhibitor of the PI3K/AKT pathway (6). Furthermore, mutation and amplification of

AKT 1, 2 and 3 together with ERBB2, FGFR1, MET and IGF-1R have also been associated with

activation of this signalling axis.

It is clear, that the ER can become involved with the PI3K/AKT/mTOR pathway in BC cells,

with evidence for both genomic and non-genomic cross-talk between this signalling pathway

and ER accounting for endocrine resistance (7-9). Growth factor mediated activation of AKT

can potentiate ER classical transcriptional activity via phosphorylation at serine 167 in the AF1

domain leading to ligand-independent transactivation (10-12). Similarly, elevated levels of AKT

have been shown to change the genome-wide binding pattern of ER, effectively altering the ER-

program (13). These bi-directional interactions between hormonal and kinase signalling

pathways create self-reinforcing synergistic loops that potentiate pro-survival signals and may

allow BC cells to escape normal endocrine responsiveness.




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Targeting the PI3K/AKT/mTOR pathway in association with endocrine therapy has seemed a

sensible strategy to explore. However, blockade of a single protein in a complex signalling

cascade is unlikely to provide total or prolonged growth inhibition. An important negative

feedback loop exists downstream in the PI3K/AKT/mTOR pathway that may limit the

effectiveness of mTOR inhibitors in BC. The mTOR activated kinase, S6K1, phosphorylates and

destabilises the IRS1 and IRS2 proteins in insulin-like growth factor (IGF) responsive cells (14).

In these cells, mTORC1 inhibition can lead to a reduction in S6K1 activity, which in turn allows

IRS1/2 expression to be increased with associated enhanced activation of IGFR1-dependent

AKT activity. Concern has been raised that activation of this negative feedback loop may

overcome the anti-tumour activity of mTORC1 blockade and limit the effectiveness of

rapamycin analogues (14,15).

In recent years, it has been suggested that direct blockade of AKT in combination with

endocrine therapy, may provide a better rationale for treatment of endocrine resistant BC,

impacting on both cell survival/apoptosis and ER ligand-independent signaling. A novel pan-

AKT kinase catalytic inhibitor (AZD5363) has been developed and shown to inhibit the growth

of a range of human tumor xenografts, either as monotherapy or in combination with HER2

inhibitors or with docetaxel (16). A recent study has also reported that combinations of AKT and

IGF-1R/InsR inhibitors can be effective in the treatment of hormone-independent ER+ BC (17).

On the basis of these data, AZD5363 is currently being investigated in phase I clinical trials.

Further investigations on intelligent combinations of signal transduction inhibitors are urgently

required both in pre-clinical models and in clinical trials. In this manuscript, we describe for the

first time the effectiveness and molecular consequence of combining AZD5363 with the four




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most widely used approaches to endocrine therapy in the clinic, namely E-deprivation,

tamoxifen, fulvestrant and AI in pre-clinical models of endocrine sensitive and resistant ER+ BC,

as well as in a patient-derived xenograft (PDX) model of ER+ BC resistant to E-deprivation..

Material and methods

Antibodies and reagents

Primary antibodies against phospho-AKTSer473, phospho-AKTThr308, total-AKT, phospho-

p70S6Thr389, total-p70S6, phospho-S6 ribosomal protein (RP)Ser235/236, total-S6RP, phospho-

EGFRTyr1068, total-EGFR, phospho-ERBB2Tyr1248, phospho-ERBB3Tyr1222, phospho-

ERBB4Tyr1284, phospho-ERαSer167, phospho-4EBP1Thr37/46, total-4EBP1, phospho-RbSer807/811,

total-Rb, phospho-RAFSer259, phospho-RAFSer338, total-RAF, phospho-PRAS40Thr246, total-

PRAS40, Cyclin D1, Cyclin D3, total-ERK1/2, 14-3-3 were purchased from Cell Signalling

Technology, UK; total-IGF1R, cleaved-PARP, total-ERα and total-ERBB3 were purchased from

Santa Cruz; phospho-ERK1/2 and Actin from Sigma; GAPDH (Abcam); PgR (Novocastro or

Dako); IRS-1 and total-ERBB2 (Millipore); IRS-2 (Upstate) and finally total-ERBB4

(Neomarkers). Secondary antibodies (anti-mouse and anti-rabbit horseradish peroxidase) were

obtained from Dako or Cell signalling. Antibodies used for chromatin immunoprecipitation were

purchased from Santa Cruz: ERα clone HC20 and CBP clone A22. 17-β-estradiol and 4-

hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich; fulvestrant (ICI182780),

letrozole and exemestane from Tocris Bioscience; AZD5363 was synthesised and supplied by

AstraZeneca UK.

Tissue culture

Human BC cell lines MCF7, T47D, ZR75.1 and HCC1428 were obtained from the American

Type Culture Collection, Rockville, USA between 2000 and 2012 and were cultured in phenol




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red-free RPMI1640 medium supplemented with 10% fetal bovine serum and 1nM estradiol (E2).

All cell lines were banked in multiple aliquots to reduce the risk of phenotypic drift and identity

confirmed using iPLEX-pro (Agena Bioscience). LTED cells modeling resistance to an AI were

derived from all four parental cell lines by long-term culture in the presence of RPMI1640

medium containing 10% dextran charcoal stripped serum (DCC), as described previously (18).

The 1%MCF7 and TamR cell lines were cultured in DMEM/F-12 medium, as previously stated

(19). MCF7-2A and BT474-A3 cells stably expressing CYP19 (AROM) were derived from

parental MCF7 and BT474 cells, respectively and were maintained in phenol red-free

RPMI1640 medium containing 10% fetal bovine serum supplemented with 1nM E2 and 1mg/ml

G-418 (Sigma-Aldrich, UK) (20). All cell lines were stripped of steroids for 48-72-hours prior

to the start of experiments.

Proliferation and colony formation assays

Cells were seeded in 10% DCC into 96-well tissue culture plates and allowed to acclimatize

overnight. Monolayers were then treated with RPMI1640 + 10% DCC containing increasing

concentrations of AZD5363, with or without the presence of 0.01nM of E2. In order to assess

the effects of AZD5363 in combination with endocrine agents, cells were maintained in the

presence of GI50 values of AZD5363 along with increasing concentrations of endocrine agent 4-

OHT, fulvestrant or anastrozole. The medium was replaced after 3-days and cells were cultured

for a total of 6-days. Each experiment was performed at least 3 times with 8 replicates per

treatment. Cell viability was determined using the CellTitre-Glo® Luminescent Cell Viability

Assay (Promega), according to the manufacture’s protocol. Values were expressed as % viability

relative to the untreated control.




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For the colony formation assays, cells were seeded into 6-well plates and allowed to acclimatise

for 24-hours. Subsequently, cells were treated with the drug combinations indicated, every 3-4

days, for the duration of 14-days until colonies were evident. Cells were then fixed using ice-

cold methanol and stained using 0.5% w/v crystal violet in 90% H20/10% methanol.

