Unknown,Transcriptomics,Genomics,Proteomics

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Effects of differentiated hMADS adipocytes on BT474 and SKBR3 breast cancer cells

ABSTRACT: Analysis of gene expression levels of HER2-positive breast cancer cells exposed to the conditioned medium from adipocytes. The hypothesis tested in the present study was that adipocytes secrete factors that induce the resistance of cancer cells to antibody-dependent cellular cytotoxicity mediated by trastuzumab. The results provide insight into the genes that may be involved in the adipocyte-induced cancer resistance to trastuzumab treatment. BT474 cells or SKBR3 cells were exposed to the conditioned medium (CM) from differentiated hMADS (#hMADS) or to the control medium for 2 h. Total RNA was extracted and analyzed. The experiment was performed in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Minh Ngoc Duong

PROVIDER: E-GEOD-52660 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly. In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells. In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib. Keywords: HER2, breast cancer, resistance, trastuzumab, lapatinib, sorafenib Total RNA was isolated using TRIZOL reagent (Life Technologies, Gathersburg, MD) and quantified by Bioanalyzer 2100 (Agilent Technologies); following the isolation procedure, mRNA was amplified starting from 1μg using MessageAmp II aRNA Amplification kit (Ambion Inc. Austin TX, USA). Ammino-allyl modified nucleotides were incorporated in in vitro transcription step according to the manufacturer’s protocol. Labeling was performed using NHS (N-hydroxysuccinimidyl) ester Cy3 or Cy5 dies (GE Healthcare Europe GMBH, Upsala - Sweden) able to react with the modified RNA. At least 5 μg of mRNA, for each sample, were labeled and purified with columns respectively. The Dye-Swap replication procedure was applied, in order to increase the accuracy of results. Samples were hybridized on 44K glass arrays (Agilent Technologies). Arrays were scanned and the images obtained were analyzed by the Feature Extraction software Agilent (version 9.5) and the text files were then processed using the Bioconductor package Limma (Linear models for microarray analysis).

Project description:Post-transcriptional regulation of gene expression accomplished by microRNAs (miRNAs) importantly affects the complex gene regulatory network. In particular, miRNAs are known to be involved in recurrent motifs named miRNA-mediated feed forward loops (FFLs) where a transcription factor (TF) regulates a miRNA and they both regulate the expression level of a target RNA. Here, we focus on the identification of active FFLs during adipogenic differentiation. A list of putative feed-forward loops was generated based on sequence analysis of conserved and overrepresented motifs in the regulatory regions. Since this approach is not specific for adipogenesis and is known to generate false positive feed-forward loops, an experiment was designed to select active ones based on their dynamics using a model-based approach. Microarray time series of gene and miRNA expression data were collected at seven time points on human multipotent adipose-derived stem (hMADS) cells upon adipogenic differentiation. Three different dynamic models, sharing the same FFL topology but incorporating descriptions of increasing complexity of miRNA and mRNA dynamics, are identified on miRNA and mRNA expression data and compared based on identification criteria, namely: goodness of fit, precision of the estimates and comparison with submodels. 24 FFLs, able to properly reproduce data, are selected as active out of the 329 putative ones. This method considerably reduces the search space for new interactions between TFs, miRNAs and mRNAs and provides interesting biological results identifying genes known from the literature to be regulators in adipogenesis and adipocyte-related functions that can be interpreted as positive control of the validity of the apporoach. Therefore, the genes in the selected FFLs that are not yet known to be involved in this context are potential novel players in this regulatory network of adipogenesis and adipocyte function. Two independent cell culture experiments were performed as biological replicates during adipogenic differentiation of human mesenchymal stem cell. Cells where harvested at the pre-confluent stage as reference (day -3) and at seven subsequent time points during human adipogenic differentiation: day -2 and 0 before, and 1, 2, 5, 10, 15 days after induction of differentiation. All hybridizations were repeated with reversed dye assignment (dye-swap).

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Effects of differentiated hMADS adipocytes on BT474 and SKBR3 breast cancer cells

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