Effect of myeloid specific rictor knockout on B16-F10 tumors grown in mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE300928 | GEO | 2025/07/01
REPOSITORIES: GEO
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Project description:Effect of myeloid specific rictor knockout on B16-F10 tumors grown in mice
| PRJNA1282685 | ENA
Project description:RNA-Seq of B16-F10 mouse melanoma cell line and gene expression profiling
2014-01-31 | E-MTAB-2271 | biostudies-arrayexpress
Project description:Nearly 50% of cutaneous melanomas carry activating mutations on the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used for their clinical management. One major drawback of BRAF/MEKi targeted therapy is the rapid development of therapeutic resistance, which can occur via multiple mechanisms, including the metabolic rewiring of cancer cells that often involves the upregulation of mitochondrial bionergetics and NAD+ biosynthetic pathways. mTORC2 is a signaling complex that requires the presence of its essential RICTOR subunit to play its regulatory functions in cell growth and metabolism. mTORC2 is believed to play mostly pro-oncogenic roles in several tumor types, including melanoma. However, bioinformatics analysis of TCGA melanoma patients’ database revealed that low RICTOR levels in tumors correlate with an overall worse clinical outcome. GSEA analysis of low-RICTOR tumors evidenced also a gene expression signature suggestive of activation of mitochondrial energy producing pathways. On these bases, we have hypothesized that inhibition of mTORC2 activity may render BRAFV600E melanoma cells resistant to BRAFi/MEKi. We show here that RICTOR/mTORC2-deficient cells are intrinsically tolerant to BRAFi/MEKi, and anticipate the onset of resistance to BRAFi after sustained drug exposure both in vitro and in vivo, indicating that mTORC2 activity normally opposes the acquisition of targeted therapy resistance in BRAFV600E melanomas. Mechanistically, RICTOR-deficient cells show an enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of NAD+ salvage pathway, and pharmacological inhibition of these processes in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. Thus, our work identifies a novel role for mTORC2 in favoring the responses of BRAF-mutated melanoma cells to targeted therapy, and suggest that the evaluation of the intratumor level of RICTOR may help to predict the responses of melanoma patients to these treatments.
2024-05-23 | PXD045346 | Pride
Project description:Background Main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF- mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to define in this context the specific role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit and regarded as an oncogenic driver in several tumor types, including MM. Methods After analyzing the TCGA MM patients’ database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAF V600E MM cell lines on their response to BRAF/MEKi in vitro. We performed a proteomic screening to identify proteins modulated by changes in RICTOR expression and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Results We found that low RICTOR levels in MM correlate with an overall worse clinical outcome. GSEA of low- RICTOR tumors revealed a gene expression signature suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy producing pathway. RICTOR-deficient BRAF V600E cells are intrinsically tolerant to BRAFi/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. In RICTOR- depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, while their pharmacological inhibition restores sensitivity to BRAFi. Conclusions Our work unveils a novel tumor suppressing role for mTORC2 in the responses of BRAF V600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low- RICTOR tumors. Importantly, our findings support the concept that the evaluation of intra-tumor RICTOR levels in MM has a prognostic value, and may help predicting the response of patients to targeted therapy.
2024-06-22 | PXD050614 | Pride
Project description:RNA-seq was performed on sorted peritoneal tissue resident macrophages (CD11b+F4/80hiTIM4+) and monocytic macrophages (CD11b+F4/80loTIM4-) from IL-4c (recombinant IL-4 (5µg) and anti-IL-4 ab (12.5µg) IP injection on days 0 and 2 and sorted on day 4) treated 6-8wks old LySM Cre+ and LysM Cre+ RICTOR KO (C57BL/6 background) for expression profiling
2017-10-01 | GSE94965 | GEO
Project description:Recent work using mouse models has revealed that mTORC2, which unlike mTORC1 is not acutely sensitive to rapamycin, plays a key role in the regulation of organismal physiology. The substrates and pathways regulated by mTORC2 are at present relatively unknown Using a mouse model with a targeted deletion of hepatic RICTOR, we investigated the loss of mTORC2 on the murine liver transcriptome Rictor floxed (RKO) and control mice (n=4 per group) were fasted overnight, refed for 3 hr, then sacrificed. Livers were removed, rinsed in PBS, and flash frozen in liquid nitrogen. RNA was extracted and hybridized to Affymetrix Genechip Mouse Gene 1.0 ST arrays
2013-09-23 | E-GEOD-46515 | biostudies-arrayexpress
