Rania El Botty

Acquired Resistance to Endocrine Treatments Is Associated with Tumor-Specific Molecular Changes in Patient-Derived Luminal Breast Cancer 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.

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient‐derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT‐PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple‐negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanibs main targets.

Targeting mTOR pathway inhibits tumor growth in different molecular subtypes of triple-negative breast cancers

Triple-negative breast cancers (TNBC) are characterized by frequent alterations in the PI3K/AKT/mTOR signaling pathway. In this study, we analyzed PI3K pathway activation in 67 patient-derived xenografts (PDX) of breast cancer and investigated the anti-tumor activity of the mTOR inhibitor everolimus in 15 TNBC PDX with different expression and mutational status of PI3K pathway markers. Expression of the tumor suppressors PTEN and INPP4B was lost in 55% and 76% of TNBC PDX, respectively, while mutations in PIK3CA and AKT1 genes were rare. In 7 PDX treatment with everolimus resulted in a tumor growth inhibition higher than 50%, while 8 models were classified as low responder or resistant. Basal-like, LAR (Luminal AR), mesenchymal and HER2-enriched tumors were present in both responder and resistant groups, suggesting that tumor response to everolimus is not restricted to a specific TNBC subtype. Analysis of treated tumors showed a correlation between tumor response and post-treatment phosphorylation of AKT, increased in responder PDX, while PI3K pathway markers at baseline were not sufficient to predict everolimus response. In conclusion, targeting mTOR decreased tumor growth in 7 out of 15 TNBC PDX tested. Response to everolimus occurred in different TNBC subtypes and was associated with post-treatment increase of P-AKT.

Metabolic Response to Everolimus in Patient-Derived Triple-Negative Breast Cancer Xenografts

Patients with triple-negative breast cancer (TNBC) are unresponsive to endocrine and anti-HER2 pharmacotherapy, limiting their therapeutic options to chemotherapy. TNBC is frequently associated with abnormalities in the PI3K/AKT/mTOR signaling pathway; drugs targeting this pathway are currently being evaluated in these patients. However, the response is variable, partly due to heterogeneity within TNBC, conferring a need to identify biomarkers predicting response and resistance to targeted therapy. In this study, we used a metabolomics approach to assess response to the mTOR inhibitor everolimus in a panel of TNBC patient-derived xenografts (PDX) (n = 103 animals). Tumor metabolic profiles were acquired using high-resolution magic angle spinning magnetic resonance spectroscopy. Partial least-squares-discriminant analysis on relative metabolite concentrations discriminated treated xenografts from untreated controls with an accuracy of 67% (p = 0.003). Multilevel linear mixed-effects models (LMM) indicated reduced glycolytic lactate production and glutaminolysis after treatment, consistent with PI3K/AKT/mTOR pathway inhibition. Although inherent metabolic heterogeneity between different PDX models seemed to hinder prediction of treatment response, the metabolic effects following treatment were more pronounced in responding xenografts compared to nonresponders. Additionally, the metabolic information predicted p53 mutation status, which may provide complementary insight into the interplay between PI3K signaling and other drivers of disease progression.

Abstract 1687: Vandetanib as a potential new treatment for ER negative breast cancers

Introduction: Recent studies have shown that the receptor tyrosine kinase RET is involved in the biology of ER positive breast cancers and in the response to endocrine treatment, but its role in ER negative tumors is unknown. Here we investigated the expression of RET in BC patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib in ER negative BC PDX. Methods: RET mRNA expression was analyzed in BC of 446 patients and 57 PDX by RT-PCR analysis. The activity of Vandetanib, a tyrosine kinase inhibitor targeting RET, EGFR and VEGFR2, was tested in three PDX of triple-negative breast cancer (TNBC) and one PDX of HER2+ BC with different levels of RET expression. Protein expression of P-RET, RET, EGFR, P-EGFR and c-KIT were determined by immunohistochemistry (IHC). Analyses of PI3K and MAPK pathways and angiogenesis were performed by IHC and RT-PCR in both untreated and Vandetanib-treated tumors. Results: In both clinical samples and PDX, elevated levels of RET were found in ER+ and HER2+ tumors, and in a subgroup of TNBC tumors. In the HBCx5 (HER2+) and HBCx24 (TNBC) PDX, both with RET over-expression, treatment by Vandetanib resulted in tumor growth inhibition (TGI) of 90% and 98%, respectively. In both models, tumor regressions were observed in 50% of xenografts. The effect of Vandetanib was associated to a marked inhibition of RET phosphorylation. To determine whether the lack of RET over-expression was associate to Vandetanib resistance, we treated two additional TNBC PDX with low and no expression of RET: HBCx4B and HBCx14. In these models, treatment by Vandetanib still inhibited tumor growth with a TGI of 85%. Tumor regressions were registered in 42% of animals in the PDX model with low expression of RET (HBCx4B), while no tumor regression were observed in HBCx14. IHC analyses showed an over-expression of EGFR in the HBCx4B xenograft and inhibition of EGFR phosphorylation in treated tumors, suggesting that tumor response to Vandetanib could depend on EGFR inhibition in this tumor. Further analyses of treated tumors revealed a decreased expression of phospho-ERK in the 4 PDX models, indicating inhibition of MAPK pathway, while the phosphorylation status of the PI3K pathway markers S6 and 4EBP1 was unchanged. Finally, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the 4 PDX tested, indicating angiogenesis inhibition. Conclusions: Treatment by Vandetanib resulted in strong tumor growth inhibition in ER negative PDX with over-expression of RET. This effect was associated to inhibition of RET phosphorylation and MAPK pathway and decreased tumor vascularization. The lack of RET over-expression did not predict Vandetanib resistance, and over-expression of EGFR was also associated to a marked tumor response. These preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers expressing Vandetanib9s targets. Citation Format: Elisabetta Marangoni, Rana Hatem, Dalila Labiod, Sophie Chateau-Joubert, Rania El Botty, Jean-Luc Servely, Ludmilla De Plater, Ivan Bieche. Vandetanib as a potential new treatment for ER negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2015-1687

