Gene expression profile in IM-0223 cells transfected with KPC1 or control vector using RNA-seq
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ABSTRACT: Melanoma is a highly aggressive cancer with increasing incidence rates and a poor survival, particularly in patients with AJCC stage IV and advanced stage III. Deregulation of NF-kB is linked to different pathological states, including melanoma. To identify the involvement of NF-kB pathway regulation in melanoma progression, we manipulated NF-kB pathway activation and profiled gene expression using RNA-sequencing. mRNA profiles of IM-0223 cells overexpressing KPC1 (KPC1) or control (V0) generated by deep sequencing using Illumina HiSeq 2500.
Project description:Melanoma is a highly aggressive cancer with increasing incidence rates and a poor survival, particularly in patients with AJCC stage IV and advanced stage III. Deregulation of NF-kB is linked to different pathological states, including melanoma. To identify the involvement of NF-kB pathway regulation in melanoma progression, we manipulated NF-kB pathway activation and profiled gene expression using RNA-sequencing.
Project description:Melanoma is a highly aggressive cancer with increasing incidence rates and a poor survival, particularly in patients with AJCC stage IV and advanced stage III. Deregulation of NF-kB is linked to different pathological states, including melanoma. To identify the involvement of NF-kB pathway regulation in melanoma progression, we manipulated NF-kB pathway activation and profiled gene expression using RNA-sequencing.
Project description:The inhibitor of kB kinase (IKK) is the master regulator of the nuclear factor kB (NF-kB) pathway, involved in inflammatory, immune and apoptotic responses. In the ‘canonical’ NF-kB pathway, IKK phosphorylates inhibitor of kB (IkB) proteins and this triggers ubiquitin-mediated degradation of IkB, leading to release and nuclear translocation of NF-B transcription factors.
The data presented show that the IKK and IKK subunits recognize a YDDX docking site located within the disordered C-terminal region of IkBa. Our results also suggest that IKK contributes to the docking interaction with higher affinity as compared to IKK.
Project description:Purpose: We identified KPC1 as the ubiquitin ligase that binds to the p105 precursor of NF-kB, ubiquitinates it and mediates its proteasomal processing to generate the p50 active subunit of the transcription factor. Using U87-MG human glioblastoma xenografts, we observed that overexpression of KPC1 results in strong inhibition of tumor growth mediated via excessive generation of p50.The goal of this RNASeq study was to analyze the profile of gene expression in xenografts overexpressing control (V0), KPC1 or p50 vectors, and to further understand how the altered gene expression patterns can explain the tumor suppressive effect we observed. Results:Transcript analysis of U87-MG xenografts overexpressing control (V0), KPC1 or p50 vector mapped to the human genome revealed: • A strong similarity between overexpression of p50 and KPC1 (correlation of 0.51, p-value<10-300 ) • A specific signature of NF-kB targets [21 of the consistently changed genes are known to be regulated by NF-kB (p-value<3.4×10-9 )] • A significant (p-value<1.4×10-18) increase in the expression of 40 tumor suppressor genes, with no significant change in other classes. • A significant down regulation of a cluster of genes including LIN28B, IL-6, HMAGA2 and VEGFA. This finding links well to an established regulatory axis involving LIN28B, Let-7 microRNA, and IL-6 in inflammation and cell transformation that is regulated by NF-kB.
Project description:Purpose: We identified KPC1 as the ubiquitin ligase that binds to the p105 precursor of NF-kB, ubiquitinates it and mediates its proteasomal processing to generate the p50 active subunit of the transcription factor. Using U87-MG human glioblastoma xenografts, we observed that overexpression of KPC1 results in strong inhibition of tumor growth mediated via excessive generation of p50.The goal of this RNASeq study was to analyze the profile of gene expression in xenografts overexpressing control (V0), KPC1 or p50 vectors, and to further understand how the altered gene expression patterns can explain the tumor suppressive effect we observed. Results:Transcript analysis of U87-MG xenografts overexpressing control (V0), KPC1 or p50 vector mapped to the human genome revealed: • A strong similarity between overexpression of p50 and KPC1 (correlation of 0.51, p-value<10-300 ) • A specific signature of NF-kB targets [21 of the consistently changed genes are known to be regulated by NF-kB (p-value<3.4×10-9 )] • A significant (p-value<1.4×10-18) increase in the expression of 40 tumor suppressor genes, with no significant change in other classes. • A significant down regulation of a cluster of genes including LIN28B, IL-6, HMAGA2 and VEGFA. This finding links well to an established regulatory axis involving LIN28B, Let-7 microRNA, and IL-6 in inflammation and cell transformation that is regulated by NF-kB. Exponentially growing U87-MG cells were stably transfected with an empty vector (V0) or vectors coding for Myc-KPC1 or Flag-p50. Cells were dissociated with trypsin, washed with PBS, and brought to a concentration of 50×10^6 cells/ml. Cell suspension (5×10^6/0.1 ml) was inoculated subcutaneously at the right flank of 7-weeks old male Balb/C nude mice (n=7). Following 21 days, mRNA from U87-MG xenografts was isolated using an RNA purification kit, and analyzed using the Illumina HiSeq 2500 sequencer. The RNASeq analysis experiment was repeated twice independently. Run1 included a total of 7 samples. Samples 1-3 were isolated from V0 – control tumors (3 different tumors), samples 4-5 were isolated from KPC1-expressing tumors (2 different pools of 3 tumors each due to small tumor size), and samples 6-7 were isolated from p50-expressing tumors for (2 different pools of 2-3 tumors each, due to very small tumor size). Run2 included a total of 5 samples. Samples 8-10 were isolated from V0 (control) tumors (3 different tumors), samples 11-12 were isolated from KPC1 tumors (2 different pools of 3 tumors each due to small tumor size). Several repeated attempts to extract RNA from the p50-expressing tumors did not yield any results, as the tumors were miniscule.