Project description:Purpose: RNA-sequencing analysis defined a novel role for PRMT7 in melanoma cancer immunotherapy. Methods: PRMT7-deficient cells and control B16.F10 cells treated with mIFN-γ (100ng/ml) for 24 hours. B16.F10 melanoma cells mRNA profiles were generated by deep sequencing, in triplicate using TruSeq Stranded mRNA Sample Prep Kit with TruSeq Unique Dual Indexes (Illumina, Hiseq4000, SR75 platform located at San Diego, CA; UCSD IGM Genomics Facility, La Jolla, CA). Resulting libraries were multiplexed and sequenced with 100 base pair (bp) to a depth of approximately 30 million reads per sample. The sequence reads that passed quality filters were trimmed with Trimmomatic v0.39. STAR v2.7.1a was then used to align the reads to the mouse genome (mm10/GRCm38). Gene expression was quantified across all samples with HOMER v4.11.1, and the normalization was carried out through the regularized logarithm (rlog) transformation of DESeq2 v1.26.0. Differential expression between PRMT7 WT and knockdown samples were calculated through DESeq2 v1.26.0, and the gene expression was considered significantly different if the absolute value of the log-fold-change (LFC) was higher than 2, the base means larger than 10 and the false discovery rate (FDR) less than 0.05. Results: Differential expression analysis between the siLuc and siPRMT7 conditions yielded 185 and 251 genes with statistically significant differences and absolute fold change greater than 2 in the IFN– and IFN+ treatments respectively. Of these, 85% of genes are upregulated in the IFN– treatment, while 72% of genes are downregulated in the IFN+ treatment. Gene ontology analysis using the 185 and 251 lists of differentially expressed genes implies that the downregulated biological processes are enriched for the immune response pathways, while upregulated genes are more involved in the cell cycle process. Altered expression of some genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to better understand PRMT7 function in cancer immunotherapy. Conclusions: Our study represents the first detailed analysis of B16.F10 melanoma transcriptomes without PRMT7 expression, with biologic replicates, generated by RNA-seq technology. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of PRMT7 biologic functions in modulating melanoma transcriptome.
2022-03-01 | GSE157141 | GEO
Project description:Unleashing the immune anti-tumor response through immune checkpoint blockade (ICB) has been successful in treating many solid-tumor malignancies, including metastatic melanoma. When successful, the ICB response can be potent; however, half of patients fail to respond. ICB responsiveness is impacted by the harsh solid tumor microenvironment (TME), which is characterized by metabolic stress. The TME impacts tumor antigenicity, with ICB-responsive melanomas exhibiting increased major histocompatibility complex class I (MHC-I) expression. Further investigation of tumor immunogenicity in the context of the TME may improve cellular therapies. Here, we define and characterize an epigenetic mechanism regulating melanoma antigen presentation driven by prolonged metabolic stress. Murine and human melanoma cell lines were cultured under prolonged metabolic stress, forcing cells to adapt to the absence of glucose. Melanoma cells adapted to the absence of glucose have IFN-γ-independent increases in MHC-I and an increased sensitivity to T cell-mediated killing. Proteomic analysis revealed dysregulation of histone epigenetic modifiers under prolonged metabolic stress, specifically loss of histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2). EZH2 directly silences gene transcription via catalyzing H3K27me3. Following metabolic adaptation, ChIP-sequencing and ChIP-PCR revealed H3K27me3 loss at genes specific to MHC-I antigen presentation. Prolonged metabolic stress in melanoma cells blunt EZH2 levels and H3K27me3 levels at promoters of genes regulating MHC-I presentation, resulting in elevated MHC-I antigenicity and increased CD8+ T cell killing. This demonstrates potential for EZH2 abundance and mutational status as a prognostic indicators of ICB-responsiveness in metastatic melanoma and supports EZH2 inhibition as adjuvant for immunotherapies.
2025-05-14 | GSE297168 | GEO
Project description:We provide evidence that IFN-induced Stat-activation is defective in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2 (mTORC2). Our studies show that transient or stable knockdown of Rictor leads to decreased expression of several IFN-inducible genes that mediate important biological functions, including antiproliferative and pro-apoptotic responses. Rictor+/+ and Rictor-/- MEFs were treated with 2500 U/ml of mouse IFNα for 24 hours
2014-01-28 | E-GEOD-47896 | biostudies-arrayexpress
Project description:The effect of TIM-3 stimulation was studied on B16F10 mouse melanoma cells, in vitro. Experiment Overall Design: Cy3: B16F10 mouse melanoma cells, treated by unspecific goat IgG (control) for 2 hours. Experiment Overall Design: Cy5: B16F10 mouse melanoma cells, in vitro, treated by an agonist goat polyclonal anti-mouse TIM3 antibody (RnD Systems) for 2 hours. Experiment Overall Design: Four biological replicates were used (in this study, biological replicate means different passage numbers of the same cell line). In each case, the treated sample (Cy5) was compared to its control one (Cy3) in Agilent dual-color microarray experiments.
2009-10-15 | E-GEOD-14418 | biostudies-arrayexpress