Abstract 5566: Phosphoproteomic profiling of Met / K-Ras signaling in colorectal cancer by label-free LTQ-Orbitrap mass spectrometry
Bibliographic record
Abstract
Abstract Background: Colorectal cancer (CRC) is the second leading cause of death from cancer overall. Mutations in the K-ras oncogene are the most frequently observed mutations in CRC. Activation of receptor tyrosine kinases, including Met, is also common in CRC. Co-activation of both Met and K-Ras could result in preferential over-activation of specific downstream cascade(s). To address the potential importance of Met and K-Ras tandem activation, we use the DLD1 CRC cell line model. DLD1 cells contain one mutated K-ras allele, resulting in a constitutively active protein. These cells express a relatively low level of Met. A derivative of this cell line, DKO4, has its mutant K-ras allele inactivated by somatic recombination. Our lab has previously shown that overexpression of Met in DLD1 cells (DLD1-Met) enhances the tumorigenicity and metastatic potential of these cells. This did not occur in DKO4-Met cells, suggesting that activated K-Ras is necessary for these Met-mediated processes. We hypothesize that Met and K-Ras co-operate to activate novel tumourigenic and metastatic signaling pathways. Methods: We undertook a label-free mass spectrometry (MS)-based proteomics examination of protein-phosphotyrosine (pY) in the DLD1 CRC model. DLD1-Met and DKO4-Met cells were enriched for pY-containing peptides and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. Peptide quantification was achieved by using both peptide ion spectral counting and normalized extracted ion current methods. To determine ligand-dependent vs. -independent differences in Met signaling, the parental DLD1 and DKO4 cells were stimulated with an HGF time course. Previous results in the lab show that DLD1 cells with activated K-Ras have prolonged MAPK signaling in response to HGF compared to DKO4 cells. Results: 9 proteins were found to be differentially regulated in these DLD1-Met vs. DKO4-Met. Validation by Western blotting in the empty vector and Met-overexpressing lines confirmed MS findings, as well as uncovered some pY changes mediated by both Met and K-Ras. Results from temporal analyses of pY in response to HGF stimulation will be presented to address this occurrence and suggest mechanisms of Met and K-Ras dependent tumourigenesis. Conclusions: We have identified a phosphoproteomic network associated with Met/K-Ras signaling. It may include novel therapeutic targets or prognostic markers for CRC. Supported by the CIHR MOP-64345 and the CIHR Banting and Best Doctoral Research Graduate Scholarship to SO Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5566.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".