Selumetinib-based therapy in uveal melanoma patient-derived xenografts

The prognosis of metastatic uveal melanoma (UM) is among the worst of all human cancers. The identification of near-ubiquitous GNAQ/GNA11 mutations and the activation of MAPK signaling in UM have raised hopes of more effective, targeted therapies, based on MEK inhibition, for example. We evaluated the potential of drug combinations to increase the efficacy of the MEK inhibitor selumetinib (AZD6244, ARRY-142886), in UM cell lines and Patient-Derived Xenografts. We first evaluated the combination of selumetinib and DTIC. We found that DTIC did not improve the in vitro or in vivo antitumor efficacy of selumetinib, consistent with the outcome of the SUMIT clinical trial assessing the efficacy of this combination in UM. We then tested additional selumetinib combinations with the chemotherapy agent docetaxel, the ERK inhibitor AZ6197, and the mTORC1/2 inhibitor, vistusertib (AZD2014). Combinations of selumetinib with ERK and mTORC1/2 inhibitors appeared to be the most effective in UM PDX models.

Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer in combination to PARP inhibition

Breast cancer is a complex disease in which each patient could present several genetic alterations that are therapeutically relevant in cancers. Here we explored the therapeutic benefit of combining PARP and mTOR inhibitors in a context of DNA repair deficiency and PI3K pathway activation. The combination of everolimus and olaparib was tested in BRCA2-mutated patient-derived xenografts (PDX) carrying alterations in the PI3K/AKT/mTOR pathway. An RPPA analysis of different signalling pathways was performed in untreated and treated xenografts. Everolimus and olaparib showed marked anti-tumor activities in the monotherapy setting and high efficacy when given in combination with 100% of mice showing tumor regressions. The fraction of P-H2AX positive cells was increased in both monotherapy arms and strongly increased in the combination setting. Everolimus given as monotherapy resulted in downregulation of different proteins involved in DNA damage repair, including FANCD2, RAD50 and SUV39H1. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA damage repair components. In conclusion, our results suggest that combining mTOR and DNA repair inhibition could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway.

Capecitabine efficacy is correlated with TYMP and RB expression in PDX established from triple-negative breast cancers

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments. Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX. Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment. Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605–15. ©2018 AACR.

