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Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC.Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays.Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival.Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Supplementary Table S7. Upstream regulators identified by the Upstream Regulator Analysis in HBCx22 TamR and HBCx34 TamR xenografts.

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Background. Activation of the PI3K pathway is associated with resistance to endocrine treatments (ET) in luminal breast cancer (LBC). Everolimus, an mTOR inhibitor, has been approved in combination with exemestane in endocrine resistant breast tumors. However, no definite signature of ET resistance or predictive factors of sensitivity to everolimus (EVE) treatment have been evidenced so far. In order to further decipher these molecular patterns, we used patient derived LBC xenografts (LBC-PDX) treated with various ET and EVE based combinations. Methods. 3 LBC-PDX (named HBCx-3, HBCx-21 and HBCx-34) were treated with ET during several months to establish hormono-resistant (HR) xenografts. Both parental and HR models were treated by tamoxifen, estrogen deprivation, fulvestrant, EVE alone, and EVE combined with all three ET modalities. Tissue Micro Arrays from control and treated tumor samples were generated for IHC analyses. RT-PCR analyses were conducted on genes related to proliferation, ER, PI3K, IGF-1R and angiogenesis pathways. Results. In primary PDX, ET showed a poor level of sensitivity for HBCx-3 and a high and sustained efficacy of ER targeting and/or ER deprivation for HBCx-21 and HBCx-34 tumors. In vivo resistance to ET was confirmed in all HR variants with tumor growth rates faster than parental xenografts. ER expression was conserved in HR tumors, but expression of the ER-responsive genes PS2, PR and MYB was strongly decreased, indicating impaired ER transcriptional activity. Acquired resistance to tamoxifen and ovariectomy was also associated to a strong decrease of IGF-1R signaling in HBCx-21, and was related to higher P-AKT expression, although P-pS6 was highly expressed in both sensitive and resistant tumors. Furthermore, in vivo experiments showed that EVE alone was highly efficient in all models independently of P-AKT expression/PTEN loss. EVE combined with fulvestrant yielded the most complete and durable tumor growth inhibition. In HBCx-3, proliferation and pS6 levels were markedly decreased only by the EVE-fulvestrant combination. In HBCx-21 and HBCx-34 HR models, EVE-based treatments decreased proliferation, expression of angiogenesis markers, as well as P-pS6 expression with no variation in (P-)AKT nor (P-)mTOR levels. A high IGF-1R protein expression was found only in the HBCx-34 TAM-R tumor, which was strongly decreased in the fulvestrant-treated tumors. Conclusions. LBC-PDX with confirmed HR status have been established and may serve as models to study alternate therapies. Of note, in the HBCx-21 and -34 models, activation of the PI3K pathway is poorly correlated with baseline sensitivity to ET, suggesting other pathways of resistance. EVE alone is highly efficient in all settings, and combination with fulvestrant is promising at the in vivo and molecular levels. Proteomic, genomic and gene expression studies are ongoing. Citation Format: Paul H. Cottu, Thomas Bagarre, Jean-Jacques Fontaine, Franck Assayag, Sophie Richon, Sophie Chateau-Joubert, Aurélie Thuleau, Patricia de Cremoux, Khemaies Slimane, Didier Decaudin, Anne Vincent-Salomon, Ivan Bièche, Elisabetta Marangoni. Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 85. doi:10.1158/1538-7445.AM2013-85

Background. Most ER+ tumors will eventually escape endocrine treatments. PI3K/mTOR pathway targeting has improved clinical results in endocrine treatments resistant (ETR) patients, however with unsustained responses. Our laboratory has developed several models of ETR luminal breast cancer (LBC) patient derived xenografts (PDX) suitable for biological studies (Cottu et al, AACR 2012 & IMPAKT 2012). The aims of the present study were 1) to analyze the biological factors associated with acquired ETR in relevant models of LBC; 2) to determine the therapeutic interest of combining everolimus with different ET models resistant to different endocrine treatments (ET). Methods. Implementation of LBC models has been reported (Cottu et al, BCRT 2012). ETR models were derived by selecting tumors growing under continuous ET, subsequently re-engrafted and rechallenged with endocrine therapies (tamoxifen, letrozole, fulvestrant), and with everolimus alone and combined with ETs. When possible, initial sensitive tumors were simultaneously treated with the same protocols. At progression, or after at least 120 days of continuous therapies, tumor tissue was retrieved. Tissue microarrays (TMA) were performed to analyze the biological modifications associated to ET and mTOR targeting. Triplicate immunohistochemistry of P-AKT/AKT, P-mTOR/mTOR and P-S6 were performed in addition to standard markers. Transcriptomic and RTPCR analyses are also being conducted. Results. Three models resistant to tamoxifen and 2 models resistant to surgical ovariectomy used a surrogate to estrogen deprivation, were developed from 3 initial PDX, and were maintained as independent hormone-resistant variants. All these models retained a luminal phenotype, based on IHC and transcriptomic analyses and had a wt PI3KCA. ETR models had an expression profile very similar to the initial PDX, suggesting that minor changes may be associated with the acquisition of an ETR phenotype. Three of these models have been tested. One tamoxifen resistant model confirmed a resistance specific to tamoxifen only and a high level of sensitivity to a fulvestrant everolimus combination. A second PDX yielded 2 other models resistant to tamoxifen and ovariectomy. These 2 models developed a resistance to all ET modalities. Therapeutic testing with combinations of everolimus and ETs were highly efficient, but everolimus alone was as efficient in these 2 models with complete and durable responses. Activation of the PI3K pathway was demonstrated in all cases by IHC analyses (expression of pAKT and pS6), and complementary ongoing RTPCR analyses. Everolimus based effects were associated with tumor fibrosis, a strong inhibition of Ki67 and pS6, but not always pAKT. The expression of ER was not modified by treatments, except in the fulvestrant-treated mice where it was strongly inhibited. Conclusion. We have developed and validated a platform of ETR models derived from our LBC original PDX. These models retained a luminal phenotype although highly resistant to ET. Activation of the PI3K pathway has been confirmed in ETR models, and everolimus alone, or combined with ET confirms high and durable efficacy. Updated mechanistic analyses performed at different time points will be presented at the meeting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-14.