Drug interaction analysis

To determine the nature of the interaction between AZD5363 and endocrine agents, combination

studies were carried out using Chou and Talalay's constant ratio combination model (21). Cells

were treated with increasing doses of AZD5363 alone, endocrine agent alone, or an equipotent

combination of AZD5363 and endocrine agent for 6 days. The GI50 for each drug alone was used

to define the ratio of equipotent doses and serial dilutions of the combinations used. Interactions

were calculated using Calcusyn software (BIOSOFT), based on the combination index (CI)

equation of Chou and Talalay. The CIs for a growth inhibition of 50%, 75%, and 90% were

obtained using mutually non-exclusive Monte Carlo CI simulations. Drug interaction was scored

using the CI so that: CI=1 is additive, CI <1 is synergistic, CI >1 is antagonistic.

Transcriptional assays

Cell lines were seeded in 24-well plates in 10% DCC medium and left to acclimatize for 24-

hours, following transfection with Fugene 6 at a ratio of 6:1 (Promega) with 0.1μg of estrogen

response element linked luciferase (EREIItkluc) and 0.1μg of β-galactosidase (pCH110) reporter

constructs. The following day, cells were treated with the drugs combinations specified and left

for 24h. Luciferase (Promega) and β-galactosidase (GalactoStar, Applied Biosystems) activity

were measured using a luminometer. Each experiment was performed 3 times with 3-4 replicates

per treatment.




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Real-time quantitative PCR

mRNA from treated cells was extracted with RNeasy Mini Kit (Qiagen), as per the

manufacturer's instructions, and quantification performed using the Agilent 2100

Bioanalyzer (Expert Software version B.02.03) with RNA Nano LabChip Kits (Agilent

Technologies). Total RNA was reverse transcribed using SuperScript III (Invitrogen) and

random primers, in accordance with the manufacturer's instructions. cDNA was subjected

to quantitative PCR using 10ng of cDNA in triplicate using the ABI Perkin-Elmer Prism

7900HT Sequence detection system (Applied Biosystems). Taqman gene expression assays

(Applied Biosystems) were used to quantify TFF1 (Hs00907239_m1), PGR

(Hs01556702_m1) CDK7 (Hs00361486_m1); EIF2A (Hs00230684_m1); FOXO3

(Hs00818121_m1); IGF1R (Hs00609566_m1); IRS2 (Hs00275843_s1); MAP2K5

(Hs00177134); MYC (Hs00153408); NRIP1 (Hs00942766_s1); NR2F2 (Hs00819630_m1);

PIK3R1 (Hs00381459_m1); PTEN (Hs02621230_s1) and GREB1 (Hs00536409_m1),

together with FKBP15 (Hs00391480_m1) as housekeeping gene, to normalise the data.

The relative quantity was determined using ΔΔct, according to the manufacturer's

instructions (Applied Biosystems).

Chromatin immunoprecipitation

MCF7-LTED cells were cross-linked in 1% formaldehyde at room temperature for 10-min

and then quenched with 125mM glycine. Samples were then lysed, sonicated and

chromatin was immunoprecipitated by overnight incubation at 4°C with ER, CBP or IgG

antibodies pre-bound with Protein G magnetic dynabeads (Invitrogen). Samples were then

washed vigorously with RIPA buffer and reversed cross-linked by an overnight incubation

in elution buffer at 65°C. DNA was digested with RNase and Proteinase K, purify

precipitated with phenol chloroform and eluted in Tris-HCl pH8.0. Real-time qPCR was




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performed using the following oligonucleotides TFF1 forward (FWD): 5′ GGC CAT CTC

TCA CTA TGA ATC ACT TCT GCA 3′ and reverse (REV): 5′ GGC AGG CTC TGT

TTG CTT AAA GAG CGT TAG 3′, GREB-1 FWD 5’ GAAGGGCAGAGCTGATAACG

3’, GREB-1 REV 5’ GACCCAGTTGCCACACTTTT 3’, PGR FWD 5’

GCTCTGCCTTGGAATGAGGT 3’ and PGR REV 5’ AAGAATTTGGGGGTGTTGGT

3’.

Western blotting

Cells were seeded into dishes and allowed to attach overnight. Monolayers were treated with the

desired drug combinations for the required length of time. Whole-cell extracts were generated,

as described previously (18). For the xenograft samples, tumours were lysed on ice,

homogenized and sonicated. The resulting protein fraction was subjected to western blotting as

previously described (18). The numerical data for each biomarker was determined using

Genetools software and normalized to GAPDH control.

Cell cycle analysis

Cells were seeded into 10cm dishes and 24-hours later treated with the drug combinations

indicated for 48-hours. Cells were fixed 90% ethanol and stained with propidium iodide. Cell

cycle analysis was carried out using fluorescence-activated-cell sorting (FACS).

Gene expression data and microarray analysis

Gene expression analysis was performed in triplicate using RNA derived from MCF7 and their

LTED derivatives after treatment with or without AZD5363 for 24-hours. mRNA extraction and

quantification was performed, as previously stated. The 12 RNA samples were amplified,

labelled, and hybridized on HumanHT-12_V4 Expression BeadChip, according to the




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manufacturer’s instructions (Illumina). Raw expression data were extracted using GenomeStudio

(www.illumina.com) software; the data were then transformed and normalized using variance-

stabilizing transformation and robust spline normalization method in the Lumi package in R

(http://www.bioconductor.org) (Accession number: GSE69893). Probes for which expression

levels were not reliably detected in one of the samples (detection p.value > 0.01) were filtered,

the remaining differentially expressed genes were identified by using the Class Comparison

(http://linus.nci.nih.gov/BRB-ArrayTools.html) using the thresholds of FDR < 5%. The

significantly expressed gene lists were subject to further pathway analysis using Ingenuity

Pathway Analysis (IPA) to identify altered pathways due to the AZD5363 treatment and the

altered pathways with p-value <0.05 were considered as significant.

Human tumour xenografts

In vivo efficacy studies were performed in 8-12-week old female Swiss nude mice

implanted with the PDX HBCx22OvaR, as previously described (22), in accordance with

the French Ethical Committee. Firstly, a pharmacodynamic (PD) study was performed for

4-days to assess biomarker changes with samples removed 2 and 4-hours post-treatment.

Between 5 and 7 mice were included in each group. Secondly, a long-term study to assess

changes in tumour volume and progression over 90-days was initiated. In order, to address

the ability of the combination to delay tumour progression, mice were followed for a

further 40 days post-drug withdrawal. The treatment groups received AZD5363 solubilized

in a 10% DMSO 25% w/v Kleptose HPB (Roquette) buffer by oral gavage, or fulvestrant

suspended in corn oil by subcutaneous injection into the flank. For the combination groups,

fulvestrant was dosed 2-hours before AZD5363. The control groups received both vehicles.

