De Novo proteome analysis of genetically modified tumor cells by a metabolic labeling/azide-alkyne cycloaddition
Seda Ballikaya1, Jennifer Lee1, Uwe Warnken2
1From the ‡Department of Applied Tumor Biology, Institute of Pathology, University Hospital Heidelberg, INF 224, 69120 Heidelberg, Germany; §Cancer Early Detection, German Cancer Research Center (DKFZ), INF 280, 69120 Heidelberg, Germany;
Abstract:
Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.
Insights
Loss of Activin receptor type II (ACVR2) drives colorectal cancer. This study developed a novel proteomic approach to identify proteins affected by ACVR2 loss in colon cancer cells, revealing key cancer-related targets.
Area of Science:
- Molecular Biology
- Proteomics
- Cancer Research
Background:
- Activin receptor type II (ACVR2) is a tumor suppressor involved in cell growth and differentiation.
- ACVR2 inactivation is a known driver of colorectal tumorigenesis.
- Understanding proteomic changes upon ACVR2 loss is crucial for cancer research.
Purpose of the Study:
- To develop and apply a novel experimental strategy for comprehensive proteomic analysis of ACVR2 loss in colorectal cancer.
- To identify specific proteins and pathways affected by the functional loss of the ACVR2 tumor suppressor.
- To validate the utility of the developed approach for studying tumor suppressor gene function.
Main Methods:
- Generation of an inducible ACVR2 expression system in an ACVR2-deficient colon cancer cell line (HCT116) using recombinase-mediated cassette exchange.
- Metabolic labeling of newly synthesized proteins with azidohomoalanine followed by Click-iT chemistry for biotinylation.
- Streptavidin-based capture of labeled proteins, on-bead tryptic digestion, and analysis by ultra-high-performance LC-LTQ Orbitrap XL mass spectrometry.
Main Results:
- Successfully reconstituted ACVR2 expression in a doxycycline-dependent manner, confirming functional expression.
- Identified 513 proteins in total, with 25 proteins found to be differentially expressed between ACVR2-deficient and ACVR2-proficient cells.
- Several differentially expressed proteins were linked to colorectal cancer, cell growth, or apoptosis, validating the approach.
Conclusions:
- The novel experimental strategy combining inducible gene expression, metabolic labeling, and mass spectrometry is effective for analyzing proteomic changes associated with tumor suppressor loss.
- The identified differentially expressed proteins provide insights into the molecular mechanisms of ACVR2-driven colorectal tumorigenesis.
- This adaptable strategy can be applied to study the proteomic impact of any gene of interest in various cellular contexts.
