Abstract
Cellular proteins are subject to frequent methylation on lysine residues, introduced by specific methyltransferases, and each lysine residue can receive up to three methyl groups. Histone methylations, which are key determinants of chromatin state and transcriptional status, have been subject to particularly intense studies, but methylations on non-histone protein substrates are also abundant and biologically significant. Numerous studies have addressed lysine methylation in the realm of cancer biology. A recent study used an antibody-based approach to investigate the methylation of Lys-561 of the stress-inducible Hsp70 protein HSPA1, focusing exclusively on dimethylated HSPA1, concluding that it was elevated in cancer [Cho et al. (2012), Nat. Commun.,3, 1072]. In the present study, we have performed a more extensive analysis of HSPA1 methylation status in cancer samples, using protein mass spectrometry. We found that the four methylation states of Lys561 on HSPA1 (un-, mono-, di- and trimethylated) could be measured accurately and reproducibly in samples from carcinomas. We investigated HSPA1 methylation in 70 effusions, representing 53 high-grade serous ovarian carcinomas and 17 breast carcinomas. Notably, we found the trimethylated form of HSPA1 to be predominant in the cancer samples. HSPA1 methylation was studied for association with clinicopathologic parameters, including chemotherapy response and survival. The trimethylated form was more prevalent in breast carcinoma effusions (p = 0.014), whereas the dimethylated (p = 0.025), monomethylated (p = 0.004) and unmethylated (p = 0.021) forms were overrepresented in the ovarian carcinomas. For the ovarian carcinomas, the monomethylated (p = 0.028) and unmethylated (p = 0.007) forms were significantly related to the presence of higher residual disease volume, while the unmethylated form was significantly associated with poor overall (p = 0.015) and progression-free (p = 0.012) survival. In conclusion, lysine methylation of HSPA1 differs between metastatic breast and ovarian carcinoma, and unmethylated HSPA1 shows potential as a prognostic marker in high-grade serous carcinoma.
Highlights
The altered properties of cancer cells, compared with normal cells, mainly result from perturbation of various cellular signaling pathways, mediated by mutation or altered expression of genes encoding signaling-associated proteins
The four possible methylation states of the Lys561-containing Asp-N-generated peptide encompassing residues 555–565 in HSPA1 were analyzed by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS)
From the resulting extracted ion chromatograms, the areas under the relevant peaks were used to calculate the relative amounts of the four methylation states, as well as the number of methyl groups on Lys-561 in HSPA1
Summary
The altered properties of cancer cells, compared with normal cells, mainly result from perturbation of various cellular signaling pathways, mediated by mutation or altered expression of genes encoding signaling-associated proteins. Lysine methylation has been intensively studied in the context of histone proteins, which are important components of chromatin. Histone lysine methylation and protein phosphorylation share several notable features: i) the modifications can be reversed by specific enzymes, i.e. lysine demethylases and protein phosphatases, respectively; ii) so-called reader domains can recognize the modified (or sometimes unmodified) residue; iii) genes that encode proteins responsible for introducing, recognizing or removing such modifications are frequently mutated or over-expressed in cancer, and have attracted considerable attention as drug targets and diagnostic/prognostic markers
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