Marketa Zvelebil

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90β at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action. [Cancer Res 2007;67(7):3239–53]

Validation of proteomic data showing expression of ER and known associated proteins. Protein abundance was measured using dimethyl labelling from wt-MCF7 (M) and MCF7 LTED (L). Changes are represented as fold change (wt-MCF7/MCF7 LTED). Negative values indicate inverted ratios for those

Aims: HER2 gene amplification is observed in up to 15% of breast carcinomas. In a rare subset of breast cancers classified as HER2-positive by immunohistochemistry and in situ hybridisation, HER2 overexpression and gene amplification are restricted to a subset of >30% but not all cancer cells. Here we sought to characterise the repertoire of gene copy number aberrations and somatic mutations in the HER2-positive and HER2-negative components of cases with heterogeneous HER2 overexpression and gene amplification. Material and methods: Cases diagnosed as HER2 positive but with >30% but <100% of cells displaying HER2 overexpression were retrieved from the authors' institutions. HER2 heterogeneity status was re-assessed using immunohistochemistry and chromogenic and/ or fluorescence in situ hybridisation. For cases with confirmed HER2 gene amplification heterogeneity, HER2-positive and HER2-negative components were microdissected from tissue sections stained with the Hercept...

Background When breast carcinomas remain confined to breast tissue, cure rates exceed 90% (http://seer.cancer.gov/csr/1975_2006/). However, if the cancer disseminates through the body, long-term survival decreases depending upon the extent of, and the sites of, colonisation Genes that control the different stages of the metastatic process need to be identified to better delineate the process, and to aid in the development of metastatic biomarkers and provide potential targets for the treatment of metastatic disease. Genetic screens, such as those that exploit RNA interference (RNAi), provide an unbiased approach to the identification of genes associated with a phenotype of interest. Although cell-based screens have been highly informative in identifying genes involved in tumour cell survival, migration and invasion, these in vitro approaches are largely unsuitable for interrogating the later stages of the metastatic process, in particular the processes of cell dissemination, tumour cell extravasation from the circulation and colonisation of secondary sites. Method Using the 4T1 mouse model an in vivo functional metastasis screen that integrates RNAi technology and massively parallel sequencing was implemented to rapidly discover and validate metastasis genes. Results Using this approach, 12 ‘hits’ that suppress tumour cell colonisation of the lungs were identified. 3 of the top 5 hits have been validated as novel metastasis suppressor genes in both mouse and human cell lines and further studies have been undertaken to elucidate the mechanism of action of the top hit. Critically, these findings are clinically relevant in primary breast cancers where there is a significant correlation between elevated expression levels of these suppressor genes and reduced frequency of metastatic events. Conclusion This study demonstrates the value of adopting an unbiased methodology to discover novel metastatic genes and establishes, for the first time, that an in vivo metastasis screen can be combined with next generation sequencing to identify novel components of the metastatic process. 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 3417. doi:1538-7445.AM2012-3417

Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested ...

