Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach:A multi-institutional research study

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EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>The epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.</p> <p>Methods</p> <p>Agilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of <it>snail </it>and <it>slug </it>was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.</p> <p>Results</p> <p>Morphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including <it>snail, slug, twist2 </it>and <it>zeb2</it>. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of <it>snail </it>and <it>slug</it>, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to <it>in vitro </it>drug effects.</p> <p>Conclusions</p> <p>This work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.</p

BRCA1 haploinsufficiency leads to altered expression of genes involved in cellular proliferation and development.

The assessment of BRCA1 and BRCA2 coding sequences to identify pathogenic mutations associated with inherited breast/ovarian cancer syndrome has provided a method to identify high-risk individuals, allowing them to seek preventative treatments and strategies. However, the current test is expensive, and cannot differentiate between pathogenic variants and those that may be benign. Focusing only on one of the two BRCA partners, we have developed a biological assay for haploinsufficiency of BRCA1. Using a series of EBV-transformed cell lines, we explored gene expression patterns in cells that were BRCA1 wildtype compared to those that carried (heterozygous) BRCA1 pathogenic mutations. We identified a subset of 43 genes whose combined expression pattern is a sensitive predictor of BRCA1 status. The gene set was disproportionately made up of genes involved in cellular differentiation, lending credence to the hypothesis that single copy loss of BRCA1 function may impact differentiation, rendering cells more susceptible to undergoing malignant processes

Comparison of qPCR and gene expression microarray results.

<p>Distribution of relative target expression levels between BRCA1 mutation carrier and non-carrier cell lines. Dashed lines represent mean relative expression values of each group (BRCA1 mutation carriers and non-carriers). Relative expression levels determined by microarray (black lines) and qRT-PCR (grey lines), for CXCR3, TBX21 and IFIT1.</p

List of BRCA1 mutations used in this study.

<p>Class abbreviations are N: Nonsense; F: Frameshift; M: Missense; S: Splicing; O: Other. Test indicates the samples present in the test set; all other samples were used in the training set.</p

Graphical representation of the interactions of a subset of the genes identified by both the NSC and SVM approaches.

<p>A total of 22 genes were input into the Ingenuity pathway analysis program, and 11 are represented in this 35 gene output pathway. A detailed key to the analysis output can be found at <a href="https://analysis.ingenuity.com/pa/info/help/help.htm" target="_blank">https://analysis.ingenuity.com/pa/info/help/help.htm</a>; which includes the following: Direct (solid lines) and indirect (dashed lines) Interaction; Inhibitory (bar at line end), Activating (arrow at line end) or Undefined (no line end) Binding; Regulation via Expression (E), Transcription (T) and Protein-protein interaction (P-P); Gene functions including Transcription regulators (wide ovals), cytokines (squares), complexes (double circles), enzymes (tall diamonds), and non-classified (circles).</p

Heat map showing classification in training and test sets using the SC approach.

<p>Samples from the training set presented on the left (53) and those from the test set are presented to the right (16). <i>BRCA1</i> status is indicated at the top of the heat map; <i>BRCA1^+/−</i> (0), <i>BRCA^−/−</i> (1). Genes used in the predictor are listed at the right. These genes were sorted according to relatedness using the GENESIS program, and a dendrogram of relatedness is presented at the left of the figure. Samples that were mis-classified are indicated by arrows at the top of the figure. Misclassification arrows missing (as noted), remove red to green bar at top, colour code the samples to identify carriers and controls.</p

Comparison of overlap between this study, and other published gene expression microarray radiation response papers.

<p>Comparison of overlap between this study, and other published gene expression microarray radiation response papers.</p