A Comprehensive Proteomic Analysis of Metastatic Cancer Progression in a Murine Model of Tumorigenesis Using Orbitrap Tandem Mass Spectrometry

Abstract

While the subject of cancer metastasis has been extensively studied, research is typically limited to a few target proteins or signaling pathways analyzed via traditional means (Western blot, ELISA, etc.). In this study we have utilized cutting edge proteomic analysis to produce a database of protein expression data across seven serially passaged mouse cell lines to explore the progression of metastasis. We hypothesized that Orbitrap tandem mass spectrometry (OTMS) would replicate association of specific proteins to metastasis and delineate previously unidentified proteins. These cell lines included the parental embryonic fibroblasts (NIH/3T3), 3T3 cells transformed with a human oncogene, T1‐A cells from a primary tumor, T2‐A cells from a slightly metastatic tumor, and T3‐PA, T3‐HA, and T4‐PA cells derived from lung and liver metastases. OTMS yielded expression data for over 3,800 proteins across all seven cell lines. These outputs were considered to reach a threshold of change if protein expression was up‐or downregulated by at least two‐fold as compared to NIH/3T3 cells. There were 50 proteins that reached threshold in all six cancerous cell lines. Of these, 12% are directly associated with cell death and 18% with differentiation. Fourteen proteins reached threshold in only the metastatic cell lines. Correlation of protein up‐ or downregulation across these lines may assist in the elucidation or delineation of signal cascades and checkpoints that result in metastasis. Indeed, some of these proteins have been identified as tumorigenic via traditional means, such as moesin, which promotes invasion and metastasis, and PIN1, which positively correlates with proliferation and anchorage independent growth. Interestingly, 152 proteins met threshold expression only in T2‐A cells while being expressed at subthreshold levels in both non‐metastatic cells and highly metastatic cells, which is over twice the number of threshold proteins that were uniquely expressed in any single cell line. As T2‐A was the first cell line isolated from a metastatic growth, these data are evidence that these proteins are necessary for the conversion of a cell from a localized tumor to a metastatic cancer cell, but which return to normalized expression levels after this initial conversion. This study has yielded an impressive proteomic database to offer insight into the development of metastasis in embryonic mouse fibroblasts. It should provide a valuable resource to researchers examining how particular proteins of interest may act within the larger context of metastatic conversion. Furthermore it validates the efficacy of OTMS proteomic analysis by identifying proteins which had previously been recognized as tumorigenic via traditional proteomic studies, while gaining an unprecedented amount of novel information on models of cancer development.Support or Funding InformationThis research was supported by a grant from Oleander Medical Technologies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.