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Purpose: Metabolic reprogramming in breast cancer (BC) subtypes offers potential personalized treatment targets. Estrogen receptor α (ERα)-positive BC patients undergoing endocrine therapy (ET) can develop ET-resistant metastatic disease. Specific mutations, like Y537S in ERα, drive uncontrolled cell proliferation. Targeting mutant receptor levels shows promise for inhibiting growth in metastatic BC expressing ERα variants. Additionally, metabolic reprogramming occurs in ERα Y537S mutant cells. Consequently, we conducted a screen to identify metabolic proteins reducing intracellular levels of ERα Y537S and inhibiting cell proliferation., Methods: Nine metabolic proteins were identified in a siRNA-based screen, with phosphomannose mutase 2 (PMM2) showing the most promise. We measured the impact of PMM2 depletion on ERα stability and cell proliferation in ERα Y537S mutant cells. Additionally, we tested the effect of PMM2 reduction on the hyperactive phenotype of the mutant and its proliferation when combined with metastatic BC treatment drugs., Results: PMM2 emerged as a significant target due to its correlation with better relapse-free survival, overexpression in ERα-positive tumors, and its elevation in ERα Y537S-expressing cells. Depletion of PMM2 induces degradation of ERα Y537S, inhibits cell proliferation, and reduces ERα signaling. Notably, reducing PMM2 levels re-sensitizes ERα Y537S-expressing cells to certain ET drugs and CDK4/CDK6 inhibitors. Mechanistically, depletion of PMM2 leads to a reduction in ESR1 mRNA levels, resulting in decreased ERα receptor protein expression. Furthermore, the reduction of PMM2 decreases FOXA1 levels, which plays a crucial role in ERα regulation., Conclusions: Our findings establish PMM2 as an innovative therapeutic target for metastatic BC expressing the ERα Y537S variant, offering alternative strategies for managing and treating this disease., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Breast cancer (BC) is a leading cause of global cancer-related mortality in women, necessitating accurate tumor classification for timely intervention. Molecular and histological factors, including PAM50 classification, estrogen receptor α (ERα), breast cancer type 1 susceptibility protein (BRCA1), progesterone receptor (PR), and HER2 expression, contribute to intricate BC subtyping. In this work, through a combination of bioinformatic and wet lab screenings, followed by classical signal transduction and cell proliferation methods, and employing multiple BC cell lines, we identified enhanced sensitivity of ERα-positive BC cell lines to ALK and MELK inhibitors, inducing ERα degradation and diminishing proliferation in specific BC subtypes. MELK inhibition attenuated ERα transcriptional activity, impeding E2-induced gene expression, and hampering proliferation in MCF-7 cells. Synergies between MELK inhibition with 4OH-tamoxifen (Tam) and ALK inhibition with HER2 inhibitors revealed potential therapeutic avenues for ERα-positive/PR-positive/HER2-negative and ERα-positive/PR-negative/HER2-positive tumors, respectively. Our findings propose MELK as a promising target for ERα-positive/PR-positive/HER2-negative BC and highlight ALK as a potential focus for ERα-positive/PR-negative/HER2-positive BC. The synergistic anti-proliferative effects of MELK with Tam and ALK with HER2 inhibitors underscore kinase inhibitors' potential for selective treatment in diverse BC subtypes, paving the way for personalized and effective therapeutic strategies in BC management., (© 2024. The Author(s).)

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17β-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor LG declared a past co-authorship with the author FA., (Copyright © 2023 Cipolletti, Leone, Bartoloni and Acconcia.)

Previously, we found that telaprevir (Tel), the inhibitor of hepatitis C virus NS3/4A serine protease, reduces estrogen receptor α (ERα) content at the transcriptional level without binding to the receptor, prevents ERα transcriptional activity, and inhibits basal and 17β-estradiol (E2)-dependent cell proliferation in different breast cancer (BC) cell lines. Here, we further characterize the Tel action mechanisms on ERα levels and function, identify a possible molecular target of Tel in BC cells, and evaluate Tel as an antiproliferative agent for BC treatment. Tel-dependent reduction in ERα levels and function depends on a Tel-dependent decrease in FOXA1 levels and activity. The effect of Tel is transduced by the IGF1-R/AKT/FOXA1 pathway, with the antiviral compound interacting with IGF1-R. Tel prevents the proliferation of several BC cell lines, while it does not affect the proliferation of normal nontransformed cell lines, and its antiproliferative effect is correlated with the ratio of FOXA1/IGF1-R expression. In conclusion, Tel interferes with the IGF1-R/AKT/FOXA1 pathway and induces cell death in ERα-expressing BC cells. Thus, we propose that this antiviral could be repurposed for the treatment of ERα-expressing BC., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs., Methods:  ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan-Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed., Results: A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC., Conclusions: CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs., (© 2022. The Author(s).)

