Abstract
Hypoxia is an environmental cue that is associated with multiple tumorigenic processes such as immunosuppression, angiogenesis, cancer invasion, metastasis, drug resistance, and poor clinical outcomes. When facing hypoxic stress, cells initiate several adaptive responses such as cell cycle arrest to reduce excessive oxygen consumption and co-activation of oncogenic factors. In order to identify the critical novel proteins for hypoxia responses, we used pulsed-SILAC method to trace the active cellular translation events in A431 cells. Proteomic discovery data and biochemical assays showed that cancer cells selectively activate key glycolytic enzymes and novel ER-stress markers, while protein synthesis is severely suppressed. Interestingly, deprivation of oxygen affected the expression of various epigenetic regulators such as histone demethylases and NuRD (nucleosome remodeling and deacetylase) complex in A431 cells. In addition, we identified PHF14 (the plant homeodomain finger-14) as a novel hypoxia-sensitive epigenetic regulator that plays a key role in cell cycle progress and protein synthesis. Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14ARF, p15INK4b, and p16INK4a, which are responsible for G1-S phase transition and decrease of AKT-mTOR-4E-BP1/pS6K signaling pathway, a master regulator of protein synthesis, in response to environmental cues. Analysis of TCGA colon cancer (n=461) and skin cancer (n=470) datasets revealed a positive correlation between PHF14 expression and protein translation initiation factors, eIF4E, eIF4B, and RPS6. Significance of PHF14 gene was further demonstrated by in vivo mouse xenograft model using PHF14 KD cell lines.
Highlights
Growing tumor cells that outgrow their vascular supply often encounter oxygen-deficient “hypoxia” condition
Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14ARF, p15INK4b, and p16INK4a, which are responsible for G1-S phase transition and decrease of AKTmTOR-4E (eIF4E)-binding protein 1 (4E-BP1)/pS6K signaling pathway, a master regulator of protein synthesis, in response to environmental cues
Since pSILAC data showed dysregulation of glycolysis/tricarboxylic acid (TCA) cycle enzymes and suppression of protein translation under reduced oxygen/nutrients availability, we examined if translation of epigenetic regulators such as DNA or histone modifiers accounting for gene transcription were changed. pSILAC proteomic data, acquired from two independent biological replicates, Figure 3: Quantitative pSILAC data reveals primary hypoxia responsible proteins in cancer cells. (A) Hypoxia induced protein synthesis of oxidative stress markers and endoplasmic reticulum (ER) stress response proteins in A431 cells grown in serum free medium
Summary
Growing tumor cells that outgrow their vascular supply often encounter oxygen-deficient “hypoxia” condition. Decades of cancer research have focused on tumor hypoxia as it triggers oncogenic property of cancer cells and its profound effects on clinical outcome in cancer patients. Hypoxia upregulates the expression of several proangiogenic factors that induce the neo-vascularization of tumor mass [6, 7] and drives metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis which leads to activation of proteases, resulting in an acidic microenvironment and induces tumor metastasis [3, 8]. HIF1α, a master regulator of hypoxia [11], has been known to mediate the transcription of cell cycle regulators, such as cyclin-dependent kinase (CDK) inhibitors, p21 and p27, by displacement of c-Myc from its DNA binding site [10, 12]. Through HIF-1α-mediated suppression of c-Myc protein or disruption of c-Myc-Max transcription factor complex, c-Myc-dependent transcription of target genes such as cyclin D2 and E2F1 (the target of retinoblastoma protein) are interrupted [15]
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