In the combination study, treatments were started when tumors reached a volume of 40-65

mm3. Each group included between 11 and 13 xenografts. Tumour volumes (measured by




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caliper), animal body weight and tumour condition were recorded twice weekly for the

duration of the study. Tumor volumes were calculated as V=axb2/2, “a” being the largest

diameter, “b” the smallest. Growth inhibition from the start of treatment was assessed by

comparison of the differences in tumour volume between control and treated groups.

Because the variance in mean tumour volume data increases proportionally with volume

(and is therefore disproportionate between groups), data were log-transformed to remove

any size dependency before statistical evaluation. Tumor volumes were reported to the

initial volume as relative tumor volume (RTV). Percent of tumor growth inhibition (TGI)

was calculated at the end of treatments (day 88) as follow: (1-RTVtreated/ RTVcontrol)

x100. Statistical significance was evaluated using a one-tailed, two-sample t-test.

Fluidigm Analysis

Tumour samples were homogenised and RNA was extracted as described above. cDNA was

synthesised using a high-capacity cDNA reverse transcription kit (Applied Biosystems). ER-

dependent genes listed (AQP3 (Hs01105469_G1); AREG (Hs00950669_m1); BMF

(Hs00372937_m1); C3 (Hs00163811_m1); CASP9 (Hs00609647_m1); CCND1

(Hs00765553_m1); CCNG2 (Hs00171119_m1); CXCL12 (Hs03676656_m1); ESR1

(Hs00174860_m1); GATA3 (Hs00231122_m1); LAMB2 (Hs00158642_m1); MUC1

(Hs00159357_m1); MYBL1 (Hs00277143_m1); RET (Hs01120030m_1); SPRY1

(Hs01083036_s1); TFF3 (Hs00902278_m1) and ZEB1 (Hs00232783_m1)) were selected and

analysis was performed using the Fluidigm Biomark System, as per manufacture’s instructions

(Fluidigm). Data was analysed using a Real-Time PCR analysis software (version 3.1.3) and

ΔΔct calculated. All genes were normalised to the housekeeper PPIA (Hs04194521_s1). Linear

fold changes were calculated based upon the relative difference in mRNA expression compared




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to vehicle control. Statistical analysis was subsequently performed using JMP 11.0 to determine

significant changes with a p-value of < 0.05.

Results

Effect of AZD5363 on cell growth of endocrine sensitive and resistant BC cell lines

A panel of endocrine sensitive and resistant BC cell lines were assessed for their sensitivity to

the AKT inhibitor AZD5363, a pyrrolopyrimidine-derived compound (Fig. 1A). MCF7, ZR75,

T47D, HCC1428 cell lines and their LTED derivatives were assessed both in the presence and

absence of E2. TamR and their parental cell line (1%MCF7) were assessed in 1% FBS and

aromatase transfected MCF7-2A and BT474-A3 cell lines were tested in the presence of

androstenedione.

The cell lines tested showed varying degrees of sensitivity to AZD5363. Of note, HCC1428 and

their LTED derivative were resistant to the antiproliferative effect of the drug, even at

concentrations in excess of 10μM (Fig. 1B & Supplementary Figure S1). T47D and their LTED

derivative together with ZR75-LTED showed the highest degree of sensitivity (GI50 c. 100nM).

MCF7 in the absence of E2, showed over a two-fold increase in sensitivity to AZD5363 (GI50

200nM) compared to in the presence of E2 (GI50 500nM). Assessment of MCF7-LTED and

ZR75 showed little effect of E2 on the sensitivity to AZD5363 (GI50 500nM). Of note, TamR

cells, similar to MCF7-LTED, have a GI50 of c. 400nM, showing less sensitivity compared to

MCF7 cells in the absence of E2. MCF7-2A showed a slightly higher degree of sensitivity to

MCF7 in the presence of E2 (400nM vs. 500nM, respectively). BT474-A3 revealed a GI50 of

approximately 600nM (Fig. 1B & Supplementary Figure S1).




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Combination of AZD5363 and endocrine agents on cell proliferation

To assess the efficacy of combining AZD5363 with current endocrine therapies, all cell lines

were grown in the presence of their specific GI50 values for AZD5363 and increasing doses of 4-

OHT, fulvestrant or anastrozole. HCC1428 and their LTED derivatives were excluded from

future studies due to their resistance to AZD5363.

In the presence of 4-OHT, MCF7, MCF7-LTED and 1% MCF7 demonstrated increased drug

sensitivity when treated with AZD5363 compared to 4-OHT alone (c. 2-fold increase in

sensitivity) (Fig. 2A). In contrast, ZR75, T47D and their LTED derivative cell lines

demonstrated no added benefit when combining AZD5363 with 4-OHT, however, AZD5363 re-

sensitised the TamR cell line to the antiproliferative effect of tamoxifen (GI50 10nM).

Combination of fulvestrant with AZD5363 also increased drug sensitivity by c.2-fold in MCF7

and 1%MCF7 and by c.10-fold in MCF7-LTED and TamR compared to fulvestrant alone. ZR75,

T47D and their LTED derivatives showed no added benefit from the combination (Fig. 2B).

Finally, we assessed the effect of the combination of AZD5363 with increasing doses of

anastrozole in MCF7-2A and BT474-A3 cells. Here again, the combination of AZD5363 with

anastrozole versus anastrozole alone was superior (c. 5-10 fold increase in sensitivity to

anastrazole) (Fig. 2C). These data were further confirmed using a colony formation assay as an

orthogonal approach in MCF7 and MCF7-LTED cells (Fig. 2D).

Subsequently, we conducted formal assessment of the interaction between 4-OHT, fulvestrant or

anastrozole with AZD5363, using Calcusyn software (Supplementary Figure S2). T47D-LTED

and ZR75-LTED were precluded from this analysis, as they loose expression of ER.

Furthermore, as the TamR cell line is resistant to 4-OHT formal analyses could not be assessed

in response to the combination of AZD5363 with 4-OHT.




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The combination of AZD5363 and 4-OHT was synergistic in MCF7, MCF7-LTED and

1%MCF7, with combination indices (CIs) < 1, at 50% and 75% growth inhibition. In contrast,

AZD5363 plus 4-OHT showed added benefit only in T47D at 50% and 75% growth inhibition.

In contrast, ZR75 at 75% and 90% growth inhibition showed no added benefit (CI>1)

(Supplementary Figure S2A).

Combination of fulvestrant with AZD5363 was predominantly synergistic in MCF7, MCF7-

LTED, 1%MCF7 and TamR. In contrast, the combination of fulvestrant and AZD5363 provided

no further benefit in T47D and ZR75 cells (Supplementary Figure S2B). The MCF7-2A and

BT474-A3 cells showed synergy with almost all doses of anastrozole when combined with

AZD5363 (Supplementary Figure S2C).

In summary, these results suggest that the combination of AZD5363 with endocrine therapy is

superior to monotherapy.

Analysis of the differential effects of AZD5363 ± endocrine therapy on PI3K/mTOR/AKT,

ER signalling and cell cycle arrest.