Project description:The transcription factor NF-kB is an important regulator of genes involved in inflammation and cell proliferation. Several studies previously showed that NF-kB transcription factor was constitutively activated in melanoma cell lines due to the up regulated activity of inhibitor of NF-kB kinases (IKK). In the present study, we tested the activation of NF-kB pathway following TNF stimulation in 9 melanoma cell lines and the subsequent inhibition after the treatment of a small–molecule IKK inhibitor NDB peptide using Secreated Alkaline Phosphate (SEAP) reporter gene assay. The pro-apoptotic effects induced by NBD peptide treatment were determined by flow cytometry as well as by Western Blot testing of PARP’s cleavage. Our findings showed that NBD peptide was able to block IKK-NFKB pathway in all tested cell lines by inducing apoptosis as confirmed by increased levels of active caspase 3 and PARP cleavage after treatment. Moreover, our finding provides the basis for the development of a novel therapeutic approach targeting NF-kB transcription factor to treat melanoma.
Project description:Melanoma is one of the tumor types with the highest risk of brain metastasis. However, the biology of melanoma brain metastasis and the contribution of the brain immune microenvironment to the responses to therapies remain insufficiently characterized. By using preclinical models and single-cell transcriptomics, we identify a mechanism to promote antitumor immunity in melanoma brain metastasis. We show that activation of the Rela/NF-kB pathway in microglia promotes melanoma brain metastasis and that targeting this pathway elicits microglia reprogramming towards a proinflammatory phenotype that enhances antitumor immunity and reduces brain metastatic burden. Additionally, proinflammatory microglial markers in melanoma brain metastasis correlate with better responses to immune checkpoint inhibitors in patients and we show that Rela/NF-kB targeting improves responses to these therapies in the brain. Thus, we propose targeting Rela/NF-kB in activated microglia as a strategy to promote antitumor immunity and responses to immune checkpoint inhibitors in melanoma brain metastasis.
Project description:Melanoma is one of the solid tumor types with the highest risk of brain metastasis. However, the biology of melanoma brain metastasis and the contribution of the brain immune microenvironment to the responses to therapies remain insufficiently characterized. By using preclinical models and single-cell transcriptomics, we identify microglia, as critical regulators of melanoma biology in the brain. We show that activation of the Rela/NF-kB pathway in microglia promotes melanoma brain metastasis and that targeting this pathway elicits microglia reprogramming towards a pro-inflammatory phenotype that enhances anti-tumor immunity and reduces brain metastatic burden. Additionally, canonical and pro-inflammatory microglial markers in melanoma brain metastasis correlate with better responses to immune checkpoint inhibitors in patients and we show that Rela/NF-kB targeting improves responses to these therapies in the brain. Thus, we propose targeting Rela/NF-kB in activated microglia as a strategy to promote anti-tumor immunity and to improve the response to immune checkpoint inhibitors in melanoma brain metastasis.
Project description:Melanoma is one of the solid tumor types with the highest risk of brain metastasis. However, the biology of melanoma brain metastasis and the contribution of the brain immune microenvironment to the responses to therapies remain insufficiently characterized. By using preclinical models and single-cell transcriptomics, we identify microglia, as critical regulators of melanoma biology in the brain. We show that activation of the Rela/NF-kB pathway in microglia promotes melanoma brain metastasis and that targeting this pathway elicits microglia reprogramming towards a pro-inflammatory phenotype that enhances anti-tumor immunity and reduces brain metastatic burden. Additionally, canonical and pro-inflammatory microglial markers in melanoma brain metastasis correlate with better responses to immune checkpoint inhibitors in patients and we show that Rela/NF-kB targeting improves responses to these therapies in the brain. Thus, we propose targeting Rela/NF-kB in activated microglia as a strategy to promote anti-tumor immunity and to improve the response to immune checkpoint inhibitors in melanoma brain metastasis.
Project description:<p>Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression, and rational therapy design. Here, we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-kB pathway in a total of 41% of cases including CYLD, TRAF3, NFKBIA and NLRC5. Functional analysis confirmed novel inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent member protein 1 (LMP1) functions to constitutively activate NF-kB signaling, and we observed mutual exclusivity among somatic NF-kB pathway aberrations and LMP1-overexpression, suggesting that NF-kB activation is selected for by both somatic and viral events during NPC pathogenesis.</p>