Summary of Study ST002241
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 PR001430. The data can be accessed directly via it's Project DOI: 10.21228/M8W69F This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST002241 |
| Study Title | ACSS2 Regulates HIF-2α Degradation through the E3-Ubiquitin Ligase MUL1 in Clear Cell Renal Cell Carcinoma |
| Study Type | untargeted metabolomics analysis |
| Study Summary | Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer driven by VHL loss and aberrant HIF-2α signaling. Acetate metabolism may contribute to this axis by ACSS2-dependent acetylation of HIF-2α and may provide opportunities to intervention. Here we tested the effects of pharmacological and genetic manipulation of ACSS2 on HIF-2α, ccRCC cells, and tumors. ACSS2 inhibition led to HIF-2α degradation and suppressed ccRCC growth in vitro, in vivo, and in primary cell cultures of ccRCC patient tumors. This treatment resulted in reduced glucose and cholesterol metabolism, mitochondrial biogenesis and altered cristae deformation, that are consistent with loss of HIF-2α. Mechanistically, HIF-2α protein levels are regulated through proteolytic degradation and we found, in parallel to VHL, HIF-2α stability was dependent on ACSS2 activity to prevent direct interaction with the E3 ligase MUL1. These findings highlight ACSS2 as a critical upstream regulator of pathogenically stabilized HIF-2α, and provides a mechanism that may be exploited to overcome resistance to HIF-2α inhibitor therapies. |
| Institute | Vanderbilt University |
| Department | Chemistry |
| Laboratory | Center for Innovative Technology |
| Last Name | CODREANU |
| First Name | SIMONA |
| Address | 1234 STEVENSON CENTER LANE |
| SIMONA.CODREANU@VANDERBILT.EDU | |
| Phone | 6158758422 |
| Submit Date | 2022-07-26 |
| Num Groups | 2 |
| Total Subjects | 10 |
| Num Males | 10 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-08-01 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001430 |
| Project DOI: | doi: 10.21228/M8W69F |
| Project Title: | ACSS2 Regulates HIF-2α Degradation through the E3-Ubiquitin Ligase MUL1 in Clear Cell Renal Cell Carcinoma |
| Project Type: | Untargeted Metabolomics analysis |
| Project Summary: | Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer driven by VHL loss and aberrant HIF-2α signaling. Acetate metabolism may contribute to this axis by ACSS2-dependent acetylation of HIF-2α and may provide opportunities to intervention. Here we tested the effects of pharmacological and genetic manipulation of ACSS2 on HIF-2α, ccRCC cells, and tumors. ACSS2 inhibition led to HIF-2α degradation and suppressed ccRCC growth in vitro, in vivo, and in primary cell cultures of ccRCC patient tumors. This treatment resulted in reduced glucose and cholesterol metabolism, mitochondrial biogenesis and altered cristae deformation, that are consistent with loss of HIF-2α. Mechanistically, HIF-2α protein levels are regulated through proteolytic degradation and we found, in parallel to VHL, HIF-2α stability was dependent on ACSS2 activity to prevent direct interaction with the E3 ligase MUL1. These findings highlight ACSS2 as a critical upstream regulator of pathogenically stabilized HIF-2α, and provides a mechanism that may be exploited to overcome resistance to HIF-2α inhibitor therapies. |
| Institute: | Vanderbilt University |
| Department: | Chemistry |
| Laboratory: | Center for Innovative Technology |
| Last Name: | CODREANU |
| First Name: | SIMONA |
| Address: | 1234 STEVENSON CENTER LANE |
| Email: | SIMONA.CODREANU@VANDERBILT.EDU |
| Phone: | 16158758422 |
Subject:
| Subject ID: | SU002327 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | NOD-scid IL2Rgnull (NSG) |
| Age Or Age Range: | 6 weeks |
| Gender: | Male |
| Animal Animal Supplier: | Jackson Laboratory (catalog number 005557) |
| Animal Housing: | Mice were housed five to a cage in a temperature- and humidity-controlled space (20-25°C, 45%–64% humidity) with regulated water and lighting (12 h light/dark) within the animal facility. |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA214135 | RCC_KD05 | doxycycline-inducible shRNA |
