Lidija Milković

Chronic Kidney Disease (CKD) is a global health crisis, projected to be the fifth leading cause of death by 2040. Its progression is driven by a reinforcing loop of mitochondrial dysfunction, oxidative stress, and chronic inflammation. The AMPK-NRF2–FOXO axis serves as a central “redox-metabolic rheostat” that maintains renal homeostasis but is commonly dysfunctional in CKD. Herein, we explore the molecular crosstalk within this network, where AMPK acts as a metabolic and redox sensor, NRF2 governs the cytoprotective response, and FOXO isoforms regulate autophagy, antioxidative defense, and senescence. We highlight the functional paradoxes within the axis and evaluate the benefits and drawbacks of nutraceuticals and pharmacological agents, such as NRF2 inducer bardoxolone methyl, underscoring the necessity for context-dependent modulation. Furthermore, we examine the AMPK–NRF2–FOXO axis within the current clinical management, according to the 2024/2026 KDIGO guidelines. These guidelines reflect a shift toward a multi-targeted pharmacological approach involving metformin, SGLT2 inhibitors, GLP-1 receptor agonists, finerenone, and hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors.

Background: The thymus is the central hub of T-cell differentiation, where epithelial cells guide the process of their maturation. Objective: Our goal was to identify and describe progenitor cells within the human thymus that can differentiate into epithelial cells. Methods: When we plated enriched thymic cells in 3D culture conditions, rare individual cells capable of self-renewal and differentiation formed spheroids. Results: Both neonatal and adult thymuses produced similar numbers of spheroids, suggesting that progenitor potential remains consistent across age groups. Some cells within the spheres express genes typical of mature epithelial cells, while others express genes associated with the immature compartment active during thymic organogenesis. However, there were also cells expressing PDGFRβ. We treated the tissues with 2-deoxyguanosine before digestion, which improved the yield of progenitor cells. We also cultured the enriched stromal thymocytes with Cyr61 and Interleukin-22, which affected the spheroid size. Conclusions: Our efforts towards thymic reconstitution are ongoing, but our research uncovers previously unknown characteristics of the elusive epithelial progenitor population.

Breast cancer is a heterogeneous disease with varying responses to therapies. While targeted treatments have advanced, conventional therapies inducing oxidative stress remain widely used. H₂O₂ has emerged as a therapeutic candidate due to its role in signaling and cell-function regulation. Its transport is tightly regulated through peroxiporins such as AQP5, expression of which is linked to poor prognosis and metastatic spread, and its role in therapy resistance remains underexplored. This study examined AQP5’s role in the acute oxidative stress response. We overexpressed AQP5 in breast cancer cell lines with low basal levels—HR+ (MCF7), HER2+ (SkBr-3), and TNBC (SUM 159)—and exposed them to H₂O₂ for 24 h. We assessed cell viability, intracellular ROS, changes in AQP3 and AQP5, and key antioxidative and cancer-related pathways (NRF2, PI3K/AKT, FOXOs). AQP5 overexpression elicited a cell-type-specific response. H₂O₂ treatment reduced viability in SkBr-3-AQP5 and MCF7-AQP5 cells, increased ROS levels in MCF7-AQP5, and decreased ROS in SUM 159-AQP5. It also increased AQP3 in MCF7-AQP5 and differentially affected NRF2, FOXOs, and PI3K/AKT signaling, notably activating NRF2/AKR1B10 axis in MCF7-AQP5 and decreasing FOXO1 in SUM 159-AQP5. These findings highlight the need for further research into AQP5’s role in the oxidative stress response in breast cancer cells.

The thymus is the central hub of T-cell differentiation, where epithelial cells guide the process of their maturation. Our goal was to identify and describe progenitor cells within the human thymus that can differentiate into epithelial cells. When we plated enriched thymic cells in 3D culture conditions, rare individual cells capable of self-renewal and differentiation formed spheroids. Both neonatal and adult thymuses produced similar numbers of spheroids, suggesting that progenitor potential remains consistent across age groups. Some cells within the spheres express genes typical of mature epithelial cells, while others express genes associated with the immature compartment active during thymic organogenesis. However, there were also cells expressing PDGFRβ. We treated the tissues with 2-deoxyguanosine before digestion, which improved the yield of progenitor cells. We also cultured the enriched stromal thymocytes with Cyr61 and Interleukin-22, which affected the spheroid size. Our efforts towards thymic reconstitution are ongoing, but our research uncovers previously unknown characteristics of the elusive epithelial progenitor population.

