Identification of mechanisms of acquired resistance to taxanes in triple negative breast cancer using patient-derived xenografts: a step closer to clinics

Abstract

Chemotherapy is a general treatment for most breast tumors and depending on breast cancer subtype combination with targeted anti-hormonal or anti-HER2 therapy is conducted. Taxane-based regimens constitute the most common therapeutic option in patients with triple negative breast cancer (TNBC), but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived xenografts (PDX).

Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. We analyzed cancer stem cell population dynamics in paired sensitive and resistant TNBC PDX models, but also epigenomics, genomics and transcriptomics profiles to elucidate the unknown molecular mechanisms responsible for this process.

Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

Exome sequencing was also performed in the matched sensitive and docetaxel resistant TNBC PDX models. Multiple mutations, small insertion/deletions and copy number variation (CNV) changes were detected in the original human metastatic samples, and most of them were maintained in serially passaged TNBC PDX models even after long term treatment with docetaxel, with very few changes being detected between paired sensitive and resistant tumors.

However, we identified a chromosomal amplification of chr12p arm present in the metastatic sample and chemoresistant PDXs of one BRCA1 mutant models which was absent in docetaxel sensitive PDXs. Increased expression levels of genes located at chr12p correlated with amplificacion in chemoresistant tumors. Clinical data from TCGA and METABRIC studies confirmed that chr12p amplification was associated with a small subset of TNBC/basal-like breast cancer patients with increased gene expression of genes from that region and poor survival after chemotherapy. Our findings suggest that there is a subset of TNBC/basal-like breast cancer harbouring chr12p amplification that may be associated with resistance to docetaxel treatment in clinical setting.

Genome-wide DNA methylation and gene expression analysis have been performed in preclinical breast cancer patient-derived xenograft (PDX) models with primary and acquired chemoresistance. These analyses revealed that DNA methylation patterns from breast cancer PDX are closer to breast cancer clinical samples than breast cancer cell lines (BCCLs) and they maintain subtype specific methylation patterns. Triple negative PDX tumors show very stable methylation patterns accompanying chemoresistance acquisition but some critical genes/pathways were unraveled as differentially methylated. Transcriptomically, TNBC PDX tumors accumulate gene expression changes during chemoresistance acquisition with some common pathways between different triple negative PDX models. Integrative analysis reveals correlation of some differentially methylated genes as differentially expressed in resistant triple negative breast cancer PDX tumors. These findings identify a set of promising pathways that may contribute to the acquisition of chemoresistance in TNBC patients.

Receptor activator of NF-κB (RANK) is expressed in human breast tumors and has been associated with aggressive breast cancers. In this study we used breast cancer PDX models to investigate the functional role of RANK and its ligand (RANKL) in clinical human breast cancer. RANK expression in human breast cancer is more frequent in ER/PR-negative than in hormone receptor positive tumors and that is maintained in breast cancer PDX models. RANKL is generally poorly expressed in human breast cancer, but some RANKL-positive breast cancer PDX models were identified. Selection of RANK/RANKL-positive breast cancer PDX models expressing different levels of RANK or RANKL was done in order to functionally study the role of RANK signalling in human breast cancer. In vitro RANKL treatment on breast cancer cells isolated from RANK/RANKL-positive PDX models show modulation of NF-κB pathway, even in tumors where RANK expression was very low.

La quimioterapia es un tratamiento general usado comúnmente en cáncer de mama, sobretodo en tumores del subtipo triple negativo (TNBC). Éstos responden inicialmente muy bien, pero con el tiempo suelen dar lugar a recidivas o metástasis quimioresistentes. Empleando modelos preclínicos de cáncer de mama hemos estudiado los mecanismos subyacentes de la adquisición de resistencia a taxanos mediante análisis de poblaciones celulares, así como de análisis epigenómicos, genómicos y transcriptómicos Con el estudio de dinámicas poblacionales descubrimos la existencia en tumores TNBC inicialmente sensibles la existencia de una población CD49f+ con capacidad de iniciación tumoral que se expande durante la adquisición de quimioresistencia. Esta población revierte en ausencia de droga y con ella la quimioresistencia. Además, describimos una firma transcripcional de resistencia, predictivo de recidiva de la enfermedad después de la quimioterapia de TNBC. Los análisis genómicos revelaron la existencia de mútiples mutaciones y aberraciones cromosómicas en las muestras de pacientes que se mantienen de forma estable en los modelos preclínicos, aún con la adquisición de quimioresistencia. Únicamente se detectó una amplificación del cromosoma 12 en tumores resistentes no presente en sensibles. Esta amplificación se asocia en muestras clínicas a un subgrupo de tumores TNBC con peor supervivencia que el resto, pudiendo ser quimioresistentes. Los análisis epigenómicos revelaron la utilidad de estos modelos preclínicos al ser muy cercanos a muestras clínicas y mantener los patrones asociados a cada subtipo de cáncer de mama. Estos patrones son tan estables que prácticamente no varían con la adquisición de quimioresistencia, sin embargo algunos genes y rutas concretos sí lo hacen. Los análisis genómicos mostraron algunas vías comunes alteradas y un análisis integrador de los datos de metilación y expresión mostraron genes y rutas afectadas que presentaban metilación y expresión diferencial con la adquisición de resistencia.