Unknown,Transcriptomics,Genomics,Proteomics

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In vitro microRNA screening in human pancreatic adenocarcinoma

ABSTRACT: No reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma. MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses chemoresistant PDAC for its specific miR expression pattern. Gemcitabine-resistant variants of PANC-1, a mutant p53 human pancreatic adenocarcinoma cell line, were established. MicroRNA screening was investigated by microarray. Gemcitabine-resistant PANC-1 (PANC-1-GR) cell clones were produced by exposing the parental cells to repeated pulsatile gemcitabine treatment over 3 days with constant sublethal concentrations followed by recovery-periods with agent-free medium until the cells recovered exponentially. Parental PANC-1 cells were treated with 0.4ÂµM gemcitabine cycles for approximately 9 months. Affymetrix GeneChip miRNA microarrays (Affymetrix UK Ltd., High Wycombe, UK) were performed in parental and chemoresistant PANC-1 cells after 17 chemotherapy cycles using the manufacturersÂ´ protocols. The samples were prepared from 1Âµg of total-RNA in accordance with the Affymetrix FlashTag Biotin HSR RNA Labeling Kit. The targets were hybridized overnight to Affymetrix GeneChip miRNA arrays. Following hybridization, the arrays were washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G. Microarray data quality was checked as recommended by the manufacturer and by the quality metrics in the Partek Genomics Suite software (Partek Inc., St. Louis, MO).

ORGANISM(S): Homo sapiens

SUBMITTER: Sameer Dhayat

PROVIDER: E-GEOD-74562 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Chemotherapy is pivotal in the multimodal treatment of pancreatic cancer patients. In recent years, technical advances have developed experimental methods that unveiled a high degree of inter- and intratumoral heterogeneity in pancreatic cancer. We hypothesized that intratumoral heterogeneity (ITH) impacts response to gemcitabine treatment and demands specific targeting of resistant subclones. Experimental Design: We addressed the effect of ITH on response to gemcitabine treatment using single cell-derived cell lines (SCDCL) from the classical-like cell line BxPC3 and the basal-like cell line Panc-1 which were analyzed by mRNA-seq and mass spectrometry. Results: Individual SCDCLs of the parental tumor cell populations of BxPC3 and Panc-1 showed considerable heterogeneity in response to gemcitabine. Unsupervised principal component analysis (PCA) including the 1,000 most variably expressed genes showed a clustering of the SCDCLs according to their respective sensitivity to gemcitabine treatment for BxPC3, while this clustering was less clear for Panc-1. In BxPC3 SCDCLs, enriched signaling pathways EMT, TNF signaling via NfKB, and IL2STAT5 signaling correlated with more resistant behavior to gemcitabine. In Panc-1 SCDCLs MYC targets V1 and V2 as well as E2F targets were associated with stronger resistance to gemcitabine. Feature extraction of proteomes again identified less proteins whose expression was associated with the response of individual SCDCLs in Panc-1 compared to BxPC3. Based on molecular profiles, we could show that the gemcitabine-resistant SCDCLs of both BxPC3 and Panc-1 are more sensitive to the BET inhibitor JQ1 compared to the respective gemcitabine-sensitive SCDCLs. Conclusions: Our model system of SCDCLs identified gemcitabine-resistant subclones within a parental tumor population and provides evidence for the critical role of ITH for treatment response in pancreatic cancer. Through molecular profiling, we identified specific signaling pathways and protein signatures that might help to explain the differential response to treatment among clones. We exploited these molecular differences for an improved and more targeted therapy of resistant subclones of a heterogeneous tumor.

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In vitro microRNA screening in human pancreatic adenocarcinoma

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