Analysis of genomic and non-genomic signalling of oestrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor Alpelisib (BYL719) and fulvestrant

Background Endocrine therapies targeting oestrogen signalling have signicantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signalling, oestrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to conrm the prognostic value of this complex and investigated whether its targeting could improve tumour response in vivo.


Background
Breast cancer (BC) is the most common cancer among women worldwide [1]. More than 75% of breast tumours express the oestrogen receptor α (ERα) in the nucleus and are commonly categorised as luminal BCs. ERα plays a major role in BC tumorigenesis as it regulates cell cycle, cell survival and angiogenesis [2]. Interfering with the ERα pathway using anti-oestrogens (selective estrogen receptor modulators such as tamoxifen or selective estrogen downregulators such as fulvestrant) or through oestrogen deprivation (e.g., aromatase inhibitors), increases the survival of ERα-positive BC patients. Despite the high level of sensitivity of luminal tumours to endocrine therapy, treatment e cacy is limited by intrinsic and acquired resistance [3,4]. Indeed, 30-50% of patients relapse after adjuvant treatment and eventually die from metastases [5]. mutated preclinical cancer models are sensitive to PI3K inhibitors, which appear to function synergistically with endocrine therapies [9]. This was recently con rmed in patients, as treatment with alpelisib (PI3K inhibitor) combined to fulvestrant prolonged survival of PIK3CA-mutated patients [10]. At the molecular level, ERα and PI3K pathways crosstalk at different levels [3]. At the genomic level, somatic activating mutations of the PIK3CA gene lead to abnormal PI3K/AKT/mTOR pathway activation [11]. In addition, PI3K inhibition increases ERα transcriptional activity via SGK1 and a feedback mechanism that attenuates the activity of PI3K inhibitors [12]. Beyond these genomic mechanisms of action, activation of PI3K pathway in BC can occur via a non-genomic signalling pathway involving cytoplasmic ERα [13,14]. Cytoplasmic ERα when complexed to Src and PI3K activates Akt, triggering proliferation and cell survival [13,[15][16][17]. Our team reported that methylation of ERα on residue R260 by the arginine methyltransferase PRMT1 is a prerequisite for its association with Src and PI3K and the activation of Akt [18,19]. Subsequently, using the proximity ligation assay (PLA) methodology to detect in situ protein/protein interactions [20], we showed that this pathway, characterized by the formation of ERα/Src/PI3K, is present in normal breast tissue and is hyperactivated in aggressive breast tumours [21]. Moreover, we unveiled that ERα/Src and ERα/PI3K interaction is associated with resistance to tamoxifen [22].
Taken together, these data introduced the concept that the non-genomic oestrogen pathway, in addition to the presence of activating PIK3CA mutations could affect the response to PI3K inhibitors associated with endocrine treatments.
In this study, we rst evaluated ERα/Src and ERα/PI3K interaction in a large cohort of BC patients. We then treated different PDX models of PIK3CA mutated and WT breast cancer with the PI3K inhibitor BYL719 combined to fulvestrant and explored their effect on tumour growth as well on both genomic and non-genomic ERα pathways.

Materials And Methods
Human breast cancer sample collection The tumours from 440 patients of the Centre Léon Bérard (CLB) with invasive non-metastatic breast cancer, whose clinical and biological data were available from the regularly updated institutional database, were analysed. Written informed consent was obtained from each patient. The study protocol was approved by the institutional ethics committee. Patient characteristics are presented in the Additional material, (Additional File 2, Table S1). In our study, tumours exhibiting less than 10% of ERα-positive cells were considered to be ERα-negative tumours.

