The Mutational Spectrum of Pre- and Post-Neoadjuvant Chemotherapy Triple-Negative Breast Cancers.

The response of triple-negative breast cancer (TNBC) patients to pre-operative (neoadjuvant chemotherapy) is a critical factor of their outcome. To determine the effects of chemotherapy on the tumor genome and to identify mutations associated with chemoresistance and sensitivity, we performed whole exome sequencing on pre/post-chemotherapy tumors and matched lymphocytes from 26 patients. We observed great inter-tumoral heterogeneity with no gene mutated recurrently in more than four tumors besides TP53. Although the degree of response to chemotherapy in residual tumors was associated with more subclonal changes during chemotherapy, there was minimal evolution between pre/post-tumors. Indeed, gene sets enriched for mutations in pre- and post-chemotherapy tumors were very similar and reflected genes involved in the biological process of neurogenesis. Somatically mutated genes present in chemosensitive tumors included COL1A2, PRMD15, APOBEC3B, PALB2 and histone protein encoding genes, while BRCA1, ATR, ARID1A, XRCC3 and genes encoding for tubulin-associated proteins were present in the chemoresistant tumors. We also found that the mutational spectrum of post-chemotherapy tumors was more reflective of matching metastatic tumor biopsies than pre-chemotherapy samples. These findings support a portrait of modest ongoing genomic instability with respect to single-nucleotide variants induced by or selected for by chemotherapy in TNBCs.

An immune-centric exploration of BRCA1 and BRCA2 germline mutation related breast and ovarian cancers.

BACKGROUND: BRCA1/2 germline mutation related cancers are candidates for new immune therapeutic interventions. This study was a hypothesis generating exploration of genomic data collected at diagnosis for 19 patients. The prominent tumor mutation burden (TMB) in hereditary breast and ovarian cancers in this cohort was not correlated with high global immune activity in their microenvironments. More information is needed about the relationship between genomic instability, phenotypes and immune microenvironments of these hereditary tumors in order to find appropriate markers of immune activity and the most effective anticancer immune strategies. METHODS: Mining and statistical analyses of the original DNA and RNA sequencing data and The Cancer Genome Atlas data were performed. To interpret the data, we have used published literature and web available resources such as Gene Ontology, The Cancer immunome Atlas and the Cancer Research Institute iAtlas. RESULTS: We found that BRCA1/2 germline related breast and ovarian cancers do not represent a unique phenotypic identity, but they express a range of phenotypes similar to sporadic cancers. All breast and ovarian BRCA1/2 related tumors are characterized by high homologous recombination deficiency (HRD) and low aneuploidy. Interestingly, all sporadic high grade serous ovarian cancers (HGSOC) and most of the subtypes of triple negative breast cancers (TNBC) also express a high degree of HRD. CONCLUSIONS: TMB is not associated with the magnitude of the immune response in hereditary BRCA1/2 related breast and ovarian cancers or in sporadic TNBC and sporadic HGSOC. Hereditary tumors express phenotypes as heterogenous as sporadic tumors with various degree of "BRCAness" and various characteristics of the immune microenvironments. The subtyping criteria developed for sporadic tumors can be applied for the classification of hereditary tumors and possibly also characterization of their immune microenvironment. A high HRD score may be a good candidate biomarker for response to platinum, and potentially PARP-inhibition. TRIAL REGISTRATION: Phase I Study of the Oral PI3kinase Inhibitor BKM120 or BYL719 and the Oral PARP Inhibitor Olaparib in Patients With Recurrent TNBC or HGSOC (NCT01623349), first posted on June 20, 2012. The design and the outcome of the clinical trial is not in the scope of this study.

A Unique Morphological Phenotype in Chemoresistant Triple-Negative Breast Cancer Reveals Metabolic Reprogramming and PLIN4 Expression as a Molecular Vulnerability.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

Chromosome-breakage genomic instability and chromothripsis in breast cancer.

BACKGROUND: Chromosomal 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. RESULTS: We 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. CONCLUSIONS: Our 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.

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. Today's 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.

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

BACKGROUND: Molecular 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. METHODS: We 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. RESULTS: DNA 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. CONCLUSIONS: This 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.

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.

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 CoCl(2), induced the up-regulation of hypoxia-inducible transcription factor-1alpha and the down-regulation of the beta-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.

Upregulation of cellular triacylglycerol - free fatty acid cycling by oleate is associated with long-term serum-free survival of human breast cancer cells.

