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Global Stability Profling of Colorectal Cancer Chemoresistance

ABSTRACT: Almost half of the patients with advanced colorectal cancer (CRC) are resistant to oxaliplatin based therapy, the first line treatment for CRC. Therefore, predicting and understanding oxaliplatin resistance is important to improve CRC patient survival. Investigated here is the use of proteomic folding stability measurements to differentiate oxaliplatin resistant and sensitive CRCs using patient-derived CRC cell lines and patient-derived xenografts (PDXs). Three protein stability profiling techniques (including the Stability of Proteins from Rates of Oxidation (SPROX), the Thermal Protein Profiling (TPP), and Limited Proteolysis (LiP) approaches) were employed to identify differentially stabilized proteins in 6 patient-derived CRC cell lines with different oxaliplatin sensitivities and 8 CRC PDXs derived from 2 of the patient derived cell lines with different oxaliplatin sensitivity. A total of 23 proteins were found in at least 2 techniques to be differentially stabilized in both the cell line and PDX studies of oxaliplatin resistance. These 23 differentially stabilized proteins included 9 proteins that have been previously connected to cancer chemoresistance. Over-representation analysis (ORA) of all the differentially stabilized proteins identified here, revealed novel pathways related to oxaliplatin resistance. Compared to conventional protein expression level analyses, which were also performed on the cell lines and PDXs, the stability profiling techniques identified novel proteins and pathways and provided new insight on the molecular basis of oxaliplatin resistance. Our results suggest that protein stability profiling techniques are complementary to expression level analyses for identifying biomarkers and understanding molecular mechanisms associated with oxaliplatin chemoresistance in CRC and disease phenotypes in general.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Baiyi Quan

LAB HEAD: Michael C. Fitzgerald

PROVIDER: PXD036298 | Pride | 2023-04-28

REPOSITORIES: Pride

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Source:

			Action	DRS
	CRC16-159_vs_SW620_Expression_Level_SPROX.msf	Msf
	CRC16-159_vs_SW620_Expression_Level_SPROX_1.raw	Raw
	CRC16-159_vs_SW620_Expression_Level_SPROX_2.raw	Raw
	CRC16-159_vs_SW620_Expression_Level_SPROX_3.raw	Raw
	CRC16-159_vs_SW620_Expression_Level_TPP.msf	Msf

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Project description:Large-scale studies of protein phosphorylation have generally focused on determining the sites and stoichiometry of phosphorylated proteins derived from different biological specimens such as cell lines and tissue samples. While such information is important for understanding the role of phosphorylation in biological systems, it fails to expose the relationship between protein phosphorylation and protein function. Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. As part of this work, the Stability of Proteins from Rate of OXidation (SPROX) and Limited Proteolysis (LiP) techniques were utilized to identify phosphorylation-induced conformational and stability changes in proteins derived from a human breast cancer cell line MCF-7. This was accomplished by comparing the conformation properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. The SPROX technique, which probes the global unfolding/refolding properties of proteins, identified 168 proteins with dephosphorylation-induced stability changes, and the LiPs technique, which probes the more local unfolding/refolding properties of proteins, identified 251 proteins with dephosphorylation-induced stability changes. A total of 49 proteins identified here with dephosphorylation-induced conformational changes, were previously identified in phosphoproteomic studies to be differentially phosphorlyated in different human breast cancer subtypes. A total of 98 proteins identified here overlapped with protein hits previously found to be differentially stabilized in four human breast cancer phenotypes, suggesting that the phosphorylated changes observed here may be disease related. The experimental approach and results reported here create a novel approach for identifying functionally significant PTMs in proteins.

Project description:Large-scale studies of protein phosphorylation have generally focused on determining the sites and stoichiometry of phosphorylated proteins derived from different biological specimens such as cell lines and tissue samples. While such information is important for understanding the role of phosphorylation in biological systems, it fails to expose the relationship between protein phosphorylation and protein function. Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. As part of this work, the Stability of Proteins from Rate of OXidation (SPROX) and Limited Proteolysis (LiP) techniques were utilized to identify phosphorylation-induced conformational and stability changes in proteins derived from a human breast cancer cell line MCF-7. This was accomplished by comparing the conformation properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. The SPROX technique, which probes the global unfolding/refolding properties of proteins, identified 168 proteins with phosphorylation-induced stability changes, and the LiPs technique, which probes the more local unfolding/refolding properties of proteins, identified 251 proteins with phosphorylation-induced stability changes. A total of 49 proteins identified here with phosphorylation-induced conformational changes, were previously identified in phosphoproteomic studies to be differentially phosphorlyated in different human breast cancer subtypes. A total of 98 proteins identified here overlapped with protein hits previously found to be differentially stabilized in four human breast cancer phenotypes, suggesting that the phosphorylated changes observed here may be disease related. The experimental approach and results reported here create a novel perspective in understanding large-scale molecular influence of phosphorylation and a shortcut in finding possible functionally significant PTMs in cellular proteins.

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Global Stability Profling of Colorectal Cancer Chemoresistance

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