Patient-derived xenografts
Before PDX establishment, all patients had previously given their verbal informed consent for experimental research on residual tumour tissue available after histopathological analyses. PDX establishment was performed after approval of the ethics committee of the Institut Curie. According to the French rules and the ethics committee of the Institut Curie, a written consent from patients to obtain residual tumour tissues is not required.
Nine breast cancer PDX models were used in this study. They were established from surgical specimens by grafting tumour fragments into the interscapular fat pad of nude mice as previously described [22][23]. Female Swiss nude mice, 10-week-old, were purchased from Charles River (Les Arbresles, France) and maintained under speci c pathogen-free conditions. Their characteristics are described in the Additional material (Additional File 3, Table S2). Their care and housing were in accordance with institutional guidelines and the rules of the French Ethics Committee (project authorization no. 02163.02). Histological and IHC statuses (ERα, PR, and HER2) were determined for the PDXs and compared with that of the patient tumour samples, as described elsewhere [23].
When tumours reached a volume of 60 to 200 mm 3 , mice were randomly assigned to the control or treatment groups, each group consisting of seven or eight mice. Fulvestrant (Faslodex®, AstraZeneca, Maccles eld, UK) was administered by intramuscular injection at a dose of 200 mg/kg once a week.
BYL719 was purchased from Medchemexpress and was administered orally at 35 mg/kg 5 times per week. Tumour growth was evaluated by measuring two perpendicular diameters of tumours with a caliper twice a week. Individual tumour volumes were calculated as V= a X b 2 /2, a being the largest diameter, b the smallest. Tumour growth inhibition (TGI) of treated tumours versus controls was calculated as the ratio of the mean tumour volume in the treated group to the mean tumour volume in the control group at the same time (end of the experiment). Statistical signi cance of TGI was calculated using the paired Student's t test by comparing the tumour volumes in the treated and control groups. Percent change in tumour volume was calculated for each tumour using the following formula: [(Vf-V0)/V0]*100; where V0= Initial volume (at the beginning of treatment) and Vf= Final volume (at the end of treatment). Classi cation of tumour response in waterfall plots: tumour regression, stabilization and progression corresponded to a percent of volume change lower, equal or > 0, respectively.
Tumour sampling was performed 24 hr after the last experiment. No speci c toxicity was reported in the experiments, neither diarrhoea, nor rash was observed and treated mice did not display any important weight loss throughout the experiment time-course.  [20], enables the in situ visualization of protein-protein interactions and was supplied by Merck. Para n-xed tumour tissues incorporated in TMA blocks were initially sectioned and incubated in a hydrogen peroxide solution, for 5 min at room temperature, to avoid peroxidase quenching. The antibody labelling steps were similar to those described above. For antibody detection, the probes were labelled with horseradish peroxidase after two washes in high purity water. A nuclear staining solution was added to the slides and incubated 2 min at room temperature. After washing the slides 10 min under running tap water, the samples were consecutively dehydrated in ethanol and xylene. Samples were mounted in non-aqueous mounting medium and visualized under a bright-field microscope. The protocol has already been optimized for ERα/Src and ERα/PI3K interactions [18,21].

Image acquisition and analysis
The hybridized uorescent slides were viewed under a Leica DM6000B microscope. Images were acquired under identical conditions at X63 magni cation. Images of three independent zones on each tumour were acquired under identical conditions at X40 magni cation. At least, 500 cells were counted per tumour.

Statistical analysis
ERα/Src and ERα/PI3K interaction in invasive breast cancer samples (by bright eld microscopy) was quanti ed as the mean number of dots (denoting interaction) per cell. For the sake of correlation and survival analyses, a cutoff for interaction was de ned at the most discriminative difference in DFS and OS as calculated by Kaplan Meier estimates. Accordingly, ERα/Src interaction was de ned as high if mean number of dots/cell > 10 and low if ≤ 10 dots/cell, while ERα/PI3K interaction was high if > 9 dots/cell and low if ≤ 9 dots/cell. Correlations between the 2 biomarkers ERα/Src and ERα/PI3K were studied. The Pearson's correlation coe cient was presented with asterisks highlighting identify its signi cance (*: p < 0.05; **: p < 0.01; ***: p < 0.001). Associations between categorical variables were studied using Pearson's Chi square test. Overall survival (OS) de ned as time from diagnosis to death or date of last follow-up and disease-free survival (DFS) de ned as time from diagnosis to death or relapse or date of last follow-up (for censored patients) were studied.
Survival curves were estimated by Kaplan-Meier method and compared between groups with different interaction levels using the Log-Rank test.
RT-QPCR analysis RNA extraction was performed as previously described [26,27]. Quantitative values were obtained from the number of the cycle (Ct value) at which the increase in the uorescent signal associated with exponential growth of PCR products was initially detected by the laser detector of the ABI Prism 7900 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA), using PE biosystems analysis software according to the manufacturer's manuals.
For gene normalization, we used the human TATA box-binding protein (TBP, GeBbank accession no. NM_003194). We used protocols for cDNA synthesis and PCR ampli cation described in detail elsewhere [28]. Results, expressed as N-fold differences in target gene expression relative to the TBP gene and termed "Ntarget", were determined as Ntarget = 2 ΔCtsample , where the ΔCt value of the sample obtained by subtracting the average Ct value of target gene from the average Ct value of TBP gene.