In order to investigate the effect of AZD5363 in combination with endocrine agents on cellular

signal transduction pathways (Fig. 3A), MCF7 and MCF7-LTED cells were treated with

AZD5363 for 1, 8, 24 or 48-hours ± E2, 4-OHT or fulvestrant, in order to establish the most

suitable time-points for further investigation. As few early changes were evident between 1 and

8-hours post-treatment, the 1-hour time-point was selected to depict early changes. Furthermore,

as 24-hour and 48-hours also showed similar alteration, the 48-hour time-point was selected to

show later events associated with AKT blockade, in particular changes associated with feed-




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back loops discussed later in this study (Supplementary Figure S3A & B (1, 8 and 24 -hour time-

point) and Fig. 3B (48-hour time-point). Addition of AZD5363 resulted in increased

phosphorylation of AKT (S473 and T308) as early as 1-hour post-treatment and was maintained

even after 48-hours. Total AKT was suppressed in those cell lines showing the highest degree of

sensitivity to AZD5363, most notably T47D, T47D-LTED, ZR75-LTED and TamR. Treatment

with AZD5363 caused a marked reduction, or even complete loss, of pPRAS40 and S6 kinase.

Of particular note, total PRAS40 was suppressed in T47D and their LTED. Phosphorylation of

4EBP1, a downstream marker of the PI3K/AKT/mTOR pathway, was reduced in T47D, T47D-

LTED and ZR75-LTED in response to AZD5363 either alone or in combination with endocrine

therapy (Fig. 3B). AZD5363 also reduced expression of pRAF (S259), most noticeably in MCF7,

T47D and their LTED derivatives; in contrast, the TamR cell line showed an increase in pRAF

(S338). We have previously shown that inhibition of AKT can increase ERK signalling (19),

therefore we assessed the effect of AZD5363 on pERK1/2 and showed differential responses.

For instance, AZD5363 suppressed phosphorylation of ERK1/2 in T47D but caused an increase

in ZR75-LTED and TamR cells after 48-hours (Fig. 3B). As pAKT, pS6 and pERK1/2 are

known to phosphorylate and activate ER, we investigated the effect of AZD5363 ± endocrine

therapy on ER phosphorylation together with expression of the progesterone receptor (PGR), an

endogenous E-regulated gene. Assessment of ER phosphorylation indicated AZD5363 had the

clearest impact on reducing phosphorylation of ERser167 in the MCF7 and MCF7-LTED and

this associated with a slight but noticeable reduction in PGR expression in the MCF7 and

MCF7-LTED, particularly in the presence of 4-OHT. Of note, whilst T47D-LTED showed

undetectable levels of ER, AZD5363 caused a marked suppression of PGR in this cell line,

suggesting some residual ER-activity may still be present.




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In order to assess the effect of AZD5363 on cell cycle arrest and apoptosis, we investigated

changes in protein expression of Cyclin D1/D3, Rb and induction of PARP cleavage (Fig. 3C).

The combination of AZD5363 ± endocrine therapy caused a decrease in expression of

phosphorylated Rb and cyclin D in the majority of cell lines tested, with a higher effect being

observed in combination with fulvestrant. Furthermore, cell cycle analysis showed a significant

reduction in S-phase in response to AZD5363 in MCF7 and MCF7-LTED (Supplementary

Figure S3C). Subtle increases in PARP-cleavage were evident in MCF7 although this did not

appear specific to treatment. In contrast, T47D-LTED showed increases in PARP cleavage

specifically in response to AZD5363 addition (Fig. 3C).

In summary, these data show AZD5363 impedes downstream AKT signalling and reduces ER-

phosphorylation in a context specific manner, leading to an increase in cell cycle arrest.

The effect of AZD5363 alone or in combination with endocrine therapy on receptor

tyrosine kinase activity and ER-transactivation

We further assessed the impact of AZD5363 on the expression and phosphorylation of receptor

tyrosine kinases (RTKs) in the target cell lines (Fig. 4A). Inhibition of AKT with AZD5363

resulted in upregulation and activation of RTKs, including EGFR, ERBB2, but also IRS2, which

was cell line specific (Fig. 4A). MCF7-LTED, ZR75 and most notably ZR75-LTED showed an

increase in pEGFR; T47D and T47D-LTED showed an increase in pERBB2 and IRS2; TamR

revealed increases pERBB3.

As cross talk between ER and RTK pathways is well documented (12), we assessed alterations

in ER-mediated transactivation after perturbation with AZD5363 alone or in combination with

endocrine therapy. MCF7, MCF7-LTED, T47D, ZR75 and TamR cells were transiently




18

transfected with an ERE-linked luciferase reporter construct and treated with E2, 4-OHT or

fulvestrant ± AZD5363 (Fig. 4B). AZD5363 showed a significant effect on ER transactivation of

MCF7, MCF7-LTED and T47D when in combination with all endocrine agents. In contrast, in

ZR75 the effect on ER transactivation was minimal, apart from when combined with fulvestrant.

TamR cells similarly showed minimal impact on ER-mediated transcription. Further analyses of

the expression of a panel of endogenous E-regulated genes (TFF1, PGR and GREB1), showed

that AZD5363 had a similar impact in keeping with the ERE-linked-luciferase assay with the

exception of T47D, which showed an increase in the expression of TFF1 when treated with

AZD5363 but a decrease in PGR and GREB1 (Supplementary Figure S4).

In order to address this further, chromatin immunoprecipitation was performed in MCF7-LTED

cells. AZD5363 caused a reduction in recruitment of ER, AIB1 and CBP to the EREs on the

TFF1, GREB1 and PGR promoters (Fig. 4C), supporting the impact of AZD5363 on ER-

mediated transactivation.

Gene expression analysis of MCF7 and MCF7-LTED identified MYC as a potential

regulator of resistance

Comparison of gene expression between MCF7 cells treated with or without AZD5363 in the

absence of E2, showed a total of 1695 genes differentially downregulated and 1751 genes

differential upregulated. MCF7-LTED cells showed fewer genes altered in response to

AZD5363 (983 genes differentially downregulated, versus 842 genes differentially upregulated).

The downregulated genes in both cell lines were mainly associated with metabolic and cell cycle

pathways (Table 1). The upregulated genes were associated with EIF2, mTOR and IGF1

signalling. Of note, ERBB2-ERBB3 signalling and big MAP kinase 1 (BMK1/ERK5) were

significantly increased in both cell lines in response to AZD5363, together with ILK1 and




19

integrin signalling. Furthermore, RAR pathway activation was also significantly upregulated in

both MCF7-LTED and MCF7 cells in response to AZD5363. In order to interrogate these

findings further, expression of selected genes representing activation of the various pathways

was tested using qRT-PCR and were shown to be significantly over-expressed (Supplementary

Figure S5) Of particular note, interrogation of genes within these pathways identified MYC,

PIK3R1, PTEN and FOXO3 as common elements (Supplementary Table S1, Supplementary

Figure S5). Further analysis revealed that MYC is upregulated over 2-fold (p<0.001) in the

MCF7-LTED and over 1.2-fold (p<0.05) in the MCF7 cell lines.

