Koll, A. A. | bioRxiv Science

bioRxiv2024

Identification of Phosphotyrosine-Mutant Desmin in Human Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC), a deadly cancer with a 5-year survival rate of [~]12%, is characterized by frequently mutated KRAS, early metastasis, and extensive desmoplasia. The latter, a formation of dense fibrotic tissue generated by pancreatic stellar cells (PSC) and tumor cells, makes up to 80% of the tumor mass and leads to treatment failure. To search for novel protein biomarkers for actionable tyrosine kinases, we used a combination of two orthogonal protein analysis methods, western blotting (WB) and mass spectrometry (MS). That is, we used 1D/2D phosphotyrosine (pTyr) WB in combination with nano liquid chromatography tandem mass spectrometry (NanoLC-MS/MS) to analyze homogenates of 6 PDAC tumor samples and 5 normal adjacent tissue controls. Surprisingly, we found a novel, abundant 55 kDa pTyr-protein in 2/6 tumor samples and identified it as mutated pTyr-desmin. Further proteomics analysis of the purified protein cut from multiple Coomassie-stained 2D gels revealed that the mutant amino acid, tyrosine (D399Y), is phosphorylated (pTyr). A possible role for mutant pTyr-desmin in PDAC metastasis is discussed, along with peptide inhibitor drugs.

cancer biology↗

bioRxiv2023

Detection of Activated Receptor Tyrosine Kinases in Human Lung Squamous Cell Carcinoma

Lung squamous cell carcinoma (LSCC) has a high mutational burden and poor prognosis, even with immunotherapy. In the Lux-Lung 8 trial, afatinib, an epidermal growth factor receptor (EGFR) inhibitor, showed a long-term benefit in 5.3% of patients with LSCC. Because activating mutations of EGFR are rare in LSCC, the response was likely due to wild-type EGFR being activated by an unknown mechanism. All receptor tyrosine kinase (RTK) proteins, both wild-type and mutated, are activated by phosphorylation of specific tyrosines which serve as binding sites for various SH2 proteins. The aim of this feasibility study was to determine whether enhanced chemiluminescent western blotting (WB) with a phosphotyrosine (pTyr) antibody is sufficiently sensitive to detect pTyr-RTK proteins in human LSCC tissues. We performed WB analysis on 25 resected human lung tissue samples, including 12 LSCC, two adenocarcinomas (LADC), and 11 control (non-tumor) lung samples. The analysis showed [~]220 kDa pTyr-protein bands in two LSCC samples that were much more abundant than the corresponding bands in controls or LADC samples. To identify pTyr-RTKs, pTyr WB patterns of the two samples were compared to those of five RTK candidates: EGFR, platelet-derived growth factor receptor beta (PDGFRB), vascular endothelial growth factor receptor, anaplastic lymphoma receptor, and mesenchymal-epithelial transition factor. The strong pTyr signal in one sample matched EGFR, whereas the other matched a combination of EGFR and PDGFRB. We conclude that pTyr WB is sufficiently sensitive to detect pTyr-RTK drivers in flash-frozen tumor tissues and might identify LSCC patient subsets responsive to RTK inhibitors.

cancer biology↗