Carbonic Anhydrase Inhibitors Induce Ferroptosis through Inhibition of AKT/FTH1 Signaling in Ewing Sarcoma Tumor Cells.

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.

Distinct Traits of Structural and Regulatory Evolutional Conservation of Human Genes with Specific Focus on Major Cancer Molecular Pathways.

The evolution of protein-coding genes has both structural and regulatory components. The first can be assessed by measuring the ratio of non-synonymous to synonymous nucleotide substitutions. The second component can be measured as the normalized proportion of transposable elements that are used as regulatory elements. For the first time, we characterized in parallel the regulatory and structural evolutionary profiles for 10,890 human genes and 2972 molecular pathways. We observed a ~0.1 correlation between the structural and regulatory metrics at the gene level, which appeared much higher (~0.4) at the pathway level. We deposited the data in the publicly available database RetroSpect. We also analyzed the evolutionary dynamics of six cancer pathways of two major axes: Notch/WNT/Hedgehog and AKT/mTOR/EGFR. The Hedgehog pathway had both components slower, whereas the Akt pathway had clearly accelerated structural evolution. In particular, the major hub nodes Akt and beta-catenin showed both components strongly decreased, whereas two major regulators of Akt TCL1 and CTMP had outstandingly high evolutionary rates. We also noticed structural conservation of serine/threonine kinases and the genes related to guanosine metabolism in cancer signaling: GPCRs, G proteins, and small regulatory GTPases (Src, Rac, Ras); however, this was compensated by the accelerated regulatory evolution.

Identification of Factors Driving Doxorubicin-Resistant Ewing Tumor Cells to Survival.

BACKGROUND: Ewing sarcoma (ES) cells exhibit extreme plasticity that contributes to the cell's survival and recurrence. Although multiple studies reveal various signaling pathways mediated by the EWSR1/FLI1 fusion, the specific transcriptional control of tumor cell resistance to doxorubicin is unknown. Understanding the molecular hubs that contribute to this behavior provides a new perspective on valuable therapeutic options against tumor cells. METHODS: Single-cell RNA sequencing and LC-MS/MS-based quantitative proteomics were used. RESULTS: A goal of this study was to identify protein hubs that would help elucidate tumor resistance which prompted ES to relapse or metastasize. Several differentially expressed genes and proteins, including adhesion, cytoskeletal, and signaling molecules, were observed between embryonic fibroblasts and control and doxorubicin-treated tumor cell lines. While several cancer-associated genes/proteins exhibited similar expression across fibroblasts and non-treated cells, upregulation of some proteins belonging to metabolic, stress response, and growth pathway activation was uniquely observed in doxorubicin-treated sarcoma cells, respectively. The novel information on differentially expressed genes/proteins provides insights into the biology of ES cells, which could help elucidate mechanisms of their recurrence. CONCLUSIONS: Collectively, our results identify a novel role of cellular proteins in contributing to tumor cell resistance and escape from doxorubicin therapy and contributing to ES progression.

MicroRNA 345 (miR345) regulates KISS1-E-cadherin functional interaction in breast cancer brain metastases.

Brain metastases manifest the advanced stage of breast cancer disease with poor prognosis for patient survival. Recent reports demonstrate that some therapeutic agents can activate the expression of several breast cancer-associated genes, whose products are involved in the onset and development of brain metastases. In this study, we discovered a functional link between KISS1 and E-cadherin that could be observed in both primary brain metastatic lesions and paired cell lines, such as parental CN34TGL and MDA-MB-231 and their respective brain metastatic subclones CN34Brm2Ctgl and MDA-MB-231Br. Remarkably, expression of KISS1 and E-cadherin genes consistently showed an inverse correlation in all of the above cell/tissue types. While E-cadherin expression was strongly upregulated in metastatic clones isolated from blood and brain, the levels of this protein in parental MDA-MB-231 cell line was low. Furthermore, E-cadherin upregulation can be artificially induced in MDA-MB-231Br and CN34Brm2Ctgl cell populations by knocking down KISS1 expression directly or through overexpressing the miR345 mimic. In the aggregate, our data suggest that the tumor microenvironment, which controls breast cancer spreading via miR345-regulated KISS1 expression, might modulate metastatic spreading by a mechanism(s) involving upregulation of E-cadherin production.