Circulating tumor cells (CTCs) are essential for the establishment of distant metastasis. Numerous studies have characterized CTCs as metastatic precursors; however, the molecular nature of CTCs has not been completely revealed yet due to the low number of CTCs in the blood stream. As an alternative approach, we developed a long-term suspension cell culture model using human breast cancer cell lines to mimic CTCs. We found that more than 40 passaged suspension cells acquired the ability to enhance metastasis like cancer stem cells. To identify molecular changes acquired during the suspension cell culture, we analyzed metabolic and lipidomic profiles as well as transcriptome in MDA-MB-468 suspension cells. Glutamate and leucine levels increased in suspension cells, and cholesterol synthesis pathway was altered. The inhibition of glutamate metabolic pathway decreased the proliferation of suspension cells compared to that of adherent cells. In the lipidomic profile, PC species containing long chain and polyunsaturated fatty acids increased in suspension cells and these species could be authentic and specific biomarkers for highly metastatic cancers. As this CTC-mimicking suspension cell culture model may easily apply to various types of cancer, we suggest this model as a great tool to develop therapeutic targets and drugs to eradicate metastatic cancer cells.
IK depletion leads to an aberrant mitotic entry because of chromosomal misalignment through the enhancement of Aurora B activity at the interphase. Here, we demonstrate that IK, a spliceosomal component, plays a crucial role in the proper splicing of the ATM pre-mRNA among other genes related with the DNA Damage Response (DDR). Intron 1 in the ATM pre-mRNA, having lengths <200 bp, was not spliced in the IK-depleted cells and led to a deficiency of the ATM protein. Subsequently, the IK depletion-induced ATM protein deficiency impaired the ability to repair the damaged DNA. Because the absence of SMU1 results in IK degradation, the mechanism underlying IK degradation was exploited. IK was ubiquitinated in the absence of SMU1 and then subjected to proteolysis through the 26S proteasome. To prevent the proteolytic degradation of IK, a deubiquitinating enzyme, USP47, directly interacted with IK and stabilized it through deubiquitination. Collectively, our results suggest that IK is required for proper splicing of the ATM pre-mRNA and USP47 contributes toward the stabilization of IK.
In most mammalian cells, the primary cilium is a microtubule-enriched protrusion of the plasma membrane and acts as a key coordinator of signaling pathways during development and tissue homeostasis. The primary cilium is generated from the basal body, and cancerous inhibitor of protein phosphatase 2A (CIP2A), the overexpression of which stabilizes c-MYC to support the malignant growth of tumor cells, is localized in the centrosome. Here, we show that CIP2A overexpression induces primary cilia disassembly through the activation of Aurora A kinase, and CIP2A depletion increases ciliated cells and cilia length in retinal pigment epithelium (RPE1) cells. CIP2A depletion also shifts metabolism toward the glycolytic pathway by altering the expression of metabolic genes related to glycolysis. However, glycolytic activation in CIP2A-depleted cells does not depend on cilia assembly, even though enhanced cilia assembly alone activates glycolytic metabolism. Collectively, these data suggest that CIP2A promotes primary cilia disassembly and that CIP2A depletion induces metabolic reprogramming independent of primary cilia.
Hypoxia-induced interleukin-32β (IL-32β) shifts the metabolic program to the enhanced glycolytic pathway. In the present study, the underlying mechanism by which hypoxia-induced IL-32β stability is regulated was investigated in ovarian cancer cells. IL-32β expression increased under hypoxic conditions in ovarian cancer cells as it did in breast cancer cells. The amount of IL-32β was regulated by post-translational control rather than by transcriptional activation. Under normoxic conditions, IL-32β was continuously eliminated through ubiquitin-dependent degradation by the von-Hippel Lindau (VHL) E3 ligase complex. Oxygen deficiency or reactive oxygen species (ROS) disrupted the interaction between IL-32β and VHL, leading to the accumulation of the cytokine. The fact that IL-32β is regulated by the energy-consuming ubiquitination system implies that it plays an important role in oxidative stress. We found that IL-32β reduced protein kinase Cδ (PKCδ)-induced apoptosis under oxidative stress. This implies that the hypoxia- and ROS-stabilized IL-32β contributes to sustain survival against PKCδ-induced apoptosis.
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