Transcriptome profiling of radiation-transformed breast cells after TRPS1 siRNA knockdown.
Ontology highlight
ABSTRACT: TRPS1 was recently identified as a radiation marker in breast cancer. In order to characterise the molecular phenotype that is associated with TRPS1 we knocked out the gene in two radiation-transformed breast cell lines.
Project description:Thyroid carcinoma (TC) is generally associated with good prognosis, nevertheless no effective treatments are available for aggressive forms not cured by current therapies. We previously identified the coatomer protein complex zeta 1 (COPZ1), as a new putative therapeutic target for TC, since its depletion impairs the viability of tumor cells, leads to abortive autophagy, ER stress, unfolded protein response and apoptosis, and reduces the tumor growth of TC xenograft models. In this study, by combining genomic, proteomic and functional approaches, we provided evidence that COPZ1 silencing stimulates a type I IFN-mediated viral mimicry response, boosts the production of several inflammatory molecules and finally induces immunogenic cell death, which, in turn, promotes dendritic cell maturation and subsequent activation of T cells. Collectively, our findings support the notion that COPZ1 targeting can be exploited as a new strategy to kill cancer cells with the subsequent involvement of an anti-tumor immune response.
Project description:Ehf is a transcriptional regulator that is highly expressed and enriched in corneal epithelium. To gain insights into the role of Ehf in the corneal epithelium, we performed siRNA knockdown of Ehf in primary human corneal epithelial cells. Primary human corneal epithelial cells were transfected with siEhf or si controls, plated, and harvested at 72 hr.
Project description:Exposure to genotoxic stresses such as cosmic radiation and second-hand tobacco smoke may increase the risk of breast cancer formation. Towards an understanding of how exposure to these genotoxic agents affect breast cancer biogenesis, we have shown that treating non-tumorigenic immortalized breast MCF 10A cells with low doses (0.1 Gray) of radiation as well as cigarette smoke condensate can generate a neoplastic breast cancer phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases, and increased invasion and motility. In addition, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. Furthermore, differential gene expression profiles were generated from the individual and combination treatment. Overall, the results indicate that when normal breast cells are exposed to low dose radiation in combination with cigarette smoke condensate a phenotype is generated that exhibits traits indicative of neoplastic transformation. Taken together, these results provide a new insight into a possible etiology for breast cancer formation in individuals exposed to cosmic radiation and second-hand smoke. To study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray) or cigarette smoke condensate (Csc - 10 microgram/ml of medium) or a combination of Rad + Csc). Following treatments, the cells were incubated for 72 hr, RNA extracted and analyzed for differential gene expression pattern.
Project description:The transcription factors PAX3 and MITF are required for the development of the neural crest and melanocyte lineage, and both proteins play important roles in melanoma cell growth and survival. PAX3 transcriptionally activates MITF expression during neural crest development, but the relationship between these transcription factors during melanocyte development and in melanoma cells is currently poorly understood. This study aimed to further our understanding of the interaction between transcriptional networks controlled by PAX3 and MITF by assessing the effect of siRNA-mediated knockdown of PAX3 and MITF in metastatic melanoma cell lines. The goals of this study were to determine (i) if PAX3 is required for maintaining expression of MITF in melanoma and melanocyte cell lines; (ii) whether PAX3 and MITF independently, or redundantly, influence growth and survival in melanoma cell lines; and (iii) to investigate the respective roles of PAX3 and MITF expression in melanoma cell differentiation. Microarrays were used to measure global changes in transcript expression in response to siRNA-mediated knockdown of PAX3 or MITF compared to non-targeting controls in two metastatic melanoma cells lines. RNA was isolated from two different metastatic melanoma cell lines 30 hours after one of four different treaments: (i) transfection with siRNA targeting PAX3; or (ii) transfection with siRNA targeting MITF; or (iii) or transfection with siRNA targeting luciferase (non-targeting negative control); or (iv) treatment with media only (control). Therefore, eight samples were used for gene expression profiling by using GeneChip arrays, with one replicate per cell line per treatment.
