14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC.

Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins produce a mixture of aspartyl and isoaspartyl residues, the latter being involved in protein aging and inactivation. Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are known to be able to distinguish the isoaspartyl peptides by unique fragments of cn* + 58.0054 (C2H2O2) and z(l-n)-56.9976 (C2HO2), where n is the position of the aspartyl residue and l is the peptide length. In the present study, we tested the specificity of isoAsp detection using the accurate masses of the specific fragments. For this purpose, we analyzed 32 whole and partial proteomes obtained from human cells as well as tissue samples and identified by ECD 466 isoaspartyl peptide candidates. Detailed inspection revealed that many of these candidates were unreliable. To increase the isoAsp detection specificity, additional criteria had to be used, for example, adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak. Most stringent filtering of candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates for l-isoaspartyl methyltransferase, an important repair enzyme converting isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.