RNA sequencing of Normal Human Fibroblasts and Patient Fibroblasts with Novel SCP2 Variant
ABSTRACT: The purpose of this study was to identify differentially expressed genes beween normal and SCP2 mutant fibroblasts Overall design: Gene expression profile of normal human fibroblasts and patient fibroblasts
Project description:UNLABELLED:Cancer-associated fibroblasts (CAF) are a major constituent of the tumor stroma, but little is known about how cancer cells transform normal fibroblasts into CAFs. microRNAs (miRNA) are small noncoding RNA molecules that negatively regulate gene expression at a posttranscriptional level. Although it is clearly established that miRNAs are deregulated in human cancers, it is not known whether miRNA expression in resident fibroblasts is affected by their interaction with cancer cells. We found that in ovarian CAFs, miR-31 and miR-214 were downregulated, whereas miR-155 was upregulated when compared with normal or tumor-adjacent fibroblasts. Mimicking this deregulation by transfecting miRNAs and miRNA inhibitors induced a functional conversion of normal fibroblasts into CAFs, and the reverse experiment resulted in the reversion of CAFs into normal fibroblasts. The miRNA-reprogrammed normal fibroblasts and patient-derived CAFs shared a large number of upregulated genes highly enriched in chemokines, which are known to be important for CAF function. The most highly upregulated chemokine, CCL5, (C-C motif ligand 5) was found to be a direct target of miR-214. These results indicate that ovarian cancer cells reprogram fibroblasts to become CAFs through the action of miRNAs. Targeting these miRNAs in stromal cells could have therapeutic benefit. SIGNIFICANCE:The mechanism by which quiescent fibroblasts are converted into CAFs is unclear. The present study identifies a set of 3 miRNAs that reprogram normal fibroblasts to CAFs. These miRNAs may represent novel therapeutic targets in the tumor microenvironment.
Project description:To clarify the molecular alterations triggered by miR-9 to induce the conversion of breast normal fibroblasts to a CAF-phenotype, gene expression profile of normal fibroblasts transiently transfected with miR-9 or control was performed. We performed a gene expression profiling of immortalized normal fibroblasts taken from a noncancerous region of the breast at least 2 cm from the outer tumor margin. Fibroblasts were transiently transfected with miR-9 or control for 24 hours. 3 independent biological replicates for each treatment.
Project description:We model processes of wound healing and tumor growth, by studying the effects of normal and cancer epithelial cells on normal fibroblasts and their reciprocal effect on normal keratinocytes in vitro. We find strong parallels between the two processes and compare our observations with transcriptional analysis of 24 clinical samples of squamous cell carcinoma. This dataset contains the tissue culture samples, for the clinical samples, see E-MTAB-1065. Normal human fibroblasts (HF) were cocultured with either normal HK or transformed epithelial cells (HaCaT and FaDu) and their expression profile was compared to the expression profile of HF cultured alone.
Project description:Cancer-associated fibroblasts (CAF) are a major constituent of the pancreatic cancer microenvironment and that the meaning is as intended. Pancreatic cancer cells can induce normal fibroblasts to convert into CAF and, reciprocally, CAF promote tumor invasions and proliferations. The mechanism of the conversion from normal fibroblasts (NF) to CAF remains unclear. MicroRNA are short non-coding RNA involved in the post-transcription gene regulation, which have been defined as an imperative controller in tumor invasions, proliferations and colony formations. Microvesicles (MV) have been proved to be an important mediator of intercellular communication and can selectively transport secreted microRNA from a donor cell into a recipient cell. In this study, we isolated primary pancreatic fibroblasts from wild type C57 mice and co-cultured them with pancreatic cancer cell lines, BxPC-3 and SW1990, and observed the conversion from NF to CAF, or at least CAF-like cells. This phenomenon could also be replicated in primary fibroblasts treated with MV separated from a cancer cell media. We identified that miR-155 was upregulated in PaC-derived MV and we confirmed that normal fibroblasts could convert into CAF after MV containing miR-155 had been taken up. TP53INP1 is a target of miR-155 in fibroblasts and a downregulation of TP53INP1 protein levels could contribute to the fibroblasts' activation. These results indicated that pancreatic cancer cells might reprogram normal adjacent fibroblasts into CAF by means of secreted MV containing miR-155. Targeting the circulating microRNA might be a potential therapy for malignant tumors.
Project description:Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes. Epithelial-mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium including the tumor progression and spreading. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity towards normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor associated fibroblast activity that influence normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4 respectively to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in medium conditioned by cancer associated fibroblast. Presence of these growth factors was also detected in tumor samples.
Project description:Normal fibroblasts surrounding tumor cells play a crucial role in cancer progression through formation of the tumor microenvironment. Because factors secreted from normal fibroblasts can modulate the tumor microenvironment, it is necessary to identify key factors associated with regulation of secreted factors and to investigate the molecular mechanisms contributing to the tumor microenvironment formation process. In this study, we found that radiation induced the expression and K63-linkage poly-ubiquitination of TRAF4 in normal lung fibroblasts. The K63-linkage poly-ubiquitinated TRAF4 formed complexes with NOX2 or NOX4 by mediating phosphorylated p47-phox in normal lung fibroblasts. Moreover, we showed that TRAF4 stabilized NOX complexes by decreasing lysosomal degradation of NOX2 and NOX4 after irradiation. NOX complexes increased endosomal ROS levels that were permeable into cytoplasm, leading to NF-?B-mediated ICAM1 up-regulation. Soluble ICAM1 was subsequently secreted into conditioned media of radiation-activated normal lung fibroblasts. The conditioned media from irradiated normal fibroblasts enhanced proliferation and epithelial-mesenchymal transition of non-small cell lung cancer cells both in vitro and in vivo. These results demonstrate that TRAF4 in irradiated fibroblasts is positively associated with aggressiveness of adjacent cancer cells by altering the tumor microenvironment. Thus, we suggest that regulation of TRAF4 might be a promising strategy for cancer therapy.
Project description:alpha-L-Iduronidase synthesis and maturation were analysed in fibroblasts from normal controls and from alpha-L-iduronidase-deficient mucopolysaccharidosis-type-I (MPS-I) patients. Fibroblasts were radiolabelled with [3H]leucine and alpha-L-iduronidase was isolated from cell lysates or culture medium by monoclonal-antibody affinity chromatography. Pulse-chase labelling of normal control fibroblasts showed that alpha-L-iduronidase was synthesized as an 81 kDa precursor and processed within 24 h via intermediates of 76 kDa and 70 kDa to a 69 kDa species. The incorporation of radiolabel into alpha-L-iduronidase in fibroblasts from three of four MPS-I patients was at levels that were either very low or undetectable. Fibroblasts from one MPS-I patient, however, exhibited levels of incorporation of radiolabelled amino acid into alpha-L-iduronidase similar to those shown by normal control fibroblasts, despite having undetectable alpha-L-iduronidase enzyme activity. The maturation of alpha-L-iduronidase in fibroblasts from this patient was delayed compared with normal controls and showed accumulation of the 76 kDa intermediate, as well as the major 69 kDa, form of the enzyme.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor type with extremely high mortality (up to 92%) during 5 years after diagnosis. Here, cancer associated fibroblasts (CAF) from PDAC were compared to CAF from melanoma metastases (MELF) and to normal dermal fibroblasts (DF). The analysis was performed in three biological replicates for normal fibroblasts, eight biological replicates for PDAC CAF, and four biological replicates for melanoma CAF. Further technical replicates were used to improve data quality.