Project description:Mature mRNAs undergo quality control during translation that may lead to RNA degradation by triggering the nonsense mediated decay (NMD) pathway. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction or an intron in the 3'UTR activates NMD. However, many of the features that lead to the activation of this pathway are unclear. UPF1, an RNA helicase, is the core NMD factor. UPF1 forms a multi-protein complex by recruiting a series of factors and other protein complexes in a process that depends on the UPF1 phosphorylation-dephosphorylation cycle. Among the factors recruited by UPF1, SMG5-SMG7 and SMG6 have critical importance in executing NMD. The SMG5-SMG7 heterodimer induces the exonucleolytic degradation of the mRNA, which depends on the recruitment of deadenylation factors. SMG6 has endonucleolytic activity and cleaves the targeted transcript close to the stop codon. The redundancy between the exonucleolytic and endonucleolytic paths to achieve degradation during NMD has been previously reported in the literature. To investigate the apparent redundancy between SMG5-SMG7 and SMG6 activity and to further understand the features that lead to the activation of NMD, we have generated two clones of SMG7 knockout human cells using CRISPR-Cas9. We generated mRNA-Sequencing data for control and both SMG7 KO clones with additional siRNA-mediated knockdown of Luciferase (Luc) as control, SMG5 or SMG6.
Project description:Time course gene-expression profiling was conducted in human lymphoblastoid cell lines (LUCY) after exposure with 1Gy and 10Gy gamma irradiation. Differentially expressed genes were identified using natural cubic spline regression modeling (NCSRM) and subsequently subjected to gene regulatory network reconstruction using a partial correlation approach (GeneNet) followed by analysis of the network. Senescence-associated signalling was identified as the chief regulatory network of radiation sensitivity of normal cells.
Project description:Viral dsRNA binds to Retinoic acid Inducible Gene I (RIG-I) Like Receptors (RLRs), promoting the production of Interferon (IFN). Interferon then stimulates the innate and adaptive immune system in an autocrine and paracrine manner. Outside of conical pathways, regulators of the interferon (IFN) activation/response system are poorly characterized. In this study, we used a discovery-biased approach to identify Kinases that are part of the interferon system. Differential changes in phosphorylation sites, in the context of dsRNA RIG-I stimulation, were identified with unbiased mass-spec biased phospho-proteomics. We then computationally identified several Kinases upregulated after RIG-I stimulation from phospho-proteomics data. A Chemoproteomics screen was then used to characterize the altered interferon response in the presence of Kinases inhibitors for the upregulated kinases. Combining unbiased phosphoproteomics with a chemoproteomics screen, we identified several potentially novel regulators of the Interferon system whose inhibition blocked the production of Interferon Stimulated Genes.
Project description:Endometrial cancer is the most commonly diagnosed gynecologic malignancy in women after breast, lung and colorectal cancer. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer is on the rise. Considerable research effort has therefore been placed on understanding the pathogenesis of this disease to combat this growing issue. There is now emerging evidence to suggest a putative role for dysregulation of the renin angiotensin system (RAS) and in particular the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. Support for this notion arises from previous literature implicating (P)RR in cancer pathophysiology (e.g., breast cancer and pancreatic carcinoma) by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. In view of these data, we aimed to investigate the functional role of (P)RR in human endometrial cancer progression and development. To this end, we employed an siRNA-mediated knock down approach to abrogate (P)RR expression in the immortalized endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1A to explore the role of (P)RR in cellular proliferation and cellular viability. To further extend these analyses we also carried out a sophisticated proteomic screen, that investigated the potential pathways via which (P)RR is acting in endometrial cancer physiology. These data confirmed that (P)RR is critical for endometrial cell cancer development, contributing to both its proliferative capacity and in the maintenance cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown. These putative protein interactions/pathways, which rely on the presence of (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets in the treatment of this cancer. Finally we contend that (P)RR, in its soluble form (s(P)RR) in blood, may have substantial potential as a novel biomarker for cancer diagnosis and prognosis prediction going forward.
Project description:WNT2 is important for placenta vascularization and acts as a pro-angiogenic factor for liver and other endothelial cells (ECs). WNT2 induction has been shown in many carcinomas and is associated with tumor progression. In colorectal cancer (CRC) WNT2 is selectively elevated in cancer associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer angiogenesis has not been addressed so far. Here, we demonstrate that WNT2 enhances EC migration and invasion, while it induces ß catenin dependent signaling in only a small subset of HUVECs. We show that siRNA-mediated knockdown of WNT2 in CAFs reduced the growth of vessel-like structures significantly in a co-culture assay, while the overexpression of WNT2 in skin fibroblasts otherwise being devoid of WNT2 led to increased angiogenesis in vitro. In a xenograft model, overexpression of WNT2 in HCT116 led to enhanced tumor volume and vessel density. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs revealed that proteins related to angiogenesis and extracellular matrix (ECM) remodeling are regulated by WNT2. Thus, stroma-derived WNT2 positively affects angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.