The expression of the α-subtype of Estrogen Receptor (ERα) characterizes most breast cancers (more than 75%), for which endocrine therapy is the mainstay for their treatment. However, a high percentage of ERα+ breast cancers are de novo or acquired resistance to endocrine therapy, and the definition of new targets for improving therapeutic interventions and the prediction of treatment response is demanding. Our previous data identified the ERα/AKT/neuroglobin (NGB) pathway as a common pro-survival process activated in different ERα breast cancer cell lines. However, no in vivo association between the globin and the malignity of breast cancer has yet been done. Here, we evaluated the levels and localization of NGB in ERα+ breast ductal carcinoma tissue of different grades derived from pre-and post-menopausal patients. The results indicate a strong association between NGB accumulation, ERα, AKT activation, and the G3 grade, while no association with the menopausal state has been evidenced. Analyses of the data set (e.g., GOBO) strengthen the idea that NGB accumulation could be linked to tumor cell aggressiveness (high grade) and resistance to treatment. These data support the view that NGB accumulation, mainly related to ER expression and tumor grade, represents a compensatory process, which allows cancer cells to survive in an unfavorable environment.

Aims: Nuclear factor (erythroid-derived 2)-like-2 factor (NRF-2) is a transcription factor well known to provide an advantage for cancer growth and survival regulating the cellular redox pathway. In breast cancer cells, we recently identified the monomeric heme-globin neuroglobin (NGB) as part of a new mechanism induced by the steroid hormone 17β-estradiol (E2) against oxidative stress. While there is mounting evidence suggesting a critical role of NGB as a sensor of oxidative stress, scarce information is available about its involvement in NRF-2 pathway activation in breast cancer cells. Results: Although NGB is not involved in the rapid E2-induced NRF-2 stability, E2 loses the capacity to regulate the expression of NRF-2-dependent genes in NGB-depleted MCF-7 cells. These data strongly sustain a role of NGB as a compensatory protein in the E2-activated intracellular pathway devoted to the increase of cancer cells tolerance to reactive oxygen species (ROS) generation in stressing conditions acting as key regulator of NRF-2 pathway activity in a time-dependent manner. Innovation: In this study, we identified a new role of NGB in the cell response to oxidative stress. Conclusion: Altogether, reported results open new insights on the NGB effect in regulating intracellular pathways related to cell adaptive response to stress and, as consequence, to cell survival, beyond its direct effect as ROS scavenger, opening new prospective in cancer therapeutic intervention.

Neuroglobin (NGB), an antiapoptotic protein upregulated by 17β-estradiol (E2), is part of E2/estrogen receptor α (ERα) pathway pointed to preserve cancer cell survival in presence of microenvironmental stressors including chemotherapeutic drugs. Here, the possibility that resveratrol (Res), an anticancer plant polyphenol, could increase the susceptibility of breast cancer cells to paclitaxel (Pacl) by affecting E2/ERα/NGB pathway has been evaluated. In MCF-7 and T47D (ERα-positive), but not in MDA-MB 231 (ERα-negative) nor in SK-N-BE (ERα and ERβ positive), Res decreases NGB levels interfering with E2/ERα-induced NGB upregulation and with E2-induced ERα and protein kinase B phosphorylation. Although Res treatment does not reduce cell viability by itself, this compound potentiates Pacl proapoptotic effects. Notably, the increase of NGB levels by NGB expression vector transfection prevents Pacl or Res/Pacl effects. Taken together, these findings indicate a new Res-based mechanism that acts on tumor cells impairing the E2/ERα/NGB signaling pathways and increasing cancer cell susceptibility to chemotherapeutic agent., (© 2018 Wiley Periodicals, Inc.)

Neuroglobin (NGB) is a relatively recent discovered monomeric heme-protein, which behave in neurons as a sensor of injuring stimuli including oxidative stress, hypoxia, and neurotoxicity. In addition, the anti-apoptotic activity of overexpressed NGB has been reported both in neurons and in cancer cell lines. We recently demonstrated that, NGB functions as a compensatory protein of the steroid hormone 17β-estradiol (E2) protecting cancer cells against the apoptotic death induced by oxidative stress. However, the E2-induced signaling pathways at the root of NGB over-expression and mitochondrial re-localization in breast cancer cells is still elusive. By using a kinase screening library, here, we report that: i) There is a strong positive correlation between NGB and ERα expression and activity in breast cancer cells; ii) The E2-activated phosphatidyl-inositol 3 kinase (PI3K)/protein kinase B (AKT) and protein kinase C (PKC) pathways are necessary to modulate the NGB protein levels; iii) The E2-induced persistent activation of AKT drive NGB to mitochondria; iv) Reactive oxygen species (ROS)-inducing compounds activating rapidly and transiently AKT does not affect the NGB mitochondrial level; and v) High level of NGB into mitochondria are necessary for the pro-survival and anti-apoptotic effect of this globin in cancer cells. As a whole, these results underline the E2 triggered pathways in E2-responsive breast cancer cells that involve NGB as a compensatory protein devoted to cancer cell survival., (© 2017 Wiley Periodicals, Inc.)

