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PDF file 43K, Phenotype of BCLs after abexinostat treatment

PDF file 86K, supplementary figure legend

PDF file 109K, Conventional molecular parameters do not predict drug-response to abexinostat

PDF file 35K, Expression level of Xist in patient-derived xenograft

PDF file 164K, Cell cycle progression was differentially altered by abexinostat according to drug-response profile

PDF file 123K, HDAC activity is impaired by treatment independently of drugresponse profiles

Purpose: Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies.Experimental Design: We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers.Results: We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population.Conclusions: Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment. Clin Cancer Res; 19(23); 6520–31. ©2013 AACR.

PDF file 126K, The effect of abexinostat on CSC is achieved through the specific inhibition of HDACs

PDF file 54K, Karyotypes of BCLs

PDF file 166K, Identification of two drug-response groups using a Gaussian mixture model (GMM)

PDF file 68K, Supplementary materials and methods

PDF file 44K, Clinical and pathological features of the breast cancer cell lines (BCLs)

PDF file 85K, Histone deacetylase inhibitors target the CSC population in lowdose sensitive BCLs

PDF file 192K, HDACi treatments define two drug-response profiles in BCLs

PDF file - 56K, Table SII.CSC frequency in three independent PDXs (CRCM226 x, CRCM168 x, CRCM174 x).

Table SI. Histoclinical and molecular parameters of the 74 primary tumors injected

PDF file - 87K

PDF file - 3065K, Figure S1. Average delay of growth of PDXs among passages (up to P5). Figure S2. DNA copy number variations found in the original primary tumors are well maintained in PDXs. Figure S3. Example of stromal dilution in parental primary tumor CRCM184 PT compared to paired CRCM 184 X. Figure S4. ALDEFLUOR phenotype of our 20 PDX models. Figure S5. Outgrowth kinetic of each sorted cell population isolated from three PDXs and injected in limited dilutions in fat pads of NSG mice. Figure S6. Univariate and multivariate analysis identified primary tumor engrafment as independent parameter associated with metastasis-free survival (MFS) in the series of 74 patients. Figure S7. Identification of parameters that predict successful engraftment. Figure S8. Global gene expression profiling of 53 PDXs. Figure S9. Supervised analyses of aCGH data comparing copy number variations in 17 paired primary tumors/PDXs. Figure S10. Representative network of genes isolated from the BCSC-GES and involved in DNA damage repair. Figure S11. Representative network of genes isolated from the BCSC-GES and involved in cell cycle control. Figure S12. The ?common? stem cell core transcriptional program (CE-4SC) is associated with clinical outcome. Figure S13. Functional validation of the CE-4SC in two different breast cancer cell lines (BCLs). Figure S14. Knock-down of genes from the CE-4SC affect tumorsphere-forming efficiency (SFE) in S68 BCL.

PDF file - 97K

Table SVI: Correlation between histoclinical and molecular parameters and our BCSC-GES in 2609 patients from public datasets

Table SIII. Breast cancer stem cell gene expression signature (BCSC-GES)

Table SIII. Breast cancer stem cell gene expression signature (BCSC-GES)

Table SV. Public datasets description utilised for the BCSC-GES study

Although advances have been made in the treatment of localized breast cancer, there has been less progress in the treatment of advanced metastatic disease. This disease is an oncoming therapeutic challenge for breast cancer since it is always fatal. Conventional therapies may fail to target cancer stem cells (CSC). Metastatic breast cancer may thus benefit from current intensive research in the CSC field. Several studies suggest that CSCs are the only cancer cells responsible for metastasis formation. CSCs may have the ability to disseminate in blood circulation as circulating tumor cells (CTCs) and to colonize new sites. To better understand the biological processes that link CSCs and metastasis, we have generated a xenobank of 50 patient-derived xenografts (PDXs) of primary tumors grafted in the humanized mammary fat pad of immunodeficient mice. These models reflect the heterogeneity of primary tumors both at molecular and cellular level. Using a lentiviral vector, we infected our PDXs to obtain GFP-luciferase-positive models designed to monitor metastasis development from the orthotopic site. To mimick the patient therapeutic management, metastasis formation is monitored during tumor growth but also after tumor surgical removal. Metastases are collected and analyzed for histology and phenotype. The different metastatic patterns are correlated with the mutational profile of PDX. CTCs are also monitored at different time of the metastatic process using a specific filter (ISET) for cell characterization and phenotype. PDXs can be classified in tumor groups with similar metastatic tropisms. To depict the role of CSC from primary tumors in the tropism of their metastases, we collected and genomic profiled the ALDEFLUOR-positive CSCs in orthotopic tumors from different models with distinct metastatic pattern. These different PDX models lead to a better understanding of the role of breast CSCs in metastasis formation from CTCs to well-constituted metastasis and their study may identify targets able to design future therapeutic strategies. Citation Format: Christophe Ginestier, Danae Lartigue, Olivier Cabaud, Aurelie Malzac, Julien Wicinski, Emmanuelle Josselin, Pascal Finetti, Jose Adelaide, Francois Bertucci, Max Chaffanet, Daniel Birnbaum, Emmanuelle Charafe-Jauffret. Patient-derived xenograft (PDX) models to study the role of breast cancer stem cells in metastasis formation. [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 3020. doi:10.1158/1538-7445.AM2014-3020

