Ewa Przybytkowski

Nanoparticles can induce changes in the intracellular metabolism of lipids without compromising cellular viability.

There is growing concern about the safety of engineered nanoparticles, which are produced for various industrial applications. Quantum dots are colloidal semiconductor nanoparticles that have unique luminescence characteristics and the potential to become attractive tools for medical imaging. However, some of these particles can cause oxidative stress and induce cell death. The objective of this study was to explore quantum dot‐induced metabolic changes, which could occur without any apparent cellular damage. We provide evidence that both uncoated and ZnS‐coated quantum dots can induce the accumulation of lipids (increase in cytoplasmic lipid droplet formation) in two cell culture models: glial cells in primary mouse hypothalamic cultures and rat pheochromocytoma PC12 cells. Glial cells treated with CdTe quantum dots accumulated newly synthesized lipids in a phosphoinositide 3‐kinase‐dependent manner, which was consistent with the growth factor‐dependent accumulation of lipids in PC12 cells treated with CdTe and CdSe/ZnS quantum dots. In PC12 cells, quantum dots, as well as the hypoxia mimetic CoCl2, induced the up‐regulation of hypoxia‐inducible transcription factor‐1α and the down‐regulation of the β‐oxidation of fatty acids, both of which could contribute to the accumulation of lipids. On the basis of our results, we propose a model illustrating how nanoparticles, such as quantum dots, could trigger the formation of intracellular lipid droplets, and we suggest that metabolic measurements, such as the determination of fat oxidation in tissues, which are known sites of nanoparticle accumulation, could provide useful measures of nanoparticle safety. Such assays would expand the current platform of tests for the determination of the biocompatibility of nanomaterials.

The use of ultra-dense array CGH analysis for the discovery of micro-copy number alterations and gene fusions in the cancer genome

BackgroundMolecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.MethodsWe performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.ResultsDNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.ConclusionsThis is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.

Chromosome-breakage genomic instability and chromothripsis in breast cancer

BackgroundChromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.ResultsWe performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.ConclusionsOur results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor’s biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

Imaging and organelle distribution of fluorescent InGaP/ZnS nanoparticles in glial cells

AIM To assess the effects of oleic acid treatment on subcellular distribution of indium gallium phosphide-zinc sulfide (InGaP/ZnS) nanoparticles in microglia and astrocytes. MATERIALS & METHODS The extent of colocalization between the nanoparticles and organelles was assessed by confocal microscopy, spectrofluorometry and cell sorting. RESULTS Cell treatment with a common fatty acid (oleic acid) within the range of physiological concentrations markedly enhanced the InGaP/ZnS uptake by microglia and afforded their colocalization within lipid droplets/lysosomes but not with mitochondria. CONCLUSION These results suggest that the availability of mono-unsaturated fatty acids, such as oleic acid, in different cells could significantly alter nanoparticle uptake and localization, which can in turn affect the functions of cells and tissues coexposed to nanoparticles.

Ultradense array CGH and discovery of micro-copy number alterations and gene fusions in the cancer genome.

The characterization of molecular alterations specific to cancer facilitates the discovery of predictive and prognostic biomarkers important to targeted therapeutics. Alterations critical to cancer therapeutics include copy number alterations (CNAs) such as gene amplifications and deletions as well as genomic rearrangements resulting in gene fusions. There are two genome-wide technologies used to detect CNAs: next generation sequencing (NGS) and dense microarray based comparative genomic hybridization, termed array CGH (aCGH). aCGH is a mature robust technology of lower cost and more accessible than NGS. This chapter describes the protocol steps and analysis required to obtain reliable aCGH results from clinical samples. Technical options and various necessary compromises related to the nature of clinical material are considered and the consequences of these choices for data analysis and interpretation are discussed. The chapter includes brief description of the data analysis, even though analysis is often performed by bioinformaticians. Todays cancer research requires collaboration of clinicians, molecular biologists, and mathematicians. Acquaintance with the basic principles related to the extraction of the data from arrays, its normalization and the algorithms available for analysis provides a baseline for mutual understanding and communication.

Abstract 709: Resistance to paclitaxel in triple negative breast cancer cells is associated with ABCB1 gene rearrangement

