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Updated: Sep 13, 2025
An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA
Published on: February 17, 2023
Proteomic analysis of tumor cell nuclear expulsion reveals significant cell adhesion and RNA binding programs in
Justin M Gray1,2, Woo Yong Park3, Ronald J Holewinski4
1Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Abstract:
Understanding mechanisms of cancer cell death and the resulting effect on disease progression is crucial in cancer biology and the insight will likely offer better options for therapeutic treatment. Nuclear expulsion occurs in apoptotic cancer cells in a peptidylarginine deiminase 4 (Padi4) dependent manner. The resulting tumor cell nuclear expulsion product (TuNEP) promotes the outgrowth of neighboring cancer cells through chromatin-bound protein complexes. It is not clear what the protein compositions and functionalities are in these TuNEPs. In this study, we performed extensive proteomic profiling and identified key TuNEP protein components from mouse and human breast cancer cells as well as human lung cancer cells (4T1, MDA-MB-231, and PC9). We further compared TuNEP- specific proteins with those from apoptotic bodies or NETs from neutrophils. We found an enrichment of cellular adhesion molecules as well as increased citrullination of proteins associated with RNA binding. We showed that cellular adhesion molecules integrin and basigin (BSG) promote the growth of tumor spheroids. Our work revealed the unique TuNEP protein components distinct from neutrophil-derived NETs and shed light on potential mechanisms by which these cancer cell-derived TuNEPs promote tumor progression.
Insights
Cancer cells expel nuclear material (TuNEPs) that promote tumor growth. This study identified unique proteins in TuNEPs, revealing novel mechanisms of cancer progression and potential therapeutic targets.
Area of Science:
- Cancer Biology
- Cell Death Mechanisms
- Tumor Progression
Background:
- Understanding cancer cell death is vital for developing effective therapies.
- Nuclear expulsion in apoptotic cancer cells, dependent on peptidylarginine deiminase 4 (Padi4), generates tumor cell nuclear expulsion products (TuNEPs).
- TuNEPs, containing chromatin-bound protein complexes, are known to promote the outgrowth of neighboring cancer cells, but their composition and function remain unclear.
Purpose of the Study:
- To identify the protein components and functionalities of TuNEPs.
- To compare TuNEP proteins with those from apoptotic bodies and neutrophil extracellular traps (NETs).
- To elucidate the role of TuNEPs in promoting tumor progression.
Main Methods:
- Proteomic profiling of TuNEPs from mouse and human breast cancer cells (4T1, MDA-MB-231) and human lung cancer cells (PC9).
- Comparative analysis of TuNEP proteins against apoptotic bodies and neutrophil-derived NETs.
- Functional assays to assess the role of identified cellular adhesion molecules (integrin, basigin/BSG) in tumor spheroid growth.
Main Results:
- Identification of key TuNEP protein components unique to cancer cells.
- Found enrichment of cellular adhesion molecules and increased citrullination of RNA-binding proteins in TuNEPs.
- Demonstrated that integrin and basigin (BSG) promote tumor spheroid growth.
Conclusions:
- TuNEPs possess unique protein compositions distinct from neutrophil-derived NETs.
- Cellular adhesion molecules within TuNEPs play a significant role in promoting tumor progression.
- These findings offer insights into novel mechanisms of cancer cell-driven tumor growth and potential therapeutic strategies.
