Abstract Osteosarcoma stem cells (OSCs) are characterized by their self-renewal and multilineage differentiation abilities, contributing to osteosarcoma (OS) malignancy. The Warburg effect describes cancer cells’ preference for glycolysis over mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Unlike differentiated cancer cells, cancer stem cells exhibit unique and diverse metabolic properties depending on the context. This study investigated the metabolic reliance of OSCs and related genes through in silico analyses of clinical OS specimens and in vitro and in vivo genetic and pharmacological analyses. Glycolysis and OXPHOS pathways were more active in OSCs than in non-OSCs at single-cell resolution. Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme balancing glycolysis and OXPHOS, was upregulated in OSCs and correlated with poor prognosis in patients with OS. Genetic inhibition of PDK1 via RNA interference reduced OSC stemness, tumorigenicity, and glycolysis. Pharmacological inhibition of PDK1 mirrored these genetic effects. Activating transcription factor 3 (ATF3) was identified through screening as a downstream factor of PDK1-regulated OSC properties. Silencing ATF3 reduced OSC stemness, while ATF3 overexpression reversed the stemness reduction caused by PDK1 deficiency. ATF3 expression, glycolysis, and stemness were significantly induced by wild-type PDK1 overexpression but not by a kinase-dead PDK1 mutant in OSCs. Pharmacological inhibition of glycolysis counteracted the upregulation of ATF3 expression and increased stemness in OSCs by PDK1 overexpression. These findings indicate that PDK1 fine-tunes metabolic balance to govern OSC stemness and tumorigenicity through ATF3, suggesting a potential therapeutic approach for targeting OSCs in OS.

Although multimodality therapy has recently advanced, patients with glioblastoma, one of the most aggressive and deadly types of central nervous system cancer, have a very poor prognosis and rare long-term survival. Vitamins are essential organic nutrients that play a pivotal role in maintaining homeostasis, and various studies have demonstrated the implication of vitamins in the pathophysiology of gliomas. Herein, we aimed to investigate the association of the vitamin metabolic pathway and the corresponding candidate genes for the malignancy, aggressiveness, and poor prognosis of gliomas using The Cancer Genome Atlas database of patients with gliomas. We demonstrated that fat-soluble vitamin metabolic processes are prominently associated with glioma grade, molecular biomarkers, molecular subtypes, and clinical outcomes. Moreover, we identified the key genes related to the fat-soluble metabolic pathway in gliomas using differentially expressed gene analysis. Among them, the expression of the vitamin K epoxide reductase complex subunit 1 (VKORC1), encoding VKOR essential for the vitamin K-dependent γ-carboxylation of target proteins, was prominently associated with not only malignancy, aggressiveness, and poor prognosis of gliomas but also the representative signal pathways related to glioma pathogenesis. Moreover, the inactivation of Vkorc1 by RNA interference decreased the proliferation and migration potential of glioma cells in vitro. Collectively, these findings reveal the pivotal role of fat-soluble vitamin and vitamin K metabolic processes in the pathophysiology of gliomas, thereby identifying a potential target for drug development for the treatment of malignant gliomas.

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR–mediated signaling during T cell development determines the diversity of the TCRβ repertoire available for positive and negative selection, and hence of the final αβTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling–dependent and repertoire–shaping role for β-selection beyond selection of in-frame rearranged TCRβ chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

Prostate cancer (PCa) is the fifth leading cause of cancer related deaths worldwide, in part due to a lack of molecular stratification tools that can distinguish primary tumours that will remain indolent from those that will metastasise. Amongst potential molecular biomarkers, microRNAs (miRs) have attracted particular interest because of their high stability in body fluids and fixed tissues. These small non-coding RNAs modulate several physiological and pathological processes, including cancer progression. Herein we explore the prognostic potential and the functional role of miRs in localised PCa and their relation to nodal metastasis. We define a 7-miR signature that is associated with poor survival independently of age, Gleason score, pathological T state, N stage and surgical margin status and that is also prognostic for disease-free survival in patients with intermediate-risk localised disease. Within our 7-miR signature, we show that miR-378a-3p (hereafter miR-378a) levels are low in primary tumours compared to benign prostate tissue, and also lower in Gleason score 8-9 compared to Gleason 6-7 PCa. We demonstrate that miR-378a impairs glucose metabolism and reduces proliferation in PCa cells through independent mechanisms, and we identify glucose transporter 1 (GLUT1) messenger RNA as a direct target of miR-378a. We show that GLUT1 inhibition hampers glycolysis, leading to cell death. Our data provides a rational for a new PCa stratification strategy based on miR expression, and it reveals that miR-378a and GLUT1 are potential therapeutic targets in highly aggressive glycolytic PCa.
© 2022. The Author(s).

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.