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Dual role of catechol-O-methyltransferase in the context of breast cancer heterogeneity

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JANÁČOVÁ Lucia FAKTOR Jakub ČÁPKOVÁ Lenka KNOPFOVÁ Lucia BENEŠ Petr FABIAN Pavel BOUCHAL Pavel

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Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Popis Introduction: Catechol-O-methyltransferase (COMT) plays an essential role in detoxification of catechols by transferring the methyl group from S-adenosyl-L-methionine to the substrate. In breast cancer, it catalyzes methylation of estrogen metabolites to block their estrogenicity which prevents their oxidation to carcinogenic quinones. In this study we asked whether this tumor suppressor role is limited to estrogen receptor (ER) dependent breast cancer, or whether it has a general validity. Material and methods: Analysis of migration and invasiveness of MCF-7 cells, a luminal A model, stably transfected with COMT coding sequences (MCF-7 COMT cells) was performed using Transwell assay. Atomic force microscopy was performed to evaluate cell stiffness in response to COMT expression. Pull-down assay with mass spectrometry identification was used to identify protein-protein interactions of COMT. Effect of potential anti-metastatic inhibitors on COMT expression was determined by crystal violet assay. A differential cell surface proteome analysis via SILAC-LC-MS quantification was performed on MDA-MB-231 breast cancer cell clone selected for higher migration capacity compared to the maternal cell line. The protein levels of COMT in different breast cancer subtypes were determined by sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry (96 patients) and by immunohistochemistry (422 patients). Results and discussions: Overexpression of COMT in MCF-7 cells decreased cell migration and invasiveness, supporting tumor suppressor role in estrogen receptor dependent breast cancer. MCF-7 COMT cells also exhibited anti-metastatic phenotype in AFM measurements showing higher cell stiffness than parental MCF-7 cells. Analysis of protein-protein interactions revealed 7-dehydrocholesterol reductase, a protein involved in cholesterol and subsequently estrogen synthesis, as COMT interacting partner in MCF-7 COMT cells. On the other hand, COMT deregulation after cell treatment with STAT3, TNFalpha; and IL-1 inhibitors suggested its sensitivity to potential anti-metastatic inhibitors even in MCF-7 cells. Moreover, a quantitative cell surface proteomics identified the membrane-bound isoform of COMT as a cell surface protein overexpressed in a more migrating clone of MDA-MB-231 cells compared to maternal MDA-MB-231 cells, indicating the association of COMT with cell motility in triple negative breast cancer. Analysis of 96 primary breast tumors using SWATH-MS proteomics showed increased COMT protein levels in more aggressive tumors: in grade 3 vs. grade 1, in luminal B vs. luminal A, in triple negative vs. luminal A, and in triple negative lymph node positive vs. triple negative lymph node negative tumors. Association between increased COMT protein levels and tumor grade was also confirmed by immunohistochemistry. Conclusions: Based on our results, we hypothesize a dual role of COMT in breast cancer: while known tumor suppressor role associated with the detoxifying role in estrogen metabolism is further supported by our data in ER+ breast cancer cells, in ER- cells and generally in tissues COMT seems to be associated with more aggressive phenotype. It is thus confirmed that the dominance of catechol estrogen methylation activity is limited to ER dependent breast cancer. The newly evidenced association of COMT with more aggressive phenotype, irrespectively of ER status, may alter the perspective how COMT is viewed as a potential therapeutic target in the context of breast cancer heterogeneity.
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