Over 80% of breast cancers (BC) at primary diagnosis express the estrogen receptor (ER+). Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ BC but resistance remains a major clinical problem. Data from large studies such as TCGA indicate that other than a small number of high frequency mutations such as TP53, PIK3CA and GATA3 that have little association with endocrine resistance, primary ER+ BC shows very low frequency of individual mutations making targeting difficult. In contrast, expression profiling of primary ER+ BC samples have identified several promising signatures/networks for targeting. In order to identify common adaptive mechanisms associated with resistance to aromatase inhibitors, we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44, MDA-MB-361 and ZR75.1) or 3D on collagen to model the stromal compartment (MCF7). At the point of resistance MCF7-LTED, HCC1428-LTED and SUM44-LTED retained ER expression whilst T47D-LTED, ZR75.1-LTED and MDA-MB-361-LTED reduced or lost ER. In order to identify common adaptive mechanisms, ingenuity pathway was used. Genes submitted for analysis were selected on the basis of a p-value of 0.001, FDR 5% and fold change 1.5 relative to the parental cell lines. Strikingly, the cholesterol biosynthesis pathway was the common up-regulated pathway in the ER+ LTED cell lines but not in the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Further interrogation showed increased expression of MSMO1 (x2.27), EBP (x1.57), SQLE (x2.37) and IDI1 (x2.74) in the MCF7-LTED. As mRNA does not always correlate with protein, we applied quantitative proteomics in wt-MCF7 and MCF7-LTED cells using dimethyl labelling followed by LC-MS/MS. Several enzymes within the cholesterol biosynthesis pathway were higher in abundance in the MCF7-LTED such as EBP (x1.54), SQLE (x3.24) and LBR (x1.86). Evidence suggests that patients with cancer accumulate cholesteryl esters as a result of loss of PTEN or PI3K/AKT/mTOR activation, stimulating SREBP, ACAT1, HMGSCR and LDLR. As 40% of ER+ BC harbor PTEN loss or PIK3CA mutations, we assessed levels of SREBP, HMGCR, LDLR or ACAT1 in our data sets. Neither SREBP nor HMGCR mRNA or protein levels changed in the ER+ LTED or ER- LTED. Furthermore, ACAT1 was downregulated in the ER+ LTED. Moreover, levels of free and esterified cholesterol in wt-MCF7 and MCF7-LTED cell lysates were unchanged. Previous studies have suggested oxysterols can act as selective ER-modulators (SERM). We subsequently investigated the function of these cholesterol metabolites in our LTED models. Treatment with 25-hydroxycholesterol (HC) or 27-HC increased ER mediated-transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a c. 30-60% drop in proliferation and concomitant drop in TFF1 expression in the ER+ LTED cells together with a reduction in recruitment of the ER and CREB binding protein (CBP) to the TFF1 promoter. Gene expression data from 704 primary ER+ BC from patients treated with adjuvant tamoxifen showed that MSMO1 (p=0.047), EBP (p=0.043), SQLE (p=0.0001), DHCR7 (p=0.002) and IDI1 (p=0.0005), enzymes required for cholesterol synthesis and up-regulated in our in-vitro models, were associated with poor relapse free survival. Of note, DHCR7 forms part of the EndoPredict gene signature. Taken together, these data provide support for the role of the cholesterol biosynthesis pathway and its metabolites as a novel mechanism of resistance to endocrine therapy in ER+ BC and its potential as a therapeutic target. Citation Format: Nikiana Simigdala, Qiong Gao, Sunil Pancholi, Marketa Zvelebil, Ricardo Ribas, Mitch Dowsett, Lesley-Ann Martin. Cholesterol biosynthesis pathway as a novel mechanism of resistance identified in ER+ long-term estrogen deprived cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A37.

Introduction: Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents.

Measures of similarities and distances among nucleotide or amino acid sequences have been used to find related regions in long sequences, to test for homology, to assess phylogenetic and functional relationships, and to estimate divergence time between pairs of evolutionarily related sequences. This last use especially invokes a model, possibly implicit and including a random component, of sequence change through nucleotide (or amino acid) replacement and insertion or deletion of single nucleotides or small blocks of contiguous nucleotides. Modeling genetic events by these mechanisms, however, cannot reflect the more macroscopic processes of evolutionary divergence of organisms, such as duplication, inversion, and transposition (shuffling) of parts of the genome. Although little comprehensive data are available as yet to study evolution at the level of entire genomes, the megasequencing projects now being set up will be producing genomic sequences in the near future. In this chapter...

ABSTRACT An advanced computational framework was developed for 3D modelling of semiconductor detectors and medical imaging systems. In our work, pixellated CdZnTe detectors with pixel size between 0.04 and 1.6 mm and thickness in the range of 0.5 to 10 mm were modeled and investigated in terms of charge transport effects, detection efficiency and energy resolution across the energy range used in medical imaging applications.

Human serine racemase (hSR) is a cytosolic pyridoxal-5′-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer’s disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR’s promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structur...

<b>Copyright information:</b>Taken from "pSTIING: a 'systems' approach towards integrating signalling pathways, interaction and transcriptional regulatory networks in inflammation and cancer"Nucleic Acids Research 2005;34(Database issue):D527-D534.Published online 28 Dec 2005PMCID:PMC1347407.© The Author 2006. Published by Oxford University Press. All rights reserved The sequence similarity between orthologous proteins (percent identity, BLAST score and -value) for each query protein is displayed alongside their interaction information and when consolidated across species, these interactions are mapped onto human proteins. As a comparison, the corresponding interaction map without orthology mapping is shown. () pSTIING supports querying by human disease, facilitating the exploration of interaction and transcriptional regulatory associations between proteins implicated in disease (e.g. breast cancer). () Linking of expression analysis to molecular interaction networks (represented by solid edges) and transcriptional regulatory modules (dashed lines) using pSTIING-CLADIST. The CLADIST tool associated with pSTIING provides various clustering algorithms including SOM, which was used to identify a cluster of co-ordinately expressed genes (represented by nodes with red labels) from MPSS expression analyses of tumour and non-tumour samples. These were then cross-correlated directly with interaction and transcriptional information in pSTIING to detect any underlying physical or functional associations.