A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis

Abstract

Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. in this study, we applied a parallel-reaction monitoring (pRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis.

Quantitative Proteomic Analysis Revealed Broad Roles of N6-Methyladenosine in Heat Shock Response

“…The above-described data-dependent acquisition (DDA) data set led to the quantification of the expression levels of 103 kinases (Figure d), among which 7 and 5 were down- (YES1, BTK1, LOC651610, EIF2AK2, STK39, PCK2, POLR2A) and upregulated (CDK6, PKN1, TK1, UCK2, CCND3), respectively, upon heat shock. Considering the superior sensitivity and throughput of the PRM-based targeted proteomics over discovery proteomics, ,− we further employed scheduled LC-PRM, in combination with SILAC, to examine the differential expression of kinases in response to heat shock (Figure a).…”

Blueprint for cancer research: Critical gaps and opportunities

“…For example, in the context of prostate cancer treatment with androgen receptor antagonists, the receptor gene can undergo amplification 173 or increased expression to overcome growth inhibition. 174 This dynamic and widespread cellular response, which contributes to therapeutic resistance, has been documented in many cancer types, including breast cancer, 175 melanoma, 176 and glioblastoma. 177 Pathway reactivation associated with drug resistance can also result from upstream or downstream induction that effectively bypasses the block created by the inhibitor.…”

Targeted Proteomic Analysis Revealed Kinome Reprogramming during Acquisition of Radioresistance in Breast Cancer Cells

“…To unveil potential functions of kinases in acquired radioresistance, we employed a PRM-based targeted proteomic method, − in combination with stable isotope labeling by amino acids in cell culture (SILAC), to screen for differentially expressed kinase proteins in two matched pairs of breast cancer cells, i.e., parental (WT) MCF-7 and MDA-MB-231 cells, as well as their corresponding radioresistant C6 and C5 clones (Figure a).…”

“…Herein, we employed our previously developed parallel-reaction monitoring (PRM)-based LC-MS/MS method − to examine the differential expression of kinase proteins in two pairs of breast cancer cell lines, i.e., the MDA-MB-231 and MCF-7 breast cancer cells and their corresponding radioresistant subclones. Our results facilitated the measurements of the relative expression levels of 300 and 281 kinase proteins in the MDA-MB-231 and MCF-7 pairs of breast cancer cells, respectively.…”