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Breast cancer represents a heterogeneous group of tumors that exhibit a wide spectrum of clinical, pathologic, and molecular features. Of these tumors, triple-negative breast cancer (TNBCs), shows one of the most aggressive clinical behaviors with distinctive metastatic patterns and very poor prognosis. Due to the “triple negative” status of receptor expression, TNBC patients do not benefit from hormonal therapies or treatments targeted against HER2. Therefore, chemotherapy and, in particular, taxane-based therapy (e.g. paclitaxel) remains the treatment of choice for patients with TNBC. Paclitaxel (PTX) is among the most effective anti-cancer agents developed in the past decades, which is widely used in the treatment of patients with locally advanced and metastatic breast cancer. TNBCs are initially highly responsive to PTX however; the majorities of TNBC patients acquire resistance and develop progressive disease. Therefore, development of resistance to chemotherapeutic agents is the major obstacle to the successful treatment of TNBC patients. In vitro studies on cell lines with acquired resistance provide models for characterization of the biological mechanisms of resistance. We have developed five resistant TNBC cell lines by exposure of cells to increasing concentrations of PTX. We used an integrative analysis of array CGH and gene expression data to gain insights into the interplay of functional changes of the genome in TNBC resistant cell lines. Integrative analysis of aCGH and gene expression revealed deregulated genes whose expression was correlated with their copy number alterations. The most highly up-regulated gene in all 5 resistant cell lines was ABCB1. This up-regulation was accompanied by gene amplification in three resistant cell lines: BT20R, SUM149R and MDA-468 but not in 2 other lines, MDA-MB-231 and MDA-MB-436. The overexpression of ABCB1 at protein level was confirmed only in the three resistant cell lines with the gene amplification, suggesting that changes in ABCB1 copy number may result in gene activation and acquisition of PTX resistance. Array CGH analysis of 7q21.12 in BT20R, SUM149R and MDA-468R revealed similar, but not identical, breakpoints in ABCB1. Using 5′RACE, we identified novel inter-chromosomal translocations of ABCB1 in 2 drug resistance cell lines containing ABCB1 breakpoints. These genomic rearrangements occurred in the 5′ un-translated region (UTR) of ABCB1. The deletion of the 5′UTR of ABCB1 in these resistant cell lines may serve to overcome a translation initiation block, which would then favor the expression of ABCB1. Thus the detection of ABCB1 genomic rearrangements may be a candidate biomarker to predict resistance to paclitaxel in triple negative breast cancers. To our knowledge, we are the first to report ABCB1 amplification and ABCB1 gene fusions as a potential mechanism of drug resistance in TNBC cell lines. Citation Format: Elaheh Ahmadzadeh, Ewa Przybytkowski, Regina Kiu, Adriana Aguilar-Mahecha, Mark Basik. Resistance to paclitaxel in triple negative breast cancer cells is associated with ABCB1 gene rearrangement. [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 709. doi:10.1158/1538-7445.AM2015-709

Abstract 4320: Genomic change in residual triple-negative breast cancers after neoadjuvant chemotherapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Triple negative breast cancer (TNBC) is characterized by its aggressive phenotype and its genomic instability. TNBC patients who do not respond to neoadjuvant chemotherapy have a very poor prognosis. Currently, little is known about the mechanisms of drug resistance and how to overcome it in TNBC. Our study aims at identifying molecular factors enriched for in residual TNBC tumors after standard neoadjuvant chemotherapy. Methods: We obtained specimens from 60 TNBC patients participating in a clinical trial (Q-CROC-03). Biopsies were collected prior to and after standard neoadjuvant chemotherapy and residual cancer was collected at the time of surgery. Matched tumor specimens (pre and post) from 9 patients were analyzed by array comparative genomic hybridization (CGH), gene expression microarrays and whole exome sequencing. All samples contained >50% tumor cellularity. Results: Gene expression data was used to identify the different TNBC subtypes (TNBCtype). Six of the 7 subtypes were represented in at least one sample from our cohort. In the post-chemo samples, we observed a change in TNBC subtype compared to the pre-chemo samples in 6 pairs. The most common switch was to the Immuno Modulatory subtype (IM). aCGH analysis showed relatively few differences in copy number variants (CNV) following chemotherapy in 3 out of 8 patients. Whole exome sequencing revealed increased allele frequency or appearance of de novo mutations in TP53 gene in the residual cancers of 2 of the 3 patients presenting differences in CNVs post treatment. Interestingly, pathway analyses revealed that genes involved in DNA binding, chromosomal organization and nucleosome organization were differentially expressed (>2fold) in 2 of the patients with CNV changes. Our results suggest that increased levels of TP53 mutations and altered transcriptional expression of genes involved in chromosomal functions could be associated with the presence of CNV changes in drug resistant tumors. Conclusion: In summary, the genome of TNBCs does not undergo major changes during neoadjuvant chemotherapy; however, enrichment for or de novo TP53 mutations is associated with the appearance of novel CNVs in drug resistant residual tumors. Citation Format: Adriana Aguilar-Mahecha, Ewa Przybytkowski, Josiane Lafleur, Cathy Lan, Stephanie Legare, Najmeh Alirezaie, Carole Seguin, Federico Discepola, Bojan Kovacina, Catalin Mihalcioiu, Andre Robidoux, Elizabeth Marcus, Josee Anne Roy, Manuela Pelmus, Olga Aleynikova, Sheida Nabavi, Jacek Majewski, Mark Basik. Genomic change in residual triple-negative breast cancers after neoadjuvant chemotherapy. [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 4320. doi:10.1158/1538-7445.AM2015-4320

Abstract A041: Targeting EGFR reverses paclitaxel resistance associated with ABCB1 overexpression in triple-negative breast cancer

