Summary of Study ST002218
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001408. The data can be accessed directly via it's Project DOI: 10.21228/M8QM6W This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST002218 |
| Study Title | Effect of Hira loss in the metabolic landscape of Fh1-deficient cells Part 2 |
| Study Summary | Tumour initiation and progression requires the metabolic rewiring of cancer cells. Fumarate hydratase (FH), a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle, has been identified as a bona fide tumour suppressor . FH loss predisposes to Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC), a cancer syndrome characterized by the presence of benign tumours of the skin and uterus, and a highly aggressive form of renal cancer. Its loss leads to aberrant accumulation of fumarate, an oncometabolite that drives malignant transformation . Even though the link between FH loss, fumarate accumulation and HLRCC is well-known, the associated tumorigenic mechanism is it is still not fully understood. Indeed, although HLRCC tumours metastasize even when small, Fh1-deficient mice develop premalignant cysts in the kidneys, rather than overt carcinomas. Interestingly, these cysts are positive for the key tumour suppressor p21. Since p21 expression is a central trigger of cellular senescence, it is postulated that this process could be an obstacle for tumorigenesis in Fh1-deficient cells. Consistent with this hypothesis, HLRCC patients harbour the epigenetic suppression of p16, another key player of senescence. Here, we have confirmed that additional oncogenic events independent from a senescence bypass are required to allow full-blown transformation in FH deficient cells. Moreover, a genome wide CRISPR/Cas9 screen identified HIRA as a target that, when ablated, increases proliferation and invasion in Fh1-deficient cells. Moreover, Fh1 and Hira-deficient cells lead to the development of tumours and invasive features in the kidney in vivo. Strikingly, Hira depletion in Fh1 deficient cells controls the activation of a MYC and E2F-dependent transcriptional and metabolic program, which is known to play different oncogenic roles during tumour initiation and progression. Of note, the activation of these programs is independent of H3.3 deposition into the chromatin, known to be controlled by HIRA. Overall, we have identified a novel oncogenic event occurring in FH deficient tumours, which will be instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments. Part 2 of this study emoployed a second FH-null clone to complement Part 1 of the study. |
| Institute | CECAD Research Center |
| Last Name | Yang |
| First Name | Ming |
| Address | Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany |
| ming.yang@uni-koeln.de | |
| Phone | 4922147884306 |
| Submit Date | 2022-07-06 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-07-28 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001408 |
| Project DOI: | doi: 10.21228/M8QM6W |
| Project Title: | Hira Loss Transforms Fh1-Deficient Cells |
| Project Summary: | Tumour initiation and progression requires the metabolic rewiring of cancer cells. Fumarate hydratase (FH), a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle, has been identified as a bona fide tumour suppressor . FH loss predisposes to Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC), a cancer syndrome characterized by the presence of benign tumours of the skin and uterus, and a highly aggressive form of renal cancer. Its loss leads to aberrant accumulation of fumarate, an oncometabolite that drives malignant transformation . Even though the link between FH loss, fumarate accumulation and HLRCC is well-known, the associated tumorigenic mechanism is it is still not fully understood. Indeed, although HLRCC tumours metastasize even when small, Fh1-deficient mice develop premalignant cysts in the kidneys, rather than overt carcinomas. Interestingly, these cysts are positive for the key tumour suppressor p21. Since p21 expression is a central trigger of cellular senescence, it is postulated that this process could be an obstacle for tumorigenesis in Fh1-deficient cells. Consistent with this hypothesis, HLRCC patients harbour the epigenetic suppression of p16, another key player of senescence. Here, we have confirmed that additional oncogenic events independent from a senescence bypass are required to allow full-blown transformation in FH deficient cells. Moreover, a genome wide CRISPR/Cas9 screen identified HIRA as a target that, when ablated, increases proliferation and invasion in Fh1-deficient cells. Moreover, Fh1 and Hira-deficient cells lead to the development of tumours and invasive features in the kidney in vivo. Strikingly, Hira depletion in Fh1 deficient cells controls the activation of a MYC and E2F-dependent transcriptional and metabolic program, which is known to play different oncogenic roles during tumour initiation and progression. Of note, the activation of these programs is independent of H3.3 deposition into the chromatin, known to be controlled by HIRA. Overall, we have identified a novel oncogenic event occurring in FH deficient tumours, which will be instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments. |
| Institute: | CECAD Research Center, University Hospital Cologne |
| Last Name: | Yang |
| First Name: | Ming |
| Address: | Joseph-Stelzmann-Straße 26, CECAD Research Center, Köln, Koeln, 50931, Germany |
| Email: | ming.yang@uni-koeln.de |
| Phone: | +4922147884306 |
Subject:
| Subject ID: | SU002304 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA211943 | LV17-09 | Fh1-/- CL1 | Cas9 |
| SA211944 | LV17-07 | Fh1-/- CL1 | Cas9 |
| SA211945 | LV17-08 | Fh1-/- CL1 | Cas9 |
| SA211946 | LV17-12 | Fh1-/- CL1 | sg1Hira |
| SA211947 | LV17-11 | Fh1-/- CL1 | sg1Hira |
| SA211948 | LV17-10 | Fh1-/- CL1 | sg1Hira |
| SA211937 | LV17-01 | Fh1 fl/fl | Cas9 |
| SA211938 | LV17-03 | Fh1 fl/fl | Cas9 |
| SA211939 | LV17-02 | Fh1 fl/fl | Cas9 |
| SA211940 | LV17-06 | Fh1 fl/fl | sg1Hira |
| SA211941 | LV17-04 | Fh1 fl/fl | sg1Hira |
| SA211942 | LV17-05 | Fh1 fl/fl | sg1Hira |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO002297 |
| Collection Summary: | Cells were seeded in 6-well plates and collected the following day at 70% confluency. Cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion of 0.5 ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials. |
| Sample Type: | kidney epithelial cells |
Treatment:
| Treatment ID: | TR002316 |
| Treatment Summary: | Fh1-deficient cells expressing Cas9 were transduced with gRNA against Hira (g1Hira). No further treatments were carried out. Cells were cultured using high glucose (4.5 g/L) DMEM (Gibco-41966-029) supplemented with 10% heat inactivated fetal bovine serum (FBS). |
Sample Preparation:
| Sampleprep ID: | SP002310 |
| Sampleprep Summary: | Cells were seeded in 6-well plates and collected the following day at 70% confluency. Cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion of 0.5 ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials. |
Combined analysis:
| Analysis ID | AN003627 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002682 |
| Chromatography Summary: | Chromatographic separation of polar metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. Samples were randomized and analysed with LC–MS in a blinded manner with an injection volume was 5 µl. Pooled samples were generated from an equal mixture of all individual samples and analysed interspersed at regular intervals within sample sequence as a quality control. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003378 |
| Analysis ID: | AN003627 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Metabolites were measured with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The sheath gas flow was set to 55 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder 5.0 and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling through instrument analysis. The normalized areas were used as variables for further statistical data analysis. |
| Ion Mode: | UNSPECIFIED |
