Clinicopathological characteristics of the patient cohort
Among the 440 patients, 433 had complete clinical data, 430 were assessable for ERα/Src interaction and 417 were assessable for ERα/PI3K interaction. Median age at diagnosis was 57.9 years (range: 30.4 to 87.4 years). Regarding the tumour stage, 41.8% of the patients had tumours beyond 20 mm, and 57.5% displayed axillary LN metastasis. Only 18.9% of the patients had SBR grade I tumours, 47.8% had grade II tumours and 33.3% grade III tumours. ER was positive in 87.1%, PR in 74.8% and HER2 was overexpressed in 7.2% of the 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 fig. 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.
High levels of ERa/PI3K interaction are associated with poorer breast cancer patient outcome
No significant impact on either OS (HR=1.24; 95% CI: 0.79-1.94; p=0.343) or DFS (HR=1.21; 95%CI: 0.83-1.75; p=0.325) was noted for patients displaying high or low levels of ERα/Src interactions (Fig. 1b.). Conversely, ERα/PI3K interaction predicted a trend towards poorer OS and DFS (Fig. 1c.), with an 8-year OS rate of 79.2% in patients with low levels versus 72.4% in patients with high levels of ERα/PI3K interaction (HR = 1.55; 95%CI: 0.99-2.44; p = 0.055), and an 8-year DFS rate of 79.2% in patients with low levels versus 72.4% in patients with high levels of ERα/PI3K interaction (HR = 1.35; 95%CI: 0.93-1.97; p = 0.116).
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 non-genomic 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 efficacy 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).
First, we evaluated the efficacy of fulvestrant alone, BYL719 alone, or BYL719 + fulvestrant in a PIK3CA WT hormone-sensitive PDX, HBCx-34 (Fig. 2a.). In this PDX model, treatment by fulvestrant for 3 months resulted in tumour regression in 5/8 xenografts, stable disease in 1 xenograft and complete response in 1 xenograft. Tumour response increased in the combination group (p = 0.01, Mann Whitney test) with 6/10 xenografts displaying complete responses, 3 tumour regression and 1 stable disease (Fig. 2b.).
In this PDX model, the ERα/PI3K interaction was significantly 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 significant 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 confirm BYL719 efficacy on PI3K signaling.
Two other models responded partially to BYL719 and fulvestrant alone, and the combination increased this anti-tumoral effect. In the PDX HBCx-3, the combination of BYL719 and fulvestrant did not inhibit tumour growth (Fig. 3a.), although this was significantly decreased compared to the control (TGI of 62% and 65%, respectively). The PIK3CA-mutated HBCx-86 model responded to the combination by exhibiting remarkable tumour regression (Fig. 3b.). For these models, ERα/PI3K interaction was efficiently disrupted with fulvestrant but not with BYL719 alone (Fig. 3c, d). Conversely, ERG remained unaffected irrespective of the treatment (Fig. 3e, f). IHC staining revealed that for both models, fulvestrant treatment decreased ERα expression (Fig. 3g, h, additional File 7, Fig S4, additional File 8, Fig S5). However, concerning BYL719 efficacy, it has no effect in the HBCx-3 model (Fig. 3g, additional File 7, Fig S4), whereas it significantly decreased P-S6 riboprotein (S235/6) staining (Fig. 3h, additional) in HBCx-86 model (Fig 3h, additional File 8, Fig S5) . We couldn’t confirm this result with Akt staining that was too low.
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 significantly 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 significantly decreased upon treatment, fulvestrant having an opposite effect in the HBCx-22 Tam R model by significantly increasing this interaction (Fig. 4c, d), corroborating our previous findings [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 efficiently 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 significant 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 significantly affected by the different treatments (Fig. 5c.). The IHC staining confirmed that ERα was faintly expressed in the nucleus of tumoral cells (Fig. 5d.). Fulvestrant induced a significant decrease in ERα expression and BYL719 efficiently targeted the PI3K pathway (Fig. 5d, additional File 11, Fig S8).
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 significantly 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 significantly decreased ERα/PI3K interaction whereas the combination had no significant effect (Fig. 6e.). Similarly to the previous ERα- model, BYL719 showed a non-significant decrease in p-AKT (S473) and P-S6 riboprotein (S235/236) staining (Fig. 6f, aditionnal File 12, Fig S10).
In the HBCx-90 PDX (PI3KCA mutated) treatment with fulvestrant had no effect on tumour growth, whereas BYL719 or the combination significantly decreased tumour volume. Interestingly, in this model resistant to fulvestrant, the anti-oestrogen had no effect on ERα/PI3K interactions (Fig. 6h.). Conversely, BYL719 significantly inhibited the downstream PI3K pathway, but did not affect ERα/PI3K interaction (Fig. 6i, additional File 13, Fig S11).
In conclusion, in ERα-negative tumors, fulvestrant effect on tumour growth is linked to its ability to disrupt ERα interaction with PI3K.