IHC experiments
Xenografted tumours were xed in 10% neutral buffered formalin, para n embedded, and haematoxylineosin-saffron (HES) stained. Outgrowths were analysed by immunohistochemistry (IHC) for expression of biomarkers. Immunostaining was performed on a Discovery XT Platform (Ventana Medical System, Tucson, AZ, part of Roche Diagnostics) with antigen retrieval using either EDTA buffer, pH 8.0 (CC1, Ventana Medical System) or citrate buffer 10 mM, pH 6.0, (CC2, Ventana Medical System). Primary antibodies were mostly monoclonal rabbit antibodies and paired slides immunostained with rabbit IgG were used as negative controls. Incubation and colour development involved anti rabbit multimer secondary antibody (horseradish peroxydase complex) with DAB (3,30-diaminobenzidine tetrahydrochloride) as substrate (ChromoMap Kit with Anti-rabbit OmniMap, Ventana Medical System).

Results
Clinicopathological characteristics of the patient cohort  Table S1 shows the clinico-pathological characteristics of the tested patient cohort (433 patients).
Representative micrographs of tumour cells with high (tumour#2) and low levels of interaction (tumour#1) of ERα/Src and ERα/PI3K are shown in g. 1a. ERα/Src interaction was high (> 10 dots) in 174 cases (40.5%), while 256 of cases (59.5%) showed low levels of interaction (≤ 10 dots). ERα/PI3K interaction was high (> 9 dots) in 156 cases (37.4%), while 261 of cases (62.6%) displayed low levels of interaction (≤ 9 dots). Interestingly, we observed a positive association between ERα/Src and ERα/PI3K interactions (p < 0.001) ( Table 1). We observed no correlation between high levels of interaction of either ERα/Src (Table 2) or ERα/PI3K ( Table 3) with any of the traditional prognostic parameters of breast cancer. Targeting oestrogen genomic and non-genomic signalling in ERα-positive PDX models Based on the present data and our previous results [21,22], we hypothesized that the oestrogen nongenomic pathway could represent a therapeutic target in BC and particularly in endocrine resistant ERα+ BCs. To test our hypothesis, we targeted non-genomic signalling using a combination of endocrine therapy (fulvestrant), known to degrade ERα, and a PI3K inhibitor, known to disrupt the complex containing ERα/PI3K and its downstream signalling [21], As our previous results were obtained with LY294002, not used in clinic, we studied the effect of three other PI3K inhibitors on MCF-7 cells and found that BYL719 was the most effective at decreasing the interaction of ERα with PI3K (Additional File 4, Figure S1). This inhibitor was thus selected for further in vivo experiments.
The treatment e cacy was tested in 6 PDX models of ERα+ breast cancers. The characteristics of the different PDXs are summarized in the additional File 3 Table S2. Five of these models were established from primary breast tumours and one from a bone metastasis. Three models (HBCx-86, HBCx-91 and BC1111) are PIK3CA mutated: p.E545Kand BC1111: p.H1047R).
In this PDX model, the ERα/PI3K interaction was signi cantly reduced by fulvestrant alone or combined with BYL719, but BYL719 alone had no effect (Fig. 2c.). The analysis of some oestrogen-regulated genes (ERG) showed a trend in the up-regulation of PGR, GREB1 and TFF1 gene expression in BYL719-treated xenografts and a signi cant decrease in the expression of the same genes in xenografts treated with fulvestrant or the combination (Fig. 2d.). ESR1 expression remained unchanged. IHC staining validated that ERα expression decreased upon fulvestrant treatment and that BYL719 inhibited downstream PI3K signalling only combined with fulvestrant, as evidenced by P-S6 riboprotein (S235/6) expression (Fig. 2e, additional File S6 Figure S3). This tumour does not express P-Akt (S473), so we couldn't con rm BYL719 e cacy on PI3K signaling.
These results suggest that the effects of fulvestrant on tumour growth are potentiated following PI3K inhibition in a context of oestrogen non-genomic signalling.
Next, we studied 2 models of PDX resistant to fulvestrant. The HBCx-22 TamR model did not respond signi cantly to BYL719 alone or in combination with fulvestrant (Fig. 4a.). The BC1111 model, was resistant to BYL719, however the combination strongly inhibited tumour growth (TGI 79%, P < 0.0001) ( Fig. 4b.). Interestingly, in the 2 models, ERα/PI3K interaction was not signi cantly decreased upon treatment, fulvestrant having an opposite effect in the HBCx-22 Tam R model by signi cantly increasing this interaction (Fig. 4c, d), corroborating our previous ndings [22]. The expression of ERG diminished following fulvestrant or combination treatment administration, whereas it increased with BYL719 (Fig. 4e, f). IHC staining of the HBCx-22 TamR model revealed that fulvestrant strongly inhibited ERα nuclear expression, while BYL719 had no effect on PI3K signalling (Fig. 4g, additional File 9 Fig S6).
With regards to the BC1111 model, fulvestrant triggered a decrease in ERα expression, while BYL719 e ciently inhibited the PI3K pathway (Fig. 4h, additional File 10 Fig S7).
We then investigated another ERα-positive PDX model engrafted from a patient expressing a low level of ERα and harbouring a PI3KCA mutation, HBCx-91. This model was resistant to fulvestrant alone but responded to BYL719 alone or in combination with fulvestrant, by inducing a stable low-grade disease (Fig. 5a.). We observed a signi cant increase in ERα/PI3K interaction upon fulvestrant treatment, whereas BYL719 alone or in combination had no effect (Fig. 5b.). The expression of ERG was not signi cantly affected by the different treatments (Fig. 5c.). The IHC staining con rmed that ERα was faintly expressed in the nucleus of tumoral cells (Fig. 5d.).