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H₂O₂ and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, partly due to their involvement in H₂O₂ transport. Oxidative stress is linked to breast cancer development but is also a mechanism of action for conventional chemotherapy. The aim of this study is to investigate the effects of prolonged oxidative stress on Aquaporin 3 (AQP3), Aquaporin 5 (AQP5), and signaling pathways in breast cancer cell lines of different malignancies alongside a non-tumorigenic breast cell line. The prolonged oxidative stress caused responses in viability only in the cancer cell lines, while it affected cell migration in the MCF7 cell line. Changes in the localization of NRF2, a transcription factor involved in oxidative stress response, were observed only in the cancer cell lines, and no effects were recorded on its downstream target proteins. Moreover, the prolonged oxidative stress caused changes in AQP3 and AQP5 expression only in the cancer cell lines, in contrast to their non-malignant counterparts. These results suggest peroxiporins are potential therapeutic targets in cancer treatment. However, further research is needed to elucidate their role in the modulation of therapy response, highlighting the importance of research on this topic.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

Breast cancer is still the leading cause of death in women of all ages. The reason for this is therapy resistance, which leads to the progression of the disease and the formation of metastases. Multidrug resistance (MDR) is a multifactorial process that leads to therapy failure. MDR involves multiple processes and many signaling pathways that support each other, making it difficult to overcome once established. Here, we discuss cellular-oxidative-stress-modulating factors focusing on transcription factors NRF2, FOXO family, and peroxiporins, as well as their possible contribution to MDR. This is significant because oxidative stress is a consequence of radiotherapy, chemotherapy, and immunotherapy, and the activation of detoxification pathways could modulate the cellular response to therapy and could support MDR. These proteins are not directly responsible for MDR, but they support the survival of cancer cells under stress conditions.

Breast cancer (BC) and ovarian cancer (OC) are among the most common and deadly cancers affecting women worldwide. Both are complex diseases with marked heterogeneity. Despite the induction of screening programs that increase the frequency of earlier diagnosis of BC, at a stage when the cancer is more likely to respond to therapy, which does not exist for OC, more than 50% of both cancers are diagnosed at an advanced stage. Initial therapy can put the cancer into remission. However, recurrences occur frequently in both BC and OC, which are highly cancer-subtype dependent. Therapy resistance is mainly attributed to a rare subpopulation of cells, named cancer stem cells (CSC) or tumor-initiating cells, as they are capable of self-renewal, tumor initiation, and regrowth of tumor bulk. In this review, we will discuss the distinctive markers and signaling pathways that characterize CSC, their interactions with the tumor microenvironment, and the strategies they employ to evade immune surveillance. Our focus will be on identifying the common features of breast cancer stem cells (BCSC) and ovarian cancer stem cells (OCSC) and suggesting potential therapeutic approaches.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H₂O₂ uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2–related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

It is well known that oxidative stress and lipid peroxidation (LPO) play a role in physiology and pathology. The most studied LPO product with pleiotropic capabilities is 4-hydroxynonenal (4-HNE). It is considered as an important mediator of cellular signaling processes and a second messenger of reactive oxygen species. The effects of 4-HNE are mainly attributed to its adduction with proteins. Whereas the Michael adducts thus formed are preferred in an order of potency of cysteine > histidine > lysine over Schiff base formation, it is not known which proteins are the preferred targets for 4-HNE under what physiological or pathological conditions. In this review, we briefly discuss the methods used to identify 4-HNE–protein adducts, the progress of mass spectrometry in deciphering the specific protein targets, and their biological relevance, focusing on the role of 4-HNE protein adducts in the adaptive response through modulation of the NRF2/KEAP1 pathway and ferroptosis.