Abstract 4499: Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Patients with triple-negative breast cancer (TNBC) have a poor prognosis and targeted therapies are lacking. Recent studies performed on patients tumors showed and increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway in TNBC. PI3K pathway is critical for cell growth, survival, and angiogenesis. Everolimus is a mTOR inhibitor recently showed to increase survival of patients with metastatic luminal breast cancer. The objectives of this work were to analyze the PI3K activation status in a large cohort of patient-derived xenografts (PDX) of TNBC and to investigate the therapeutic potential of mTOR inhibition. Experimental procedures: this study included a panel of 32 TNBC PDX models previously described (Marangoni et al 2007). Expression of AKT, P-AKT, P-mTOR, S6, P-S6, P-4EBP1, PTEN and INPP4B was analyzed by WB and IHC. Mutations of PIK3CA (exons 9 and 20), PIK3R1 (exons 11-15), and AKT1 (exon 4) were detected by sequencing of cDNA fragments obtained by RT-PCR amplification. The efficacy of the mTOR inhibitor everolimus was investigated in vivo on 10 PDX models with different expressions and mutational status of PI3K markers. Results: INPP4B protein expression was lost in 56% of tumors (n=18) and expressed at low levels in 31% of models, while only 2 models displayed a marked expression. PTEN expression was lost in 78% of tumors. Thirteen PDX models (40%) displayed a concomitant loss of both INPP4B and PTEN proteins. In 67% of tumors, the ratio between phosphorylated and unphosphorylated AKT was greater than 1. S6 was found to be phosphorylated in the great majority of tumors. PI3KCA and AKT1 genes were mutated only in 1 and 2 tumors, respectively. On the 10 PDX models treated with everolimus, 6 models responded to treatment with a tumor growth inhibition (TGI) comprised between 60% and 80%. Four models were classified as resistant or low responder (TGI<50%). Preliminary analysis of treated tumors from 6 models indicates increased level of P-AKT (feedback loop) to occur only in responder models, while inhibition of S6 phosphorylation occurred in treated tumors from both responder and resistant models. Finally, expression of INPP4B or PTEN alone did not predict for tumor response, while a P-AKT/AKT ratio greater than 1 predicted response to everolimus (p<0.05, Fishers exact test). Conclusions: the majority of TNBC PDX models showed loss of PTEN or INPP4B proteins or both, associated with activation of PI3K pathway. Preliminary results obtained from 10 PDX models indicate that mTOR targeting resulted in tumor growth inhibition in several models with AKT phosphorylation. Additional TNBC models will be tested in order to search for robust predictive biomarkers. This large panel of characterized PDX of TNBC models represents a clinical relevant tool to investigate the activity of PI3K-AKT-mTOR inhibitors and identify predictive biomarkers. Citation Format: Elisabetta Marangoni, Rana Hatem, Rania El Botty, Ludmilla De Plater, Dalila Labiod, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bieche. Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2014-4499

Abstract C174: Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus.

The addition of everolimus (Rad) to endocrine therapies (ET) in luminal advanced breast cancers has shown to increase overall survival in randomized studies. However drug resistance is an emerging problem and most patients eventually experience progression of disease. We aimed to establish and characterize patient-derived xenografts (PDX) of luminal breast cancers with acquired resistance in vivo to both ET and Rad. Two PDX xenografts (HBCx22 and HBCx34), established from early stage ER+ breast cancers, were treated in vivo with different ET (tamoxifen, fulvestrant, ovariectomy and letrozole) during 12 months and tumors escaping to therapies were re-engrafted and maintained under treatment. Four hormono-resistant (HR) xenografts were established, 2 resistant to Tamoxifen (HBCx22TamR and HBCx34TamR) and 2 resistant to ovariectomy (HBCx22OvaR and HBCx34OvaR). Expression of estrogen receptor (ER) was maintained, while expression of several ER-controlled genes (MYB, PS2 and PR) was decreased in HR tumors indicating modifications of ER transcriptional activity. The expression of ER co-regulators was additionally studied: GREB1 was reduced specifically in TamR, while FOXA1 were increased specifically in TamR. Expression analysis of P-AKT, P-mTOR and P-S6 showed similar levels of PI3 kinase pathway activation between parental and HR xenografts. Gene expression profiling, performed with Affymetrix Gene Expression Arrays, showed that genes differentially expressed in TamR and OvaR tumors were only partially overlapping, suggesting treatment-specific mechanisms of hormono-resistance. In addition, TamR and OvaR signatures were tumor-specific. Gene ontology enrichment and pathway analyses of gene expression datasets will be presented at the meeting. HR xenografts were then treated with Rad alone and combined to ET. In HBCx22OvaR and TamR, resistant to all ET, treatment with Rad arrested tumor growth in vivo but did not show any additive effect when combined to ET. Conversely, the HBCx34TamR model was still responding to fulvestrant, and the combination Rad+Fulvestrant resulted in tumor regressions. In the HBCx34TamR xenograft, tumor escape to Rad treatment occurred in one mouse after 8 months of continuous treatment. The tumor was re-engrafted and maintained as tumorgraft resistant to both tamoxifen and Rad (HBCx34Tam-RadR). Western blot and IHC studies showed that mTOR signaling was still inhibited in this tumor, but it was associated to MAP kinase activation and increased IGF-1R expression. In conclusion, we have developed PDX models of luminal breast cancer with cross-resistance to ET and everolimus in vivo. These models provide a valuable preclinical tool to investigate molecular mechanisms of acquired resistance and to test new targeted therapies and new combinations in patients’ tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C174. Citation Format: Rania El Botty, Paul Cottu, Franck Assayag, Pierre de la Grange, David Gentien, Aurelie Thuleau, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bieche, Elisabetta Marangoni. Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C174.