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We are grateful to Kenny Milne, Douglas Lamont and the FingerPrints Proteomics Facility at the University of Dundee for mass spectrometry. We thank Dr. Reyes Sanles-Falagan for critical reading of the manuscript. Human tissue samples were obtained with the support of the MD Anderson Foundation Biobank (record number B.0000745, ISCIII National Biobank Record). Research and publication of this work was funded by the EMERGIA 2021 program (EMERGIA21_00124) from the Consejeria de Universidad, Investigacion e Innovacion, Junta de Andalucia, Spain to J.M-F. J.M-F. was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie programme (101025429), the Ministerio de Ciencia, Innovacion y Universidades, Agencia Estatal de Investigacion (MICINN-AEI, RYC2021-032389), the Consejeria de Universidad, Investigacion e Innovacion under the EMERGIA program (EMC21_00124) Junta de Andalucia, and the VII PPIT of the University of Seville (2023/00000479). RVD and G.M-B were supported by MICINN-AEI and European Regional Development Fund (project PID2021-124251OB-I00 and PID2022-136854OB-I00, respectively). G.M-B was also funded by The Instituto de Salud Carlos III IISCIII, CIBERONC (CB16/12/00295) and ERA PerMed ERA-NET co-funded by the NextGeneration-EU (ISCIII and Fundacion cientifica AECC, AC21_2/00020). PH was supported by R+D+I grant (PID2019-104195G) from the Spanish Ministry of Science and Innovation-Agencia Estatal de Investigacion/10.13039/501100011033. CW was supported by a Welcome Trust Programme Grant.

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Radiotherapy resistance driven by Asparagine endopeptidase through ATR pathway modulation in breast cancer

Publicated to:JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH. 44 (1): 74- - 2025-02-27 44(1), DOI: 10.1186/s13046-025-03334-6

Authors: Morillo-Huesca, Macarena; Lopez-Cepero, Ignacio G; Conesa-Bakkali, Ryan; Tome, Mercedes; Watts, Colin; Huertas, Pablo; Moreno-Bueno, Gema; Duran, Raul V; Martinez-Fabregas, Jonathan

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Abstract

BackgroundTumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear.MethodsHigh-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify protein and mRNA levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings.ResultsHere we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients resulted in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients.ConclusionThe newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.

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