Rana Hatem

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.

Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer

Background:Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known.Methods:We used RT–PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome.Results:MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway.Conclusions:Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings.

BackgroundTumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.MethodsTo characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts.ResultsAs expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated.ConclusionsTaken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

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.

Rare overexpression of anaplastic lymphoma kinase gene in inflammatory and non-inflammatory breast cancer

Inflammatory breast cancer (IBC) is the least common, mostly poorly understood and most severe form of breast carcinoma. Due to this exceptional severity, treatment of IBC remains a challenge for breast cancer physicians. Despite intensive research, few molecular characteristics of IBC have been described, and none are used for specific targeted therapies. IBC patients therefore still receive common breast cancer regimens. One of the main pitfalls for IBC molecular characterisation is the small sample sizes of published IBC series. Second, IBC has been variously defined, although the breast cancer community now agrees that the diagnosis requires the presence of rapidly progressing inflammatory signs of the breast. Third, the sample size and clinical characteristics of the non-IBC comparison cohort may interfere with identification of molecular aberrations specific to IBC. Finally, most studies have focused on molecular abnormalities detected in non-IBC, while specific biological disorders are expected to be responsible for the unique phenotype and behaviour of IBC. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor with oncogenic functions. ALK is involved in non-small cell lung cancers (NSCLC) and other malignancies when altered by translocation, ampli-

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

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.

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 A9: Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy.

Background: In HER2 positive and triple-negative breast cancer subgroups, residual disease after neoadjuvant therapy is associated with higher risk of metastatic recurrence compared to patients achieving a pathological complete response. Residual tumor analysis after neoadjuvant treatment is a major and under-explored field to identify resistance mechanisms. To develop patient-derived xenografts (PDX) of residual breast cancer we started a program of residual tumor engraftment in nude mice, following the same procedures previously published for PDX of human breast cancer (Marangoni et al, 2007 and Reyal et al, 2012). Methods: 26 residual breast tumors and 2 residual metastatic axillary lymph nodes were engrafted in swiss nude mice immediately after surgery. Expression of Ki67, HER2, PTEN, P-AKT, P-S6, MET, RET and KIT were analyzed in xenografts by immunohistochemistry, western blot and RT-PCR analyses. Brain, lungs, liver and bones of xenografts were systematically formalin-fixed to search for human metastasis. The in vivo drug response of established xenografts was determined for the following treatments: adryamicin+cyclophosphamide (AC), docetaxel, capecitabine, cisplatin, irinotecan, everolimus, trastuzumab and lapatinib (for the HER2+ PDX). PDX tumors were additionally mechanically dissociated to establish cell lines. Results: Seven PDX were established (tumor take of 25%), 5 triple-negative and 2 HER2+. Six out of seven PDX were metastatic in the lungs. Two xenografts were established from lymph node metastasis. The in vivo drug responses were concordant with the response to neo-adjuvant treatments in patients. Histological analyses showed that xenografts’ tumors recapitulated the patients’ tumor morphology. Residual tumor xenografts expressed high level of Ki67 protein and tumor latency during the first tumor passages was found to be shorter when compared to tumor latency of non pre-treated breast cancers. In 5/5 triple-negative breast cancer PDX the PTEN protein was lost and the PI3 kinase pathway activated. The mTOR inhibitor Everolimus was tested in 2 triple-negative PDX: one was resistant and one was responding, with a tumor growth inhibition of 80%. Triple-negative PDX show expression of “druggable” tyrosin kinase receptors (MET, RET, KIT) providing relevant models to test new target therapies in these models. One cell line was established from a highly metastatic triple-negative breast cancer xenograft. When re-injected into mice, the cell line was tumorigenic, however the tumor architecture was changed and the xenograft was not metastatic. Conclusions: we have established a panel of metastatic PDX models of breast cancer resistant to neo-adjuvant therapies. These models provide a valuable preclinical tool to investigate mechanisms of resistance to neo-adjuvant treatments and for the preclinical testing of new targeted agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A9. Citation Format: Elisabetta Marangoni, Dalila Labiod, Franck Assayag, Rania El Botty, Rana Hatem, Sophie Richon, Sophie Chateau-Joubert, Marine Carlus, Helene Bonsang-Kitzis, Alice Pinheiro, Cecile Laurent, Ivan Bieche, Fabien Reyal. Establishment and characterization of residual breast cancer patient-derived xenografts resistant to neo-adjuvant therapy. [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 A9.