Targeting oestrogen non-genomic signalling in ERα-negative PDX models
As the oestrogen non-genomic complex is also activated in ERα-negative breast tumours [21] and the PI3K pathway is active in TNBC, we tested the combination of BYL719 + fulvestrant in 3 ERα-negative PDX models. In the HBCx-17 model (WT for PIK3CA), only the combination of BYL719 + fulvestrant inhibited tumour growth with a TGI of 64% (p = 0.03, Mann-Whitney t-test), although no tumour regression was observed (Fig. 6a.). Interestingly, fulvestrant and BYL719 alone signi cantly decreased ERα/PI3K formation, whereas the combination had no additive effect (Fig. 6b.). IHC analysis revealed a similar decrease in P-S6 riboprotein (S235/236) expression in tumours treated with BYL719 alone or combined with fulvestrant, altought it was less clear for p-Akt (Fig. 6c, additional File 11, Fig S9).
Next, we evaluated the impact of combining treatments in the HBCx-66 model. Fulvestrant had a modest effect on tumour growth, while administration of BYL719 alone or in combination led to a strong decrease in tumour volume (Fig. 6d.). BYL719 and fulvestrant signi cantly decreased ERα/PI3K interaction whereas the combination had no signi cant effect (Fig. 6e.). Similarly to the previous ERαmodel, BYL719 showed a non-signi cant decrease in p-AKT (S473) and P-S6 riboprotein (S235/236) staining (Fig. 6f, aditionnal File 12, Fig S10).
In conclusion, in ERα-negative tumors, fulvestrant effect on tumour growth is linked to its ability to disrupt ERα interaction with PI3K.

