Profiling of genes regulated by TGF-beta in lung carcinoma (A549) and immortalized lung epithelial (HPL1D)cells.
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ABSTRACT: TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results: Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell lines. Semi quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. Conclusion: These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells. Keywords: cell type comparison in response to TGF-beta
Project description:TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. The effect of TGF-beta on the regulation of gene expression is cell-type specific. In order to identify TGF-beta regulated genes in different cell-types, we followed the expression profiling approach. Keywords: cell type specific response to TGF-beta
Project description:The survival of isolated metastatic cells and expansion into macroscopic tumour has been recognized as a limiting step for metastasis formation in several cancer types yet the determinants of this process remain largely uncharacterized. In colorectal cancer (CRC), we identify a transcriptional programme in tumour-associated stromal cells, which is intimately linked to a high risk of developing recurrent disease after therapy. A large proportion of CRCs display mutational inactivation of the TGF-beta pathway but paradoxically they are characterized by high TGF-beta production. In these tumours, TGF-beta instructs a transcriptional programme in stromal cells, which confers a high risk of developing metastatic disease. We quantified the association of TGF-beta-activated fibroblasts with disease progression. To this end, we used as surrogates the gene expression programme upregulated by addition of TGF-beta to normal colon mucosa-derived fibroblasts (CCD-Co-18) in culture. CCD-Co-18 were seeded at 60% confluence and treated with TGF-β1. Gene expression profiles were measured in duplicate using HG-U133 plus 2.0. We used RMA background correction, quantile normalization and RMA summarization (Gautier et al., 2004). A TGF-β response signature was obtained by selecting genes with limma P-value < 0.05 and at least two fold up-regulation in TGF-β treated fibroblasts.
Project description:The survival of isolated metastatic cells and expansion into macroscopic tumour has been recognized as a limiting step for metastasis formation in several cancer types yet the determinants of this process remain largely uncharacterized. In colorectal cancer (CRC), we identify a transcriptional programme in tumour-associated stromal cells, which is intimately linked to a high risk of developing recurrent disease after therapy. A large proportion of CRCs display mutational inactivation of the TGF-beta pathway but paradoxically they are characterized by high TGF-beta production. In these tumours, TGF-beta instructs a transcriptional programme in stromal cells, which confers a high risk of developing metastatic disease. We quantified the association of TGF-beta-activated fibroblasts with disease progression. To this end, we used as surrogates the gene expression programme upregulated by addition of TGF-beta to normal colon mucosa-derived fibroblasts (CCD-Co-18) in culture.
Project description:The proliferation and migration of cancer cells are regulated by stimuli from the tumour microenvironment during tumour progression and metastasis. While transforming growth factor-β (TGF-β) increases migration and metastasis of various types of epithelial cancer cells via induction of epithelial-mesenchymal transition (EMT), it also inhibits their proliferation by inducing cell cycle arrest in the G1 phase. However, the correlation between the tumour promoting and suppressing effects of TGF-β remains elusive because of the lack of analysis at the single-cell level. Here, we showed that TGF-β conferred higher motility and mesenchymal phenotypes to oral cancer cells residing in the G1 phase, suggesting a correlation between TGF-β-dependent cell cycle arrest and increased cell migration. We identified keratin-associated protein 2-3 (KRTAP2-3) as a regulator of these dual effects of TGF-β and showed that the expression of KRTAP2-3 was associated with TGF-β-induced cell cycle arrest and increased migration and correlated with poor prognosis in patients with head and neck cancer. Deletion of the KRTAP2-3 gene decreased in vitro migration and in vivo metastasis of oral cancer cells via induction of mesenchymal-epithelial transition. KRTAP2-3 also induced the expression of the zinc finger BED domain-containing protein 2 and ectodermal-neural cortex 1, which suppressed proliferation and increased migration of oral cancer cells, respectively. The present findings revealed the mechanisms by which TGF-β orchestrates proliferation and migration of cancer cells by inducing the expression of KRTAP2-3 and highlighted the importance of targeting motile cancer cells under cell cycle arrest in the G1 phase to suppress metastasis. Identification of marker genes for KRTAP2-3 induced gene
Project description:We investigated TGF-beta-induced gene expression in normal human lung fibroblasts (NHLFs) under different media setting, and diffrenece in TGF-beta-induced gene expression between control human male fibroblasts and human male IPF fibroblasts cultured in HPLM using RNA-seq.