Combination of AZD5363 and fulvestrant in the luminal BC xenograft HBCx22OvaR

A high degree of redundancy in the kinase networks and cross-talk with ER is well documented

(23), suggesting destruction of ER with fulvestrant may be a better strategy than antagonism

with tamoxifen or blockade of estrogen biosynthesis with an AI. Therefore, we assessed the

combination of fulvestrant and AZD5363 compared with monotherapy in a patient derived

luminal BC xenograft HBCx22OvaR modeling acquired resistance to E-deprivation (22) (Fig. 5).

Treatments were well tolerated. Fulvestrant monotherapy treatment caused a modest but non-

significant reduction in tumour growth compared with the vehicle control group (30% inhibition,

p=0.11). Continuous dosing of AZD5363 at 50mg/kg bid resulted in a significant reduction in

tumour growth (57% inhibition, p=5.0E-03), compared with vehicle control. Combination of

fulvestrant with AZD5363 resulted in almost complete inhibition of tumour growth (80%

inhibition p< 1E-05) compared to vehicle control. The combination dosing was significantly

more active than AZD5363 and fulvestrant monotherapy groups (p=3.75E-03 and 6.3E-04,

respectively) (Fig. 5A). After 90-days of treatment, the therapies were withdrawn and tumour

volume assessed to establish the efficacy of the combination in delaying tumour progression.

Removal of AZD5363 showed a dramatic rise in tumour volume as early as 10-days post-




20

withdrawal (Fig. 5B), whilst the combination of AZD5363 plus fulvestrant showed sustained

anti-tunour effect even after 50 days post-cessation of treatment.

In order to assess dynamic changes in the PI3K/AKT/mTOR, ER, apoptotic and cell cycle

pathways, a second xenograft experiment was carried out to study tumour pharmacodynamics.

HBCx22OvaR were treated for 4-days with the combinations indicated and samples resected 2

and 4-hours post-dosing of AZD5363. As expected, western blot analysis showed significant

increases in pAKT (S473) and pAKT (T308) and decreases in pPRAS40 in response to

AZD5363. Two-hours post-treatment with AZD5363 showed a significant rise in PGR

expression compared to vehicle control and was negated by the addition of fulvestrant (Fig. 5C).

In order to assess the effect of AZD5363 on ER-mediated transcription, we used Fluidigm

analysis of several ER-dependent target genes (Fig. 5D). Fulvestrant caused a significant

reduction in expression of the majority of the genes selected whilst AZD5363 showed a trend

towards an increase. The combination of fulvestrant plus AZD5363 negated this rise. Four-hours

post-therapy showed a significant rise in ER-regulated genes in response to AZD5363

monotherapy and addition of fulvestrant did not suppress this effect with the exception of TFF1.

Analysis of CCND1 showed a decrease in response to fulvestrant and the combination with

AZD5363. However, 4-hours post-treatment showed a rise in expression. In contrast, CCNG2,

which blocks cell cycle entry and is negatively regulated by E-bound-ER and activated PI3K

(24,25), was increased in all AZD5363 treatment groups, indicative of cell cycle arrest.

Furthermore, AZD5363 caused increases in CASP9 and BMF expression indicating of a rise in

apoptosis (Fig. 5D).




21

Discussion

Activation of PI3K/AKT/mTOR pathway has been shown to be involved in endocrine resistance

in BC, most notably via interaction with ER (8,10). Hence, AKT being a central mediator within

this pathway poses a rational therapeutic target in endocrine resistance BC. In support of this

notion, we have shown that suppression of AKT activity with the catalytic AKT inhibitor

AZD5363, inhibited the growth of ER+ human BC cells modelling acquired resistance to E-

deprivation and tamoxifen and prevented the emergence of hormone independent cells in vivo.

The combination of AZD5363 with endocrine therapy showed the greatest efficacy in both ER+,

endocrine sensitive and resistant cell lines, most notably those containing activating mutations in

PIK3CA and/or loss of PTEN function (Fig.1). In contrast, HCC1428, which are wild-type (wt)

for both genes, were resistant to AKT inhibition (GI50 > 10000nM). The combination of

AZD5363 with fulvestrant provided the greatest combination effect, which was later confirmed

in a patient derived xenograft model. Nonetheless, the combination of AZD5363 with an AI was

also highly effective. Moreover, ZR75-LTED and T47D-LTED, which have suppressed

expression of ER compared to their parental cells, were the most sensitive to the antiproliferative

effects of single agent AZD5363, with GI50 values less than 100nM. It is noteworthy, that

PIK3CA mutation may not be the only governing feature of sensitivity to AKT inhibition. For

instance, both MCF7 and T47D cells and their derivatives express mutant PIK3CA and wt-PTEN,

yet T47D were at least 2-fold more sensitive than MCF7, and this is most pronounced when

comparing T47D-LTED with MCF7-LTED, where there is a 5-fold difference in sensitivity.

Furthermore, ZR75, which are wt-PIK3CA and PTEN null, are 5-fold less sensitive than ZR75-

LTED, suggesting that whilst mutation within the pathway may delineate sensitivity in some

circumstances, cell context remains a defining feature.




22

Treatment with AZD5363 reduced phosphorylation of 4EBP1, a downstream mediator of the

mTOR pathway, and increased phosphorylation of AKT itself. The latter has been shown to

occur with several ATP-competitive inhibitors of AKT, as a result of the protein being

maintained in a hyperphosphorylated but catalytically inactive form (26). In all cell lines tested,

AZD5363 caused cell cycle arrest, most notably via suppression of phosphorylation of Rb and

reduction in cyclin D and in several cell lines this was associated with elevated apoptosis, most

notably in the T47D-LTED. Furthermore, fluidigm analysis of our HBCx22OvaR PDX showed

increased expression of CASP9 and BMF in response to AZD5363. These data are in keeping

with previous studies in which AKT dependent phosphorylation of BAD suppresses apoptosis

(27).

Previous studies have shown that AKT can phosphorylate ER at serine 167 in a ligand

independent manner and is associated with resistance to endocrine therapy (10,19); furthermore,

elevated AKT has been associated with an altered ER transcriptional program (13). In this

current study, we provided further support for these observations and showed that inhibition of

AKT in MCF7-LTED reduced recruitment of ER, AIB1 and CBP to EREs. Furthermore, the

combination of endocrine therapy with AZD5363 suppressed ER-mediated transcription to a

greater extent than either agent alone. In contrast, however, although AZD5363 suppressed the

proliferation of the TamR cell line and resensitised it to the antiproliferative effects of tamoxifen,

little impact on ER-mediated transcription in the presence of 4-OHT was evident compared to 4-

OHT alone. One explanation for this finding may be due to the high degree of redundancy in the

signal transduction pathways within the TamR cell line. We have previously shown that

phosphorylation of ERser167 can occur via pAKT or pERK1/2/pp90rsk. This was confirmed in

the current study where AZD5363 had no impact on ERser167 phosphorylation. It could be

postulated, that continued signalling via the pERK1/2 pathway may, in the long-term, negate the




23

initial antiproliferative effect of AZD5363 in this setting. Previous studies have shown that

targeting a single protein in a complex pathway can cause feedback loops resulting in increased

ER-mediated transcription (28). Here, we assessed expression of several ER-mediated target

genes both in our cell line models and in our patient derived xenograft in response to AZD5363.

