In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.

Glioblastoma is a highly aggressive and lethal brain tumor, with limited treatment options. Abnormal activation of the neddylation pathway is observed in glioblastoma, and the NEDD8-activating enzyme (NAE) inhibitor, MLN4924, was previously shown to be effective in glioblastoma cell line models. However, its effect has not been tested in patient-derived glioblastoma stem cells. We first analyzed public data to determine whether NEDD8 pathway proteins are important in glioblastoma development and patient survival. NAE1 and UBA3 levels increased in glioblastoma patients; high NEDD8 levels were associated with poor clinical outcomes. Immunohistochemistry results also supported this result. The effects of MLN4924 were evaluated in 4 glioblastoma cell lines and 15 patient-derived glioblastoma stem cells using high content analysis. Glioblastoma cell lines and patient-derived stem cells were highly susceptible to MLN4924, while normal human astrocytes were resistant. In addition, there were various responses in 15 patient-derived glioblastoma stem cells upon MLN4924 treatment. Genomic analyses indicated that MLN4924 sensitive cells exhibited enrichment of Extracellular Signal Regulated Kinase (ERK) and Protein kinase B (AKT, also known as PKB) signaling. We verified that MLN4924 inhibits ERK and AKT phosphorylation in MLN4924 sensitive cells. Our findings suggest that patient-derived glioblastoma stem cells in the context of ERK and AKT activation are sensitive and highly regulated by neddylation inhibition.

Breast cancer has a high risk of recurrence and distant metastasis after remission. Controlling distant metastasis is important for reducing breast cancer mortality, but accomplishing this goal remains elusive. In this study, we investigated the molecular pathways underlying metastasis using cells that mimic the breast cancer distant metastasis process. HCC1143 breast cancer cells were cultured under two-dimensional (2D)-adherent, tumor sphere (TS), and reattached (ReA) culture conditions to mimic primary tumors, circulating tumor cells, and metastasized tumors, respectively. ReA cells demonstrated increased TS formation and enhanced invasion capacity compared to the original 2D-cultured parental cells. In addition, ReA cells had a higher frequency of ESA⁺CD44⁺CD24⁻ population, which represents a stem-cell-like cell population. RNA sequencing identified the cholesterol synthesis pathway as one of the most significantly increased pathways in TS and ReA cells compared to parental cells, which was verified by measuring intracellular cholesterol levels. Furthermore, the pharmacological inhibition of the cholesterol synthesis pathway decreased the ability of cancer cells to form TSs and invade. Our results suggest that the cholesterol synthesis pathway plays an important role in the distant metastasis of breast cancer cells by augmenting TS formation and invasion capacity.

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

(1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.