Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of human prostate carcinoma cells during a stepwise epithelial to mesenchymal transition

ABSTRACT: Background: The loss of epithelial characteristics and the acquisition of a migratory phenotype, referred to as epithelial to mesenchymal transition (EMT), is a crucial event in tumor metastasis. A better defined cell culture model for the study of EMT and its role in prostate carcinogenesis has been a challenge. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. Here, we present an extension of this model to stepwise malignant transformation in relation to EMT and gene expression reprogramming. Results: To achieve transformed prostate cells, EPT1 cells were kept growing in extended saturation density culture to select for cells overriding quiescence. Foci formed in EPT1 cells in 4 weeks. Cells from the foci can form robust colonies in soft agar suggesting malignant transformation. The transformed cells were named EPT2. In this stepwise transformation model, EPT2 cells showed much higher abilities to proliferation at confluence, higher resistance to apoptosis, and much lower dependence on serum and exogenous growth factors than EP156T and EPT1 cells. When EP156T and EPT1 cells at different passages were compared, only EPT1 cells at later passage but not EP156T cells could be induced to malignant transformation by high density culture. Microarray expression profiling showed that EMT and transformation were strongly connected at the gene expression level, suggesting that EMT makes EPT1 cells at later passage more susceptible to transformation induction. Conclusions: Our findings provide a novel stepwise transformation model in which EMT can emerge not only independent of transformation, but also can promote subsequent malignant transformation in prostate carcinogenesis. Dynamic changes of a core set of genes are involved in both EMT and subsequent malignant transformation.

ORGANISM(S): Homo sapiens

SUBMITTER: Kjell Petersen

PROVIDER: E-TABM-949 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have developed cdk4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) to study lung cancer pathogenesis. By studying the oncogenic effect of common lung cancer alterations (p53, KRAS, and c-MYC) we demonstrate the ability of this model to characterize the stepwise transformation of bronchial epithelial cells to full malignancy. Using HBECs derived from multiple individuals we found: 1) the combination of five genetic alterations (p53, KRASV12, c-MYC, CDK4 and hTERT) is sufficient for full tumorigenic conversion of HBECs; 2) high levels of KRASV12 are required for full malignant transformation of HBECs, however these levels also stimulate oncogene-induced senescence; 3) RAS-induced senescence is largely bypassed with loss of p53 function; 4) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 5) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 6) serum-induced epithelial-to-mesenchymal transition (EMT) increases in vivo tumorigenicity; 7) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation as well as sensitivity to standard platinum-based chemotherapies; and 8) an mRNA signature derived from tumorigenic and non-tumorigenic clones is predictive of outcome in lung cancer patients. Collectively, we demonstrate this HBEC model system can be used to study the effect of oncogenic mutations on malignant progression, oncogene-induced senescence, and EMT along with clinically translatable applications such as development of prognostic signatures and drug response phenotypes. Human bronchial epithelial cells (HBECs) immortalized with cdk4 and hTERT were transformed with p53 knockdown, KrasV12 and cMYC over-expression and profiled on Illumina HumanHT-12 V4.0 expression beadchips. Transformed HBECs were grown in two different growth media: KSFM (defined, serum-free medium) or R10 (RPMI with 10% FBS) as indicated. Clones were isolated from HBECs with sh-p53 + KrasV12 and sh-p53 + KrasV12 + cMYC.

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Transcription profiling by array of human prostate carcinoma cells during a stepwise epithelial to mesenchymal transition

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