Increased expression of ER-target genes appeared context-specific and time-dependent. One

explanation for these contrasting observations is the nature of the cross-talk between ER and

AKT. For instance, increased AKT signalling may lead to ligand-independent ER activity in

which the cell still relies on ER function for proliferation. Alternatively, increased AKT can

suppress ER-expression circumventing the need for ER-driven transcription. In the latter setting,

perturbation of AKT would therefore increase ER-mediated transcription. Taken together, these

data indicate that the combination of AZD5363 with a selective ER downregulator (SERD), such

as fulvestrant, may alleviate these potential ER-mediated feedback loops.

Studies have suggested that inhibition of kinases within the PI3K/mTOR/AKT pathway can lead

to upregulation of several RTKs impacting on their potential clinical utility (17,29-31). In order

to address this, we assessed the impact of AZD5363 on expression and phosphorylation of type

I/II growth factor receptors. AZD5363 caused a cell-type specific upregulation of various RTKs

including IGF1R, EGFR, ERBB2 and ERBB3. It has been suggested that altered expression of

these RTKs may impede the effectiveness of AZD5363 (17). In order to assess this further, we

used a global gene expression approach to identify networks of genes that were associated with

response and resistance to AZD5363. The significantly downregulated canonical pathways,

following treatment with AZD5363 were associated with cell cycle progression and metabolism,

confirming our observation in both proliferation and protein assays. One of the most

significantly upregulated pathways was EIF2 which was evident in both MCF7 and their LTED

derivative. The PERK-eIF2αP pathway mediates survival and facilitates adaptation to the




24

deleterious effects of the inactivation of PI3K or AKT (32). The IGF1 signaling pathway, which

encompasses IGF1R, IRS2 and FOXO3 was also significantly upregulated. Previous studies

have shown that upregulation of IGF1R maintains PI3K activity and PIP3 formation, to

counteract the inhibition of AKT which could potentially reduce the potency of AZD5363, via a

FOXO dependent transcriptional mechanism (17).

RAR signalling was also associated with inhibition of AKT activity. RAR transactivation is

suppressed by phosphorylation via AKT (33). Of note, RAR-alpha (α) is required for efficient

ER-mediated transcription and cell proliferation (34). Assessment of the effect of AZD5363 on

ER-mediated transactivation using an artificial reporter construct showed suppression of

transactivation. However, assessment of transcript levels of several endogenous E2-regulated

genes particularly in the T47D and TamR cell line as well as in our PDX model showed

increases in TFF1 and GREB1 expression, in keeping with previous studies with a dual

mTOR/PI3K inhibitor (35). Of note, assessment of genes within the RAR signalling pathways

showed increases in expression of CDK7, which has previously been shown to phosphorylate

and activate both ER (36) and RARα (37). Furthermore, high intratumoral RARα protein levels

correlate with reduced relapse free survival in ER+ patients treated with neoadjuvant tamoxifen

(38). Taken together, activation of RAR as a result of AKT inhibition may potentiate ER-activity

in certain settings.

ERK5 signaling pathway was significantly upregulated in response to AZD5363. ERK5, similar

to ERK1/2, regulates EGF-induced cell proliferation and is known to cross talk with ERBB2 and

PDGFR. Both of pathways were also elevated in response to AZD5363. ERK5 is also capable of

phosphorylating the ERK1/2 substrates MYC and AP1 (39). Moreover, ILK, HIF1 and VEGF

pathways were upregulated in response to AKT inhibition. Previous studies have shown that β-




25

parvin, which is lost in metastastic BC (40), leads to an upregulation of ILK/AKT mediated

signalling resulting in increased HIF1α and VEGF (41). Both HIF1α and VEGF contain internal

ribosome entry sites (IRES), located in their 5’ UTRs, allowing cap independent translation,

bypassing the requirement for mTOR/4EBP1 signaling (42). This may provide a survival

mechanism in response to chronic treatment with AZD5363. Of particular interest, MYC

expression was identified as a potential driver in six of the upregulated pathways in response to

AZD5363. MYC activity has been shown to abrogate response to mTOR inhibition in prostate

cancer via its interaction and upregulation of 4EBP1 (43). Notably, 4EBP1 expression did not

change in response to AZD5363 in our PDX model.

Overall, the transcriptional profiling revealed multiple mechanisms of compensation to AKT

inhibition, highlighting the complex interplay between ER and signal transduction pathways.

This global gene expression analysis did, however, use only one cell line model and its LTED

counterpart.

In conclusion, the results provide mechanistic evidence for the combination of AZD5363 and

endocrine therapy to delay the onset of resistance, as well as re-sensitise endocrine resistant

tumours to the antiproliferative effects of endocrine therapy. Most notably the combination of

AZD5363 with fulvestrant, supressing both the ER and AKT signalling axes appeared superior

to either agent alone. The current study also highlights potential routes of escape via RTK, RAR,

ERK5, MYC and PERK-eIF2αP signalling, that merit investigation for further improvements in

treatment efficacy.




26

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29

Table 1. Top upregulated and downregulated canonical pathways after treatment

with AZD5363 in (A) MCF7-LTED and (B) MCF7 cell lines in the absence of E2.

MCF7-LTED and MCF7 cells were treated in the presence or absence of AZD5363 for 24-

hours and RNA was submitted to microarray analysis

A.

