ABSTRACT
Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3ß to facilitate GSK3ß phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3ß targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.
Subject(s)
Glucose/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Hexokinase/metabolism , Neoplasms/pathology , A Kinase Anchor Proteins/metabolism , A549 Cells , Animals , CHO Cells , Carcinogenesis/pathology , Cell Line, Tumor , Cricetulus , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Deoxyglucose/pharmacology , Epithelial-Mesenchymal Transition/physiology , Female , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycosylation , HCT116 Cells , HEK293 Cells , Hexokinase/genetics , Humans , Mice , Mice, Inbred BALB C , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , NF-E2-Related Factor 2/metabolism , Neoplasm Metastasis/pathology , Phosphorylation/drug effects , Rats , Snail Family Transcription Factors/metabolismABSTRACT
S100B is frequently elevated in malignant melanoma. A regulatory mechanism was uncovered here in which elevated S100B lowers mRNA and secreted protein levels of interleukin-6 (IL6) and inhibits an autocrine loop whereby IL6 activates STAT3 signaling. Our results showed that S100B affects IL6 expression transcriptionally. S100B was shown to form a calcium-dependent protein complex with the p90 ribosomal S6 kinase (RSK), which in turn sequesters RSK into the cytoplasm. Consistently, S100B inhibition was found to restore phosphorylation of a nuclear located RSK substrate, CREB, which is a potent transcription factor for IL6 expression. Thus, elevated S100B reduces IL6-STAT3 signaling via RSK signaling pathway in malignant melanoma. Indeed, the elevated S100B levels in malignant melanoma cell lines correspond to low levels of IL6 and p-STAT3.
Subject(s)
Interleukin-6/genetics , Melanoma/genetics , Ribosomal Protein S6 Kinases, 90-kDa/genetics , S100 Calcium Binding Protein beta Subunit/genetics , STAT3 Transcription Factor/genetics , Calcium-Binding Proteins/genetics , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/genetics , Cytoplasm/genetics , Doxycycline/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/drug therapy , Melanoma/pathology , Signal Transduction/drug effectsABSTRACT
Chromatin organization has a fundamental impact on the whole spectrum of genomic functions. Quantitative characterization of the chromatin structure, particularly at submicron length scales where chromatin fractal globules are formed, is critical to understanding this structure-function relationship. Such analysis is currently challenging due to the diffraction-limited resolution of conventional light microscopy. We herein present an optical approach termed inverse spectroscopic optical coherence tomography to characterize the mass density fractality of chromatin, and we apply the technique to observe chromatin decompaction in live cells. The technique makes it possible for the first time, to our knowledge, to sense intracellular morphology with length-scale sensitivity from â¼30 to 450 nm, thus primarily probing the higher-order chromatin structure, without resolving the actual structures. We used chromatin decompaction due to inhibition of histone deacytelases and measured the subsequent changes in the fractal dimension of the intracellular structure. The results were confirmed by transmission electron microscopy and confocal fluorescence microscopy.
