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

Abstract

Radiotherapy constitutes a major therapeutic modality for early management of breast cancer. Despite the high efficacy in treating breast cancer (BC), radiation resistance and tumor recurrence are major hurdles in breast cancer radiotherapy. Herein, stable isotope labeling by amino acids in cell culture (SILAC) was employed, along with the parallel-reaction monitoring (PRM)-based targeted quantitative proteomic method, to examine the differences in kinase protein expression in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones. We quantified the relative protein expression levels of 300 and 281 kinases in C5/MDA-MB-231 and C6/MCF-7 pairs of breast cancer cells, respectively. We also showed that TAF9, which was one of the differentially expressed kinases, enhances radiation resistance in breast cancer cells. Moreover, a correlation analysis of gene expression suggested TAF9’s role in upregulating the expression of genes involved with radioresistance. Overall, our study uncovered a large number of differentially expressed kinases accompanied with the acquisition of radioresistance and revealed a role of TAF9 in promoting radioresistance in breast cancer.

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).…”

Targeted Proteomic Analysis of Small GTPases in Radioresistant Breast Cancer Cells

“…Clonogenic survival assay was performed as described previously. , Briefly, control shRNA- and shARFRP1-treated MCF-7 and MDA-MB-231 cells were plated in triplicate in six-well plates at a concentration of 300 cells per well for 0 and 1.5 Gy treatment, and 600 cells per well for 3 and 5 Gy treatment. X-rays were delivered using a Rad Source RS-2000 cabinet irradiator (Rad Source Technologies, Buford, GA) followed by a 10-day incubation.…”

Deregulations of RNA Pol II Subunits in Cancer

“…Furthermore, POLR2H mRNA levels are significantly higher in lung cancer tissue samples compared to normal samples [126], and its high expression significantly correlates with patients' survival in lung squamous cell carcinoma [127,128]. Interestingly, more recent data showed that POLR2H is upregulated in a subset of breast cancer patients resistant to radiotherapy, potentially implicating its role in the acquisition of radio-resistance [129,130].…”