MCF7-LTED upregulated pathways

p-value MCF7-LTED-Downregulated Pathways

p-value

EIF2 Signaling 2.00E-07 Mitotic Roles of Polo-Like Kinase 2.51E-11

IGF-1 Signaling 3.31E-04Cell Cycle: G2/M DNA Damage Checkpoint Regulation 3.16E-11

RAR Activation 1.07E-03Remodeling of Epithelial Adherens Junctions 7.41E-10

mTOR Signaling 1.95E-03 ATM Signaling 7.24E-09 Diphthamide Biosynthesis 4.37E-03 Hereditary Breast Cancer Signaling 8.51E-09 Endometrial Cancer Signaling 1.48E-02

Role of CHK Proteins in Cell Cycle Checkpoint Control 1.95E-08

PTEN Signaling 1.55E-02 Protein Ubiquitination Pathway 3.02E-08 ErbB2-ErbB3 Signaling 2.09E-02 GADD45 Signaling 6.03E-08

Insulin Receptor Signaling 2.57E-02DNA damage-induced 14-3-3σ Signaling 6.03E-08

Clathrin-mediated Endocytosis Signaling 2.63E-02 14-3-3-mediated Signaling 8.13E-08 Mouse Embryonic Stem Cell Pluripotency 3.09E-02 Cyclins and Cell Cycle Regulation 4.07E-07

ERK5 Signaling 3.24E-02Role of BRCA1 in DNA Damage Response 4.79E-07

Aryl Hydrocarbon Receptor Signaling 3.39E-02

Sertoli Cell-Sertoli Cell Junction Signaling 1.70E-06

Ketolysis 3.63E-02Cell Cycle Control of Chromosomal Replication 2.19E-06

HGF Signaling 3.80E-02Germ Cell-Sertoli Cell Junction Signaling 2.75E-06

DNA Methylation and Transcriptional Repression Signaling 4.07E-02 Mismatch Repair in Eukaryotes 3.72E-06 Amyloid Processing 4.37E-02 Estrogen-mediated S-phase Entry 8.13E-06

Calcium Transport I 4.47E-02Breast Cancer Regulation by Stathmin1 2.69E-05

Growth Hormone Signaling 4.79E-02Epithelial Adherens Junction Signaling 4.07E-05

ILK Signaling 4.90E-02 Glycolysis I 4.27E-05




30

B.

MCF7-Upregulted Pathways

p-value MCF7-Downregulated Pathways p-value

Hypoxia Signaling in the Cardiovascular System

1.26E-06 Mitotic Roles of Polo-Like Kinase 2.88E-07

Protein Ubiquitination Pathway

4.90E-06 Cell Cycle: G2/M DNA Damage Checkpoint Regulation

7.94E-07

EIF2 Signaling 3.39E-05 Cell Cycle Control of Chromosomal Replication

4.07E-06

Aryl Hydrocarbon Receptor Signaling

1.29E-04 Role of CHK Proteins in Cell Cycle Checkpoint Control

5.25E-06

IGF-1 Signaling 5.37E-04 GADD45 Signaling 7.08E-06 ERK5 Signaling 1.05E-03 Estrogen-mediated S-phase Entry 8.91E-06 ILK Signaling 1.20E-03 Mismatch Repair in Eukaryotes 1.41E-05 Molecular Mechanisms of Cancer

1.35E-03 ATM Signaling 1.41E-05

Integrin Signaling 1.51E-03 Remodeling of Epithelial Adherens Junctions

1.62E-05

NGF Signaling 1.78E-03 Role of BRCA1 in DNA Damage Response

1.78E-05

p53 Signaling 1.86E-03 Sertoli Cell-Sertoli Cell Junction Signaling

6.92E-05

Regulation of eIF4 and p70S6K Signaling

2.00E-03 Hereditary Breast Cancer Signaling 2.69E-04

Neurotrophin/TRK Signaling 2.19E-03 Cholesterol Biosynthesis I 3.09E-04 Estrogen Receptor Signaling 2.34E-03 Cholesterol Biosynthesis II (via

24,25-dihydrolanosterol) 3.09E-04

PDGF Signaling 2.88E-03 Cholesterol Biosynthesis III (via Desmosterol)

3.09E-04

Renal Cell Carcinoma Signaling

2.88E-03 Breast Cancer Regulation by Stathmin1

3.80E-04

Colanic Acid Building Blocks Biosynthesis

3.55E-03 Cyclins and Cell Cycle Regulation 4.37E-04

ErbB2-ErbB3 Signaling 4.27E-03 DNA damage-induced 14-3-3σ Signaling

5.13E-04

VEGF Signaling 4.37E-03 Germ Cell-Sertoli Cell Junction Signaling

7.41E-04

RAR Activation 5.25E-03 Superpathway of Cholesterol Biosynthesis

1.07E-03




31

Figure Legends

Figure 1. GI50 values for antiproliferative effect of AZD5363 in relation to PTEN,

PI3KCA and HER2 status in endocrine resistant and sensitive BC cell lines. (A)

Chemical structure of AZD5363. (B) Cells were treated in absence or presence of

exogenous E2 (0.01nM) and doubling concentrations of AZD5363. Treatments were

performed at day 1 and day 3 after seeding. After 6 days of treatment, cell viability was

analyzed by using a cell titre-glo assay and GI50 values were plotted. Genotype is depicted

by greyscale for expression of PTEN, PI3KCA and HER2

Figure 2. Antiproliferative effect of AZD5363 in combination with escalating doses of

various endocrine agents. (A) 4-OHT, (B) fulvestrant and (C) anastrozole, in endocrine

sensitive and resistance BC cell lines. Cell lines were treated with vehicle or GI50

concentrations of AZD5363 and increasing amounts of 4-OHT, fulvestrant and anastrozole.

After 6 days of treatment, cell viability was analysed using cell titre glo and data expressed

as fold-change relative to vehicle treated control. Error bars represent ± SEM. (D) Colony

forming assay of MCF7 and MCF7-LTED, following treatment with AZD5363 in the

presence or absence of endocrine agents. Cells were left for 14 days with treatment

changes every 3-4 days.

Figure 3. Effect of the combination of AZD5363 with E-deprivation (DCC), 4-OHT or

fulvestrant on PI3K/mTOR/AKT, ER and cell cycle signalling. (A) Schematic

representation of the AKT signalling pathway and cross-talk with RTKs and ER.

Endocrine resistant and sensitive breast cancer cell lines were treated for 48-hours with the

drug combinations indicated to assess (B) effect on PI3K/AKT/mTOR and ER pathways




32

and (C) cell cycle. Cyclin D3 was assessed for MCF7 derived cell lines, whilst Cyclin D1

was tested in the remaining cell lines.

Figure 4. Effect of the combination of AZD5363 with E-deprivation (DCC), 4-OHT or

fulvestrant on (A) RTK expression, (B) ER-mediated transactivation and (C)

recruitment of the ER-transcriptional machinery to the TFF1, GREB1 and PGR

promoters in response to AZD5363 in the absence of exogenous E2. (A) Endocrine

resistant and sensitive BC cell lines were treated for 48-hours with the drug combinations

indicated. Whole-cell extracts were assessed for expression on RTK markers by

immunoblotting. (B) Cell lines were co-transfected with EREIItkLuc and pCH110, and

treated for 24-hours with the drug combinations indicated. Luciferase activity was

normalized by β-galactosidase from triplicate wells and fold-changes expressed relative to

the DCC control. Error bars represent ± SEM. *p<0.05; **p<0.01; ***p<0.001. (C)

MCF7-LTED cells were treated for 24-hours in DCC in the presence or absence of

AZD5363 and ER/CBP binding to TFF1, GREB1 and PgR assessed via chromatin

immunoprecipitation studies. Error bars represent ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 5. Effect of AZD5363 alone or in combination with fulvestrant on biomarker

changes and tumour progression in HBCx22OvaR. (A) Long-term study assessing

changes in tumour volume over 90-days of treatment and (B) assessment of tumour

volume post drug withdrawal. PD study performed for 4-day was conducted to assess

biomarker changes in (C) protein expression of pertinent targets within the

PI3K/AKT/mTOR pathway and ER-signalling via measurement of PgR expression by

semi-quantitative western blotting and (D) mRNA analysis of ER-regulated, cell cycle and

apoptosis target genes using Fluidigm heatmap shows Log2 expression of samples




33

extracted 2 and 4-hours after final administration of AZD5363. Bars represent ± SEM.