Discussion
Based on our results and other existing literature, we postulated that the actors of oestrogen non-genomic signalling could constitute both new prognostic markers and new therapeutic targets. In this study, we sought to validate the activation of this pathway in aggressive breast cancers in a new cohort of breast tumour specimen. ERα/Src/PI3K being a hallmark of the non-genomic signalling, we studied ERα/Src and ERα/PI3K by in situ PLA in a cohort of 440 invasive breast tumours. Interestingly, we found that their high level of expression was correlated with a decrease in patient survival, ERα/PI3K being associated with the most pronounced effects. These data corroborate those obtained in a rst cohort of 175 BCs [21] and justify to investigate the targeting of ERα/PI3K in in vivo models of BCs.
As a proof-of-concept, we decided to target ERα/PI3K interactions using an anti-oestrogen (fulvestrant), or a PI3K inhibitor alone (BYL719) or in combination in 6 models of ERα + and 3 ERα-BC PDXs. For the ERα-positive models, we evaluated their effect on tumour growth as well as on oestrogen non-genomic signalling (by studying ERα/PI3K interaction) and on genomic signalling (by studying the expression of ERα target genes). For the ERα-negative models, we assessed the e cacy of treatments on tumour growth and on ERα/PI3K interactions. We decided to use a PI3K inhibitor acting predominantly against PI3Kα, as it has been largely shown by our team and others that treating BC cells with PI3K inhibitors disrupts ERα/PI3K interactions in ERα-positive cell lines [13,15,21]. We con rmed this result in the present study using BYL719 and showed that it was able to disrupt ERα/PI3K interactions in MCF-7 cells. However, our present work reveals that BYL719 had no signi cant effect in vivo on ERα/PI3K interactions in ERα + PDX models tested. For 3 of them, this could be explained because the inhibitor had no clear effect on PI3K signalling and for two PDX models resistant to fulvestrant, ERα/PI3K could also not be disrupted by fulvestrant. These results suggest that it would be better to nd novel molecules able to destabilize this interaction. As a proof-of-concept, Aurrichio's team recently showed that a peptide targeting the site of interaction between ERα/Src was able to disrupt ERα/Src/PI3K complex formation, as well as cell proliferation in vitro and in vivo [29]. At the opposite, in 2 ERα-negative models, BYL719 was able to decrease ERα/PI3K interaction, supporting the idea that PI3K activity is important for its binding to cytoplasmic ERα. However, this effect does not seem to be associated with response to tumour growth as BYL719 has no effect on ERα/PI3K interaction in HBCx-90 model although it has an impact on tumour growth.
In summary, of the 6 PDX of ERα + BCs tested, 4 responded to the combination of BYL719 + fulvestrant, 3 of which were PIK3CA mutated. Activation of the non-genomic ERα pathway decreased in treated tumours of 3 PDXs, due largely to fulvestrant and was not always associated with the in vivo response (HBCx-3). The combination of BYL719 and fulvestrant was more e cient than fulvestrant alone in 3 models, however this effect was not associated with decreased levels of ERα/PI3K complex in xenografts treated with the combination compared to fulvestrant-treated xenografts. Similarly, PI3K dependent regulation of ERα transcription was observed only in 3 PDXs and was not correlated to PIK3CA mutations nor to response to the PI3K inhibitor. However, in order to obtain a strong tumour response to combined therapy, it is necessary to simultaneously inhibit genomic and non-genomic signalling. Indeed, complete responses were obtained in HBCx-34 xenografts, where both pathways were inhibited. However, when only one pathway was inhibited, the response was partial, as evidenced for HBCx3 and HBCx86, in which only the non-genomic pathway was inhibited by fulvestrant, whereas for HBCx-22 TamR and BC1111 models, only the genomic pathway was inhibited. For the HBCx-91 model, the response was partial and both oestrogen signalling pathways remained unresponsive to fulvestrant. Interestingly, in the 3 models resistant to fulvestrant, ERα/PI3K was not disrupted. Inversely, in 2 cases their interaction increased, although ERα was e ciently degraded in the nucleus and ERG expression was downregulated. This is in accordance with recent results from our lab showing that ERα/PI3K interaction increases upon resistance to endocrine therapy [22]. Of note in the HBCx-22, where the increase of ERα/PI3K interaction upon fulvestrant is very strong, PI3K pathway is activated, suggesting that this interaction may activate PI3K activity and the downstream pathway, increasing resistance to treatment inducing tumor growth.
Unlike previous ndings [12], we observed no increase in ERα expression at the mRNA or the protein levels in the 6 ERα-positive models treated with BYL719, likely due to the different models investigated (in cellulo vs in vivo) Concerning ERα-negative models, in HBCx-17 and HBCx-66 tumours, fulvestrant had a modest effect on growth inhibition. Interestingly, in these models, fulvestrant alone was able to decrease PI3K pathway probably by disrupting ERα/PI3K interactions which might affect PI3K activity and then downstream signalling. Conversely, in the HBCx-90 model, where fulvestrant had no effect on tumour growth, neither ERα/PI3K interaction nor the downstream pathway were inhibited.
Altogether, our results con rm that ERα/PI3K interaction could be evaluated before associating endocrine therapy with PI3K inhibitors in BC. Moreover, targeting this interaction may improve the response to endocrine therapy in ERα-positive tumours and patient survival in ERα-negative BCs.

Conclusions
In summary, the present study identi es ERα/PI3K interaction, a hallmark of oestrogen non-genomic signalling, as a new biomarker associated with a decrease in BC patient survival. In addition, targeting this interaction may circumvent resistance to endocrine therapies in ERα-positive tumours and could contribute to decreasing tumour growth in ERα-negative tumours.