Breast cancer is a major health problem and heterogeneity of the disease has been considered as a strong limitation to find the best therapies to cure cancer, overcome recurrences and metastases. The establishment of models that reflect tumor biology and metastatic progression is critical to develop successful new therapeutic strategies. In the breast, orthotopic xenografts currently appear as the best models to study tumor growth, metastasis and develop tools for prognosis prediction. Furthermore, mouse transplant assays have been used to assess cancer stem cell (CSC) activity and demonstrate that leukemia and many solid tumors are organized along a hierarchical model. Despite the promise of the CSC model sustained by mouse transplantation assays, the clinical relevance of xenografts studies to identify determinants of stemness able to influence clinical outcome remains challenging. In breast cancer, transcriptional programs from functionally validated CSC populations remain to be deciphered. Here, we report the establishment of a bank of primary breast tumor-derived xenografts (xenobank). We showed that the xenografts retain the main features of primary tumors, that engraftment is correlated with the presence of CSC in tumors, and that engraftment in the mouse is able to predict prognosis in patients. This suggests that CSCs may govern breast cancer prognosis. We established the gene expression profiles of functionally validated ALDEFLUOR-positive CSC populations (breast CSC-GES) and demonstrated their clinical relevance. Among 2609 patients with breast cancers, we validated that he expression of the breast CSC-GES is correlated with poor outcome and metastasis in uni-and multivariate analysis (5-year MFS was 70% CI95 [67–74] in the breast CSC-positive class and 80% (CI95 [77–83]) in the breast CSC-negative class (p = 5.5E−04 with log-rank test). Furthermore, we identified a core of 19 genes commonly expressed in breast CSC, murine embryonic, neural and hematopoietic stem cells programs and demonstrated for each gene its ability to modulate breast CSC population, being implicated in self-renewing or differentiation programs. We found that the core of genes in common between four stem cell gene expression studies (CE-BCSC-3SC) displayed an adverse prognostic impact for patients with breast cancer. The core contained genes implicated in oxidative phosphorylation, detoxification, lipid metabolism, and genomic stability, and these shared determinants of stemness influenced clinical outcome. Thus, we show ed that CSCs from orthotopically engrafted primary breast tumors have clinical and biological relevance. This functionally validated CSC population is highly correlated with survival and express genes governing main stem cell functions, substantiating a major prediction of the CSC model and opening further promises for new CSC therapies using valid preclinical models. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-01.

Development of pre-clinical models that reflect tumor biology and metastatic progression is a critical issue to better develop therapeutic strategies. The xenotransplantation mouse model of human primary tumors is presented as the best model to mimic patient tumor biology in different pathologies. In breast tumors, orthotopic transplantation recently provides strong evidence of maintenance in xenotransplants of main cognate breast tumoral features. As it has recently been reported that primary breast tumor engraftment is a prognostic indicator of disease outcome for women with breast cancer, we aimed to determine parameters that influence engraftment success. Between January 2008 and June 2009, we transplanted 76 fresh primary tumors from 76 different individuals into cleared and humanized fat pad of female NOD-SCID/ gamma (c) null mice. Tumors grew from 20 out of the 76 samples (29%), and we maintained the main molecular (molecular subtypes, genomic profiles, phenotypes) and histological features of matched primary tumors in xenografts. If a high grade, an absence of hormone receptor expression, a basal-like/ERBB2 molecular subtype was correlated with engrafment rate, the presence of ALDH-expressing CSC in primary tumors was the only parameter predicting engraftment in an independent manner. Using this model, we showed that breast cancer xenografts were hierarchically organized with a subpopulation of ALDEFLUOR-positive cells that maintains tumorigenic activity. In order to identify pathways regulating tumorigenic activity of the CSC population, we derived a CSC gene expression signature (GES) from the comparison of expression profiles of the ALDEFLUOR-positive and -negative populations from our xenograft series. This CSC signature presented three mains core transcriptional programs containing DNA repair genes, suggesting a special ability to resist to DNA damage, gene of G1-S transition checkpoint control including pRb and E2F, and gene of G2-M transition checkpoint, suggesting a potential role in self-renewal and differentiation programs. Given that the presence of CSC in primary tumors is an indicator of tumor engraftment and engraftment predict prognosis, we surmised that molecular machinery governing CSC properties is likely to influence clinical outcome. Indeed, our established CSC GES was able to predict bad outcome in primary breast cancer. Thus, we can speculate that the greater retention of cancer stem cell programs observed in patients with shorter survival compared with those with better outcome may reflect greater uncoupling of self-renewal and maturation programs. Overall, our data indicate the importance of developing CSC biomarkers to contribute to personalized cancer therapy and the need to identify therapeutic targets directed toward CSCs using orthotopic xenografts models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5339. doi:1538-7445.AM2012-5339