Project description:In pancreatic cancer the survival rate is low, as the available treatment options usually only extend survival and seldom produce a cure. Drug resistance and disease reoccurrence is the typical reason for death after cancer diagnosis. 5-Fluorouracil (5-FU) is the main chemostatic used in first line therapy. However the majority of the tumors become resistant to treatment. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc03.27 cell line by long-term exposure to 5-FU. In addition to increased expression of markers associated with multidrug resistance, the 5-FU resistant clones showed alterations typical of the process of epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, which was confirmed on RNA and protein levels. Expression of the adhesion molecule L1CAM is associated with a chemoresistant and migratory phenotype of pancreatic cancer. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with monoclonal antibodies, we discovered that the increased invasiveness observed in the chemoresistant cells depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we discovered that L1CAM expression depends on Slug rather than β-catenin in the 5-FU resistant cells. We demonstrate a functional link between Slug and the expression level of L1CAM in pancreatic cancer cells having undergone EMT following long-term exposure to 5-FU. Our findings provide further insight into the molecular mechanisms leading to a chemoresistant and migratory phenotype in pancreatic cancer cells and indicate the importance of Slug-induced L1CAM in refractory pancreatic cancer. Examination of expression of 5-Fluorouracil (5-FU) Panc03.27 cell line resistant clone versus expression of 5-FU sensitive clones (NT) in 4 replicates per cell lines

Project description:Purpose: The acquisition of cellular invasiveness of breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Very little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like 21NT cells results in increased survival, growth, and invasiveness. In this study we explore the role of TBX3 in breast cancer progression pathways, focusing specifically on its involvement in the induction of EMT and the transition from DCIS to invasive mammary carcinoma. Methods: Total mRNA was isolated from 21NT transfected cell lines. Sequencing was conducted using an Illumina HiSeq 2500 sequencer, comparing the resultant transcriptional profiles with overexpression of either TBX3 isoform (TBX3iso1 or TBX3iso2) as fold changes relative to the empty vector control. RNA-Seq data was integrated with ChIP-array data downstream to assess direct targets of TBX3 isoforms and relate it to the process of EMT. TBX3, SLUG and TWIST1 expression were assessed by immunohistochemistry in two independent early-stage (Stage 0 and Stage 1) breast cancer cohorts. Results: An EMT-related transcriptional profile was observed with overexpression of TBX3iso1 and TBX3iso2. Using genome-wide bioinformatic approaches in conjunction with more conventional in vitro studies, we have identified SLUG and TWIST1 as downstream targets of TBX3 and have assessed their expression by immunohistochemistry in two different patient cohorts. Our findings suggest that TBX3 facilitates the process of early invasion in DCIS lesions by promoting the induction of EMT and tumor progression through the low-grade pathway. Finally, we propose a progression model in which SLUG is an important and necessary effector downstream of TBX3, leading to increased motility, invasiveness, and induction of key invasiveness-associated genes. Conclusions: This is the only existing study which has conducted assessment of transcriptional changes associated with TBX3 isoforms (TBX3iso1 and TBX3iso2). Our published study has identified the transcriptional regulator TBX3 as an enabler of EMT in non-high grade, pre-invasive lesions of the breast, inducing SLUG and promoting the transition from in situ (DCIS) to invasive breast cancer.

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Human cancer cells express Slug-based epithelial-mesenchymal transition gene expression signature obtained in vivo

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