Sophie Chateau-Joubert

Inhibiting Aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts

Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin–cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment. Mol Cancer Ther; 11(12); 2693–703. ©2012 AACR.

Evaluating Patient-Derived Colorectal Cancer Xenografts as Preclinical Models by Comparison with Patient Clinical Data

Development of targeted therapeutics required translationally relevant preclinical models with well-characterized cancer genome alterations. Here, by studying 52 colorectal patient-derived tumor xenografts (PDX), we examined key molecular alterations of the IGF2-PI3K and ERBB-RAS pathways and response to cetuximab. PDX molecular data were compared with that published for patient colorectal tumors in The Cancer Genome Atlas. We demonstrated a significant pattern of mutual exclusivity of genomic abnormalities in the IGF2-PI3K and ERBB-RAS pathways. The genomic anomaly frequencies observed in microsatellite stable PDX reproduce those detected in nonhypermutated patient tumors. We found frequent IGF2 upregulation (16%), which was mutually exclusive with IRS2, PIK3CA, PTEN, and INPP4B alterations, supporting IGF2 as a potential drug target. In addition to maintaining the genomic and histologic diversity, correct preclinical models need to reproduce drug response observed in patients. Responses of PDXs to cetuximab recapitulate also clinical data in patients, with partial or complete response in 15% (8 of 52) of PDXs and response strictly restricted to KRAS wild-type models. The response rate reaches 53% (8 of 15) when KRAS, BRAF, and NRAS mutations are concomitantly excluded, proving a functional cross-validation of predictive biomarkers obtained retrospectively in patients. Collectively, these results show that, because of their clinical relevance, colorectal PDXs are appropriate tools to identify both new targets, like IGF2, and predictive biomarkers of response/resistance to targeted therapies.

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.

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.

Abstract B142: Dissociation of preclinical primary human cancer xenografts for cell surface transportome profiling.

Background: Tumor cell metabolism is of growing interest in both basic and clinical cancer research. A better understanding of underlying molecular mechanisms involving metabolite transport in normal and tumor cells should help drug discovery and development. Specific exofacial ligands to metabolite transporters derived from the receptor binding domain (RBD) of retrovirus envelope proteins were developed and used to quantify cell surface metabolite transporters. While cell surface labelling can be readily performed on cultured cell lines, analysis of single cells from solid tumors is more challenging. In this study, we developed a robust method for the disaggregation of tumor cells from human breast cancers grown as xenografts in mice. This procedure was then used to analyse the expression profiles of 4 cell membrane metabolite transporters involved in cell proliferation and tumorigenesis: Glut1, ASCT2, PiT1, and PiT2. Materials and methods: Eight primary human breast cancer xenografts were used for ex vivo experiments (Marangoni et al 2007). We developed an optimized disaggregation protocol to obtain maximum viable cell recovery from the xenografts. The protocol was validated for presence of CD44+ tumor cells and for cell viability using caspase 3 and DAPI exclusion and subsequently, applied to the xenografts for flow cytometry analyses, immunohistochemistry and ex vivo cell culture. Expression profiles of 4 metabolite transporters were assessed in 5 different human breast cancer xenografts. Results: The optimal dissociation protocol developed for these tumors combined mild non-enzymatic (non-enzymatic dissociation buffer) and enzymatic (collagenase III/DNase I) steps, followed by cell purification on a dual density Ficoll gradient. Less than 10% of resulting DAPI negative tumor cells were caspase 3 positive. Dissociated cells showed sustained viability in in vitro cultures for at least 12 days. The numbers of CD44+ cells determined by flow cytometry corresponded to those observed by IHC. The expression profiles of Glut1, ASCT2, PiT1, and PiT2 were distinct for each of the five human breast cancers, and metabolite transporter profiles were highly conserved for xenografts derived from the same tumor. Conclusions: Mouse xenograft implants of human breast cancer tumors were used to optimize and validate a dissociation method for the production of viable single cells. Cell suspensions were then assessed for cell surface metabolite transporters expression by flow cytometry. The expression patterns of four metabolite transporters, Glut1, ASCT2, PiT1, and PiT2 showed distinctive signature profiles for each group of xenografts, indicative of specific metabolic adaptations that can be tracked with our ligands for each tumor. Reference: 1. Marangoni E et al, CCR 2007;13:3989–3998. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B142.

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

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

Abstract 85: Endocrine resistant luminal breast cancer xenografts are powerful models for the analysis of sensitivity to endocrine and everolimus treatments .

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, Aurelie Thuleau, Patricia de Cremoux, Khemaies Slimane, Didier Decaudin, Anne Vincent-Salomon, Ivan Bieche, 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

The iron chelator deferasirox synergises with chemotherapy to treat triple-negative breast cancers: Iron deprivation for breast cancer treatment

To ensure their high proliferation rate, tumor cells have an iron metabolic disorder causing them to have increased iron needs, making them more susceptible to iron deprivation. This vulnerability could be a therapeutic target. In breast cancers, the development of new therapeutic approaches is urgently needed for patients with triple‐negative tumors, which frequently relapse after chemotherapy and suffer from a lack of targeted therapies. In this study, we demonstrated that deferasirox (DFX) synergises with standard chemotherapeutic agents such as doxorubicin, cisplatin and carboplatin to inhibit cell proliferation and induce apoptosis and autophagy in triple‐negative breast cancer (TNBC) cells. Moreover, the combination of DFX with doxorubicin and cyclophosphamide delayed recurrences in breast cancer patient‐derived xenografts without increasing the side‐effects of chemotherapies alone or altering the global iron storage of mice. Antitumor synergy of DFX and doxorubicin seems to involve downregulation of the phosphoinositide 3‐kinase and nuclear factor‐κB pathways. Iron deprivation in combination with chemotherapy could thus help to improve the effectiveness of chemotherapy in TNBC patients without increasing toxicity. Copyright

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.