Background: Breast cancer represents a heterogeneous group of tumors that exhibit a wide spectrum of clinical, pathologic, and molecular features. Of these tumors, triple-negative breast cancer (TNBCs), shows one of the most aggressive clinical behaviors with distinctive metastatic patterns and very poor prognosis. TNBC is characterized by the absence of expression of estrogen receptor, progesterone receptor, and low levels of human epidermal growth factor (HER2). Paclitaxel (PTX) is among the most effective anti-cancer agents developed in the past decades, which is widely used in the treatment of patients with locally advanced and metastatic breast cancer. TNBCs are initially highly responsive to PTX however; the majorities of TNBC patients acquire resistance and develop progressive disease. Therefore, acquired resistance to paclitaxel has become one of the major obstacles in the successful treatment of patients with TNBC. Several mechanisms of resistance to paclitaxel has been identified, however there is little data about mechanisms of resistance to chemotherapy in TNBCs. Methods: In order to investigate the molecular mechanisms of acquired resistance to PTX in TNBCs, we developed four resistant TNBC cell lines (BT20, SUM149, MA-MB-231 and MDA-MB-436) by exposure of cells to increasing concentrations of PTX. We used an integrative analysis of array CGH and gene expression data to gain insights into the interplay of functional changes of the genome in TNBC resistant cell lines. Results: We found a novel amplification of the ABCB1 gene in BT20 and SUM149 resistant cell lines only. Gene expression analysis revealed significant up-regulation of expression of ABCB1 and EGFR ligands in SUM149 and BT20 resistant cells compared to parental cell lines. The functional activity of ABC transporters assessed using Rhodamine 123 efflux assay demonstrated a marked increase in the efflux of rhodamine in SUM149-R and BT20-R, which was reversed by verapamil. We treated resistant cells with two anti-EGFR drugs, lapatinib and neratinib, which are also known ABC transporter inhibitors, and found that both drugs inhibited rhodamine 123 efflux and restored sensitivity to PTX in these PTX-resistant TNBC cells. Conclusion: This is the first report of ABCB1 gene amplification in paclitaxel resistant triple negative breast cancer cells. Our results suggest that ABCB1 gene amplification and EGFR ligand over-expression plays a critical role in the development of PTX resistance in TNBC cells, and that this resistance can be targeted by therapy with anti-EGFR agents. Thus, ABCB1 gene amplification and EGFR ligand expression may be novel predictive biomarkers for both chemotherapy and anti-EGFR therapy in TNBCs. Citation Format: Elaheh Ahmadzadeh, Ewa Przybytkowski, Adriana Aguilar-Mahecha, Mark Basik. Targeting EGFR reverses paclitaxel resistance associated with ABCB1 overexpression in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A041.

Abstract LB-225: Resistance to paclitaxel in triple negative breast cancer cells is associated with ABCB1 overexpression and gene amplification, and can be reversed by anti-EGFR targeting.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Due to the absence of expression of estrogen, progesterone and HER2 receptors, triple negative breast cancer (TNBC) patients do not benefit from targeted therapies. Therefore, chemotherapy remains the only treatment of choice for patients with TNBC. Despite initial clinical responses to chemotherapy, the majority of TNBC patients acquire resistance and develop progressive disease. There is little data about mechanisms of resistance to chemotherapy in TNBCs. Methods: We investigated the molecular mechanisms of acquired resistance to paclitaxel (PTX) in TNBCs. Four TNBC cell lines (BT20, SUM149, MA-MB-231 and MDA-MB-436) were cultured in the presence of increasing concentrations of paclitaxel until they acquired resistance. Gene expression and aCGH analysis were performed on all parental and resistant pairs. Results: We found a novel amplification of the ABCB1 gene in BT20 and SUM149 resistant cell lines only. Gene expression analysis revealed significant up-regulation of expression of ABCB1 and EGFR ligands in SUM149 and BT20 resistant cells compared to parental cell lines. The functional activity of ABC transporters assessed using rhodamine 123 efflux assay demonstrated a marked increase in the efflux of rhodamine in SUM149-R and BT20-R which was reversed by verapamil. We treated resistant cells with two anti-EGFR drugs, lapatinib and neratinib, which are also known ABC transporter inhibitors, and found that both drugs inhibited rhodamine 123 efflux and restored sensitivity to PTX in these PTX-resistant TNBC cells. Conclusion: This is the first report of ABCB1 gene amplification in paclitaxel resistant triple negative breast cancer cells. Our results suggest that ABCB1 gene amplification and EGFR ligand over-expression plays a critical role in the development of PTX resistance in TNBC cells, and that this resistance can be targeted by therapy with anti-EGFR agents. Thus, ABCB1 gene amplification and EGFR ligand expression may be novel predictive biomarkers for both chemotherapy and anti-EGFR therapy in TNBCs. Citation Format: Elaheh Ahmadzadeh, Ewa Przybytkowski, Adriana Aguilar-Mahecha, Mark Basik. Resistance to paclitaxel in triple negative breast cancer cells is associated with ABCB1 overexpression and gene amplification, and can be reversed by anti-EGFR targeting. [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 LB-225. doi:10.1158/1538-7445.AM2013-LB-225