Project description:Microarray analysis to examine glycan-related gene expression in idiopathic pulmonary fibrosis Heparan sulfate 6-O-endosulfatases (Sulf1 and Sulf2) remove 6-O sulfate groups from heparan sulfate intra-chain sites on the cell surface and in the extracellular matrix, and modulate the functions of many growth factors and morphogens including FGF, Wnt and TGF-beta. Works from our laboratory have shown that TGF-beta 1 induces Sulf1 and Sulf2 expression in a cell-type specific manner in the lung, specifically Sulf1 in lung fibroblasts and Sulf2 in type II alveolar epithelial cells. Interestingly TGF-beta 1-induced Sulf1 and Sulf2 in turn modulate TGF-beta 1 function in culture. The aim of this study is to examine the expression of Sulf1 and Sulf2 as well as other glycan-related genes (heparan biosynthetic enzymes, TGF-beta, FGF and Wnt signaling pathway components) in human idiopathic pulmonary fibrosis (IPF) lungs compared to normal lung samples. We will examine gene expression in triplicate samples from RNA of total lung homogenates from IPF and control (normal) lungs
Project description:Proliferation and migration of cancer cells are regulated by the stimuli from tumour microenvironment during its progression and metastasis. While transforming growth factor-β (TGF-β) increases migration and metastasis of various types of epithelial cancer cells via induction of epithelial-mesenchymal transition (EMT), it also decreases their proliferation by inducing G1 cell cycle arrest. However, the correlation of these tumour promoting and suppressing effects of TGF-β remains to be elusive because of the lack of analysis at a single cell level. Here, we showed that TGF-β confers the oral cancer cells residing in G1 phase with higher motility and mesenchymal phenotypes, suggesting a correlation between TGF-β-dependent cell cycle arrest and increased migration. We identified keratin-associated protein 2-3 (KRTAP2-3) as a regulator of these dual effects of TGF-β. Expression of KRTAP2-3 was associated with both TGF-β-induced cell cycle arrest and increased migration, and also was correlated with poor prognosis of head and neck cancer patients. We found that deletion of KRTAP2-3 gene decreased in vitro migration and in vivo metastasis of oral cancer cells via induction of mesenchymal-epithelial transition (MET). KRTAP2-3 also induces the expression of Regulator of G-protein signalling 4 (RGS4) and Zinc finger BED domain-containing protein 2 (ZBED2), which suppresses proliferation and increases migration of oral cancer cells, respectively. Present findings revealed novel mechanisms how TGF-β orchestrates proliferation and migration of cancer cells by inducing the expression of KRTAP2-3, and highlighted the importance of targeting the motile cancer cells under G1 cell cycle arrest to suppress metastasis.
Project description:Mouse tumour organoids (MTOs) derived from a compound mutant (LAKTP) intestinal cancer model were orthoptopically transplanted into syngeneic C57BL/6J mice. Tumour-bearing mice were treated with TGF-beta inhibitor Galunisertib or vehicle control. Whole tumour mRNA was extracted from primary tumours and expression profiling was performed with the objective to characterize the tumour microenvironment (TME). As a TGF-beta-activated TME has been associated to a poor prognosis and the CRC consensum molecular subtype CMS4, and the mouse model was found to have such an activated TME, we used the array data to classify these tumours in this mouse model system as CMS4-like. Furthermore, treatment with TGF-beta inhibitor reduced the fibroblast- and T cell-specific TGF-beta response signatures, also associated to poor prognosis in human CRC. This treatment was associated to a strong reduction/prevention of liver metastasis, as well as a reduction of primary tumour (and local carcinomatosis) size.
Project description:In this project we evaluated the proteomic profiling with TGF-β stimuli at 24h in a CRISPR-Cas9 model for ALMS1 gene in hTERT-BJ-5ta cells. Proteomic results showed a majority inhibition of downstream regulated pathways by the TGF-β, associating the protein coding genes (PCG) with processes like TGF- β matrix regulation, epithelial mesenchymal transition (EMT), PI3K/AKT or P53. In conclusion, seems that the depletion of ALMS1 could be inhibiting the signals transduction through the TGF -β and the routes regulated downstream.
Project description:Transcriptional profiling of human umbilical vein endothelial cells following stimulation with tumour necrosis factor alpha and transforming growth factor beta singly or combined for 8 hr All stimulations were for 8 hr - TNF-α vs no cytokine; TGF-β1 vs no cytokine; TNF-α & TGF-β1 vs TNF-α alone; TNF-α & TGF-β1 vs TGF-β1 alone