Environmental factors or adverse growth conditions that may reduce cell function or viability are considered stress. The cell ability to sense and respond to environmental stresses determine its function and survival destiny. We recently defined Neuroglobin (NGB), a heme-protein, as a compensatory protein in the 17β-Estradiol (E2) anti-apoptotic activity and as a sensor of oxidative stress in both neurons and breast cancer cells. Here, the possibility that NGB levels could represent a pivotal regulator of integrated response of cancer cells to stress has been evaluated. Data obtained in neuroblastoma and in breast cancer cell lines evidence that nutrient deprivation significantly up-regulated NGB levels at different time points. However, the analysis of autophagy activation led to exclude any possible role of stress- or E2-induced NGB in the upstream regulation of general autophagy. However, the over-expression of Flag-NGB in ERα stable transfected HEK-293 cells completely affects nutrient deprivation-induced decrease in cell number. In addition, reported results indicate that modulation of the anti-apoptotic Bcl-2 level may play a key role in the protective NGB function against energetic stress. Overall, these data define a role of NGB as compensatory protein in the cell machinery activated in response to stress and as general stress adaptation marker of cancer cells susceptible to oxidative stress, oxygen and, as demonstrated here for the first time, even to nutrient willingness. Despite the lacking of any direct NGB role on autophagic flux activated by energetic stress, NGB upregulation appears functional in delaying stress-related cell death allowing an appropriate cell response and adaptation to the changing extracellular conditions.

Although paclitaxel (Taxol) is an active chemotherapeutic agent for the treatment of breast cancer, not all breast tumors are sensitive to this drug. In particular, there is a wide agreement on the low sensitivity of estrogen receptor (ER) α-positive breast cancer to paclitaxel treatment. However, the ERα-based insensitivity to paclitaxel is still elusive. Here, the effect of the E2/ERα-dependent upregulation of neuroglobin (NGB), an antiapoptotic globin, on the reduced sensitivity of breast cancer cells to paclitaxel-induced apoptosis has been evaluated in ERα-containing MCF-7 cells. The E2 pretreatment enhances the ERα activity and significantly impairs paclitaxel-induced apoptosis as evaluated by Annexin V assay and PARP-1 cleavage. NGB displays a pivotal role in the E2/ERα-induced antiapoptotic pathway to abrogate paclitaxel-induced cell death in stable NGB-silenced MCF-7 cell clones. Moreover, in the absence of the active ERα, paclitaxel significantly reduces the NGB cell content. In conclusion, these results highlight the involvement of ERα activation and of E2/ERα-dependent NGB upregulation in the insensitivity of MCF-7 to paclitaxel. These novel findings could have important implications in the development of targeted therapeutics for overcoming paclitaxel insensitivity in ERα-positive human breast cancer. © 2016 IUBMB Life, 68(8):645-651, 2016., (© 2016 International Union of Biochemistry and Molecular Biology.)

The over-expression of human neuroglobin (NGB), a heme-protein preferentially expressed in the brain, displays anti-apoptotic effects against hypoxic/ischemic and oxidative stresses enhancing neuron survival. As hypoxic and oxidative stress injury frequently occurs in fast proliferating neoplastic tissues, here, the effect of these stressors on the level, localization, and anti-apoptotic function of NGB in wild type and NGB-stable-silenced MCF-7 breast cancer cells has been assessed. The well-known endogenous NGB inducer 17β-estradiol (E2) has been used as positive control. The median pO2 present in tumor microenvironment of breast cancer patients (i.e., 2% O2) does not affect the NGB level in breast cancer cells, whereas hydrogen peroxide and lead(IV) acetate, which increase intracellular reactive oxygen species (ROS) level, enhance the NGB levels outside the mitochondria and still activate apoptosis. However, E2-induced NGB up-regulation in mitochondria completely reverse lead(IV) acetate-induced PARP cleavage. These results indicate that the NGB level could represent a marker of oxidative-stress in MCF-7 breast cancer cells; however, the NGB ability to respond to injuring stimuli by preventing apoptosis requires its re-allocation into the mitochondria. As a whole, present data might lead to a new direction in understanding NGB function in cancer opening new avenues for the therapeutic intervention.