*p<0.05; **p<0.01; ***p<0.001




Fig. 1

A

PTEN

PI3KCA

HER2

Mutation

WT HER2 (RNA expression)

A HER2-amplified

A

B

AZD5363 structure




0

150

300

450

600

750

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

100

200

300

400

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

200

400

600

800

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

100

200

300

400

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

100

200

300

400

500

600

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

200

400

600

800

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

MCF7- 2A

A

B

C

-AZD5363

+AZD5363

BT474 A3

MCF7 LTED ZR75 LTED T47D LTED TamR

MCF7 ZR75 T47D 1% MCF7

MCF7 LTED ZR75 LTED T47D LTED TamR

MCF7 ZR75 T47D 1% MCF7

Fig. 2

0

400

800

1200

1600

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

100

200

300

400

500

600

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

Anastrozole (nM)

0

100

200

300

400

500

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

Anastrozole (nM)

0

100

200

300

400

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

100

200

300

400

0 0.01 0.1 1 10 100

Re

lati

ve lu

min

esc

en

ce

(X1

00

0)

4-OHT (nM)

0

400

800

1200

1600

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

100

200

300

400

500

600

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

150

300

450

600

750

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

200

400

600

800

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

4-OHT (nM)

0

150

300

450

600

750

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

100

200

300

400

500

600

0 0.01 0.1 1 10 100

Re

lati

ve L

um

ine

sce

nce

(X

10

00

)

fulvestrant (nM)

0

150

300

450

600

750

0 0.01 0.1 1 10 100

Re

lati

ve lu

min

esc

en

ce

(X1

00

0)

fulvestrant (nM)

D

MC

F7

MC

F7-L

TED




MCF7 MCF7 LTED T47D T47D LTED ZR75 ZR75 LTED TamR

4EBP1

ER

pERK1/2

pPRAS40 (T246)

pAKT (S473)

S6RP

pAKT (T308)

pER (S167)

pRaf (S259)

Raf

AKT

ERK1/2

PRAS40

pS6RP (S235/236)

14-3-3

Actin

pRaf (S338)

p4EBP1 (T37/46)

PgR

pP70S6K (T389)

P70S6K

AZD5363 (1000nM)

E2 (0.01nM)

4-OHT (10nM)

fulvestrant (1nM)

-

-

-

-

+

-

-

-

-

+

-

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+

+

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Fig. 3

A

B

pRb (S807)

Rb

Parp total/Cleavage

Cyclin D1/D3

Actin

C




0

1

2

3

4

ERE

luci

fera

se a

ctiv

ity

fo

ld c

han

ge

T47D

0

1

2

3

4

5

6

ERE

luci

fera

se a

ctiv

ity

fo

ld c

han

ge

MCF7

0

0.5

1

1.5

2

2.5

3

3.5

ERE

luci

fera

se a

ctiv

ity

fo

ld c

han

ge

MCF7 LTED

**

***

**

**

**

** **

**

0

0.2

0.4

0.6

0.8

1

1.2

ERE

luci

fera

se a

ctiv

ity

fo

ld c

han

ge

TamR

* **

*

* * 0

2

4

6

8

10

12

ERE

luci

fera

se a

ctiv

ity

f

old

ch

ange

ZR75 B

A

**

Fig. 4

AZD5363 (1000nM)

fulvestrant (1nM)

4-OHT (10nM)

+

-

-

-

-

+

-

-

+

+

-

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+

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E2 (0.01nM)

+

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AZD5363 (1000nM)

fulvestrant (10nM)

4-OHT (10nM)

-

-

-

+

-

-

+

+

-

-

-

+

+

-

+

-

+

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AZD5363 (1000nM)

fulvestrant (1nM)

4-OHT (10nM)

+

-

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-

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+

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E2 (0.01nM)

AZD5363 (1000nM)

E2 (0.01nM)

4-OHT (10nM)

fulvestrant (1nM)

-

-

-

-

+

-

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p-ERBBB2

p-EGFR

EGFR

pERBB3

pERBB4

ERBB2

ERBB3

ERBB4

IGF-1R

IRS1

IRS2

Actin

MCF7 MCF7 LTED T47D T47D LTED ZR75 ZR75 LTED TamR

0

10

20

30

40

50

60

IgG AIB1 CBP ER IgG AIB1 CBP ER

DCC DCC+AZD5363

% o

f In

pu

t (T

FF1

)

TFF1

0

10

20

30

40

50

60


DCC DCC+AZD5363

% o

f in

pu

t (G

REB

1)

GREB1 C

** ** **

* **

**

0

10

20

30

40


DCC DCC+AZD5363

% o

f In

pu

t (P

gR)

PGR

* *




pAKT(S

473)

pAKT(T

308)

pPRAS40

(T24

6)

PGR

p4EBP1(

T37/4

6)0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

Bio

ma

rke

r S

ign

al (%

co

ntr

ol)

Vehicle 4hrs

Fulvestrant 4hrs

AZD5363 2 hrs

AZD5363 4 hrs

AZD5363+Fulvestrant 2 hrs

AZD5363+Fulvestrant 4 hrs

Fig. 5

0

2

4

6

8

10

12

14

16

18

20

0 10 20 30 40 50 60 70 80 90 100110120130140150

Rela

tive T

um

or

Vo

lum

e

Days after start of treatment

AZD5363

stop

0

2

4

6

8

10

12

14

16

18

20

0 10 20 30 40 50 60 70 80 90 100110120130140150

Rela

tive T

um

or

Vo

lum

e

Days after start of treatment

AZD5363 + Fulvestrant

stop

A

C D

B

ER dependent transcription

Apoptotic/ cell cycle related transcription

Fulv

4h

AZD5363

2h

AZD5363+Fulv

2h

AZD5363

4h

AZD5363+Fulv

4h CXCL12

C3

TFF1

MYBL1

GREB1

IGF1R

PGR

SPRY1

ZEB1

GATA3

MUC1

LAMB2

ESR1

RET

TFF3

AQP3

AREG

Fulv

4h

AZD5363

2h

AZD5363+Fulv

2h

AZD5363

4h

AZD5363+Fulv

4h

CCND1

CASP9

CCNG2

BMF

**

**

***

**

*




Published OnlineFirst June 26, 2015.Mol Cancer Ther Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, et al. fulvestrant (ICI182780) in vivoin endocrine resistant breast cancer and synergises with AKT antagonist AZD5363 influences estrogen receptor function

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