Declarations
Ethical Approval and Consent to participate Before PDX establishment, all patients had previously given their verbal informed consent for experimental research on residual tumour tissue available after histopathological analyses.
PDX establishment was performed after approval of the ethics committee of the Institut Curie.
Written informed consent was obtained from each patient. The study protocol was approved by the institutional ethics committee.

Consent for publication
All authors gave consent for the publication of the manuscript in Cancer and Metabolism.

Availability of supporting data
All data in our study are availability upon request.

Competing interests
The authors declare that they have no con ict of interest.    treated tumours embedded in para n to study the interactions between ERα and PI3K. Quanti cation was performed by counting the number of signals per cell in ve independent zones of the section (n > 500 cells counted/tumour). Signi cance (P-value) between treatments was determined using the Student t-test. ns: non-signi cant; * P < 0.05; ** P < 0.01. d Expression of estrogen-regulated genes (ERG) analysed by RT-QPCR in PDX tumour samples (N=4). e IHC staining was performed on formalin-xed para nembedded PDX tumours using anti-ERα, anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies.
Quanti cation of highly, medium and negative cells was performed as described in the method section.
Signi cance (P-value) between treatments and controls were performed using t-test. Only signifancy for strong positive cells was highlighted as * P < 0.05; **** P < 0.0001.

Figure 3
In vivo drug response to BYL719 or fulvestrant alone, and combined in the HBCx-3 and HBCx-86 PDX models. a Effect of the different treatments on HBCx-3 tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. b Effect of the different treatments on HBCx-86 tumour growth.
c, d PLA was performed and analysed as in Figure 2. ns: non-signi cant; *P < 0.5; ** P < 0.01. e, f RT-QPCR was performed from RNA extracted from frozen tumour samples using speci c primers for ERG. g, h IHC Page 24/28 staining was performed on formalin-xed para n-embedded PDX tumours using anti-ERα, anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies. Quanti cation of highly, medium and negative cells was performed as described in the method section. Signi cance (P-value) between treatments and controls were performed using t-test. Only signifancy for strong positive cells was highlighted as ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4
In vivo drug response to BYL719 or fulvestrant alone, and combined in the HBCx-22 TamR and BC1111 PDX models. a Effect of the different treatments on HBCx-22 TamR tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. b Effect of the different treatments on HBCx953 tumour growth. c, d PLA was performed and analysed as in Figure 2. e, f RT-QPCR was performed from RNA extracted from frozen tumour samples using speci c primers for ERG. g, h IHC staining was performed on formalin-xed para n-embedded PDX tumours using anti-ERα, anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies. Quanti cation of highly, medium and negative cells was performed as described in the method section. Signi cance (P-value) between treatments and controls were performed using t-test. Only signifancy for strong positive cells was highlighted as * P < 0.05; ** P < 0.01, **** P < 0.0001.

Figure 5
In vivo drug response to BYL719 or fulvestrant alone, and combined in the HBCx-91 PDX model. a Effect of the different treatments on HBCx-91 tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. b PLA was performed and analysed as in Fig. 2. c RT-QPCR was performed from frozen tumour for ERG expression. d IHC staining was performed on formalin-xed para n-embedded PDX tumours using anti-ERα, anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies.
Quanti cation of highly, medium and negative cells was performed as described in the method section.

Figure 6
In vivo drug response to fulvestrant, BYL719 alone, and combined in 3 ERα-negative models. a Effect of the different treatments on HBCx27 tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. b PLA was performed and analysed as in Fig. 2. c IHC staining was performed on formalin-xed para n-embedded PDX tumours using anti-P-S6 riboprotein (S235/6) antibody. d Effect of the different treatments on HBCx-801 tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. e PLA was performed and analysed as in Figure 2. f IHC staining was performed on formalin-xed para n-embedded PDX tumours using anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies. g Effect of the different treatments on HBCx-90 tumour growth. Each treatment included 10 mice, y-axis indicates the mean of RTV +/-SD. h PLA was performed and analysed as in Fig.  2. i IHC staining was performed on xed PDX tumours using anti-P-AKT (S473) and anti-P-S6 riboprotein (S235/6) antibodies. Quanti cation of highly, medium and negative cells was performed as described in the method section. Signi cance (P-value) between treatments and controls were performed using t-test.
Only signifancy for strong positive cells was highlighted as * P < 0.