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Abstract Background The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. Results Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. Conclusions The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase’s role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

Simple Summary: Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults and has one of the poorest survival rates of all cancers. The disease is often pre-cursed by myelodysplastic neoplasms (MDS), a more benign class of diseases that can last many years but also carry a high risk of progression to AML. Diagnosis requires histological examination of a bone marrow biopsy, an invasive procedure that can be influenced by sampling variations and subjective enumeration. A circulating biomarker that could be measured as part of a routine peripheral blood test would provide a much quicker, safer, and reliable alternative. This study examines the potential use of α-enolase (ENO1) as a potential biomarker and shows that it is highly expressed in bone marrow biopsies from patients with both MDS and AML, and that patients with these diseases also have decreased circulating levels of autoantibodies to ENO1. In solid tumours, high expression of the glycolytic enzyme, α-enolase (ENO1), predicts for poor patient overall survival (OS), and circulating autoantibodies to ENO1 correlate positively with diagnosis and negatively with advanced disease. Although ENO1 is one of the most highly expressed genes in acute myeloid leukaemia (AML), its potential role as a biomarker in AML or its precursor, myelodysplastic neoplasms (MDS), has not been investigated. A meta-analysis of nine AML online datasets (n = 1419 patients) revealed that high ENO1 expression predicts for poor OS (HR = 1.22, 95% CI: 1.10–1.34, p < 0.001). Additionally, when compared to AML in remission (n = 5), ENO1 protein detected by immunohistochemistry was significantly higher at diagnosis in bone marrow from both AML (n = 5, p < 0.01) and MDS patients (n = 12, p < 0.05), and did not correlate with percentage of blasts (r = 0.28, p = 0.21). AML patients (n = 34) had lower circulating levels of ENO1 autoantibodies detected by ELISA compared to 26 MDS and 18 controls (p = 0.003). However, there was no difference in OS between AML patients with high vs. low levels of anti-ENO1 autoantibodies (p = 0.77). BM immunostaining for ENO1 and patient monitoring of anti-ENO1 autoantibody levels may be useful biomarkers for MDS and AML. [ABSTRACT FROM AUTHOR]

Background: The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer., Results: Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins., Conclusions: The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase., (© 2024. The Author(s).)