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
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Jung Eun Park1, Shun Wilford Tse1, Guo Xue1, Christina Assisi1, Aida Serra Maqueda1, Gallart Palau Xavier Ramon1, Jee Keem Low2, Oi Lian Kon3, Chor Yong Tay4, James P. Tam1 and Siu Kwan Sze1
1Division of Structural Biology and Biochemistry School of Biological Sciences, Nanyang Technological University, Singapore 637551
2Department of Oncology, Tan Tock Seng Hospital, Singapore 308433
3Laboratory of Applied Human Genetics, Division of Medical Sciences, National Cancer Centre Singapore, Singapore 169610
4Division of Materials Technology School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798
Siu Kwan Sze, email: [email protected]
Jung Eun Park, email: [email protected]
Keywords: hypoxia; pSILAC; PHF14; cell cycle inhibition
Received: August 26, 2018 Accepted: December 27, 2018 Published: March 15, 2019
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.
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