Pulsed SILAC-based proteomic analysis unveils hypoxia- and serum starvation-induced de novo protein synthesis with PHD finger protein 14 (PHF14) as a hypoxia sensitive epigenetic regulator in cell cycle progression

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, p14 ARF , p15 INK4b , and p16 INK4a , 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.

Silencing expression of PHF14 in glioblastoma promotes apoptosis, mitigates proliferation and invasiveness via Wnt signal pathway

“…PHF14 increases mitotic defects and DNA errors in lung cancer model [26]. It also induces G1-S phase arrest in squamous carcinoma [22]. In addition, PHF14 promotes tumor cellular epithelial–mesenchymal transition (EMT) and enhances the proliferation and invasiveness of bladder cancer [23].…”

“…PHF14, a member of the PHD finger protein family, is coded by a highly conserved gene found in the 19p13.2 chromosomal region [20]. PHF14 has been described as a modulator for mesenchymal cells proliferation in alveolar tissues [20, 21], as well as a novel hypoxia-sensitive epigenetic regulator in cell proliferation [22]. However, the biological function of PHF14 in tumors differs according to different cancer types.…”

“…However, the biological function of PHF14 in tumors differs according to different cancer types. While PHF14 depletion has been shown to suppress cell proliferation in lung cancer cells and bladder cancer cells, its overexpression constrained BTC cells growth and improved prognosis in colon cancer [22–26]. The role of PHF14 in GBM has not been adequately described.…”

Application of Advanced Mass Spectrometry-Based Proteomics to Study Hypoxia Driven Cancer Progression

“…Later on, a global analysis of newly translated proteins between normoxic and hypoxic (24 h) A431 epidermoid carcinoma cells revealed an intriguing finding that only 5% of total proteins were upregulated, and more than 60% were translationally suppressed in hypoxia condition ( 66 ). In concordance with the previously mentioned study by Bousquet et al.…”

Emerging proteomic approaches to identify the underlying pathophysiology of neurodevelopmental and neurodegenerative disorders

“…3b). This allows for detection of differences in protein abundances in cell cultures and can be modified to examine de novo protein synthesis [33,34]. SILAC has been used to study neurodevelopmental and neurodegenerative disorders [35,36].…”