| SA214136 | RCC_KD04 | doxycycline-inducible shRNA |
| SA214137 | RCC_KD01 | doxycycline-inducible shRNA |
| SA214138 | RCC_KD02 | doxycycline-inducible shRNA |
| SA214139 | RCC_KD03 | doxycycline-inducible shRNA |
| SA214130 | RCC_C01 | Wild-type |
| SA214131 | RCC_C05 | Wild-type |
| SA214132 | RCC_C04 | Wild-type |
| SA214133 | RCC_C02 | Wild-type |
| SA214134 | RCC_C03 | Wild-type |
| Showing results 1 to 10 of 10 |
Collection:
| Collection ID: | CO002320 |
| Collection Summary: | Mice which were inoculated with 786-O cells transduced to express the pTRIPZ doxycycline-inducible shRNA system were provided a 200 mg/kg doxycycline rodent diet (Bio-Serv) and allowed to feed ad libitum. Tumor burden was monitored via weekly manual, digital caliper measurement with intermittent measurements taken as needed. Mice were euthanized once the first tumor reached size endpoint (1,000 mm3) or if discomfort was observed as outlined in the IACUC approved protocol. |
| Sample Type: | Tumor cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002339 |
| Treatment Summary: | Mice which were inoculated with 786-O cells transduced to express the pTRIPZ doxycycline-inducible shRNA system were provided a 200 mg/kg doxycycline rodent diet (Bio-Serv) and allowed to feed ad libitum. |
Sample Preparation:
| Sampleprep ID: | SP002333 |
| Sampleprep Summary: | Fresh tumor sample materials were flash frozen and stored at -80°C until analyzed via Liquid Chromatography-Mass Spectrometry (LC-MS and LC-MS/MS)- untargeted metabolomics in the Vanderbilt Center for Innovative Technology (CIT). Individual tissue samples were thawed on ice and lysed in 400 µl ice-cold lysis buffer (1:1:2, acetonitrile: methanol: 0.1M ammonium bicarbonate, pH 8.0) and sonicated 2 x 10 pulses using a probe sonicator at 50% power with cooling in ice in-between. Homogenized samples were normalized by protein amount (200 µg total protein per tissue sample) based on BCA assay (Thermo Fisher Scientific, Fair Lawn, NJ). Isotopically labeled phenylalanine-D8 and biotin-D2 were added to individual samples, and proteins wwere precipitated by addition of 800 µL of ice-cold methanol followed by overnight incubation at -80°C. Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min), and metabolite extracts were dried down in vacuo. Individual samples were reconstituted in 100 µl of reconstitution buffer (acetonitrile: water, 90:10, v:v) containing tryptophan-D3, valine-D8, and inosine-4N15 and cleared by centrifugation. A quality control (QC) sample was prepared by pooling equal volumes from individual samples following reconstitution. Quality control samples were used for column conditioning, retention time alignment and to assess mass spectrometry instrument reproducibility throughout the sample set and allows for sample batch acceptance. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Following lysis and standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003659 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002711 |
| Chromatography Summary: | The LC-MS and LC-MS/MS analyses were performed on a high-resolution Q-Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a Vanquish UHPLC binary system and autosampler (Thermo Fisher Scientific, Germany) using previously described methods. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) |
| Column Temperature: | 40 |
| Flow Gradient: | 45 min |
| Flow Rate: | 0.20mL/min |
| Solvent A: | 90% water, 10% acetonitrile, 5 mM ammonium formate |
| Solvent B: | 10% water/90% acetonitrile; 5 mM ammonium formate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003410 |
| Analysis ID: | AN003659 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The acquired raw data were imported, processed, normalized and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS and MS/MS sample runs were aligned against a QC sample |
| Ion Mode: | NEGATIVE |