We previously showed that exogenous oleate protects human breast cancer cells against palmitate-induced apoptosis in part by increasing esterification of this free fatty acid (FFA) into triacylglycerol (TG). Here, we studied the mechanism whereby oleate protects these cells against apoptosis induced by serum withdrawal. The metabolism of FFA, TG, and glucose, in parallel with long-term cell survival in the absence of serum, was investigated in a panel of human breast cancer cell lines and in nontransformed MCF-10A cells after treatment with exogenous oleate. Short-term (3-24 h) exposure of MDA-MB-231 human breast cancer cells to exogenous oleate resulted in a dose-dependent long-term (10 day) serum-free survival that correlated with the accumulation of TG in lipid droplets and with upregulation of lipolysis. Both effects persisted for several days after oleate removal. Rapid TG lipolysis and FFA re-esterification, supported by high rates of glycolysis that provide the glycerol backbone for TG synthesis, are consistent with the presence of very active TG-FFA cycling in human breast cancer cells. Only the cancer cell lines capable of accumulating TG showed long-term serum-free survival after oleate treatment. The results suggest that upregulation of TG-FFA cycling induced by oleate may be involved in maintenance of human breast cancer cell survival.

Widespread bimodal intrachromosomal genomic instability in sporadic breast cancers associated with 13q allelic imbalance.

Genomic instability is thought to underlie tumor progression in solid tumors, such as breast cancer. Although evidence that the hereditary breast cancer genes, BRCA1 and BRCA2, are involved in DNA repair suggests that genomic instability plays an important role in hereditary breast tumorigenesis, genomic instability remains poorly characterized in sporadic breast cancers. Using a DNA fingerprinting technique, inter-(simple sequence repeat) PCR (inter-SSR PCR), the degree of genomic instability was quantified in 47 sporadic breast cancers compared with matched adjacent normal breast tissues. Almost all sporadic breast cancers show significant genomic instability by inter-SSR PCR. The distribution of this instability is bimodal; 57% of the tumors show fewer changes, whereas 43% show striking genomic alterations. Further analysis of two inter-SSR PCR tumor-normal differences revealed a genomic amplification and probable deletion. Thus, inter-SSR PCR can detect chromosomal breakage-related genomic alterations in most sporadic breast cancers. Genomic instability as detected by inter-SSR PCR is not correlated with aneuploidy, suggesting that this technique preferentially detects intrachromosomal alterations. Chromosomal instability in breast cancer can therefore be subdivided into at least two groups: (a) intrachromosomal and (b) gross chromosomal. Allelic imbalance at markers at the 13q13 and retinoblastoma loci (13q) and not at 17q loci was significantly associated with high levels of intrachromosomal instability, suggesting genes at 13q13 and retinoblastoma loci are either selectively targeted or involved in the genesis of genomic instability in sporadic breast cancers.

Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. A role for cardiolipin.

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.

Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise.

UNLABELLED: Changes in the concentration of malonyl-CoA in many tissues have been related to alterations in the activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in its formation. In contrast, little is known about the physiological role of malonyl-CoA decarboxylase (MCD), an enzyme responsible for malonyl-CoA catabolism. In this study, we examined the effects of voluntary exercise on MCD activity in rat liver, skeletal muscle, and adipose tissue. In addition, the activity of sn-glycerol-3-phosphate acyltransferase (GPAT), which like MCD and ACC can be regulated by AMP-activated protein kinase (AMPK), was assayed. Thirty min after the completion of a treadmill run, MCD activity was increased approximately 2-fold, malonyl-CoA levels were reduced, and ACC and GPAT activities were diminished by 50% in muscle and liver. These events appeared to be mediated via activation of AMPK since: 1) AMPK activity was concurrently increased by exercise in both tissues; 2) similar findings were observed after the injection of 5-amino 4 imidazole carboxamide, an AMPK activator; 3) changes in the activity of GPAT and ACC paralleled that of MCD; and 4) the increase in MCD activity in muscle was reversed in vitro by incubating immunoprecipitated enzyme from the exercised muscle with protein phosphatase 2A, and it was reproduced by incubating immunopurified MCD from resting muscle with purified AMPK. An unexpected finding was that exercise caused similar changes in the activities of ACC, MCD, GPAT, and AMPK and the concentration of malonyl-CoA in adipose tissue. IN CONCLUSION: MCD, GPAT, and ACC are coordinately regulated by AMPK in liver and adipose tissue in response to exercise, and except for GPAT, also in muscle. The results suggest that AMPK activation plays a major role in regulating lipid metabolism in many cells following exercise. They also suggest that in each of them, it acts to increase fatty acid oxidation and decrease its esterification.