Lysine-acetylation (LysAc) and its regulatory enzymes are broadly associated with cancers. Consequently, HDAC inhibitors have been developed to modulate the LysAc status, and are currently undergoing more than 80 clinical trials as anti-tumor therapeutics. Molecular biology and genetics approaches have been attempted to characterize LysAc pathway and the anti-tumor effects of HDAC inhibitors. However, such research provides limited insight into the functions of LysAc sites and their dynamics. LysAc sites among some substrate proteins have been elegantly analyzed by a candidate approach using mass spectrometry, generating key information for downstream biological studies to establish its known functions. Nevertheless, a large number of LysAc substrate proteins and their LysAc dynamics have not been globally studied in cancer-related environments before. This knowledge gap needs to be filled to improve our understanding of LysAc in cancer and other diseases. We hypothesize that dysregulation of LysAc plays a key role in cancer progression. The long-term objective of this research plan is to comprehensively elucidate the dynamic changes of lysine acetylation in cancer, and to use this knowledge to gain a better understanding of its biological functions. We propose to study global LysAc dynamics in cancer cells by a novel, integrated proteomics approach, consisting of (i) high- resolution protein pre-fractionation, (ii) efficient purification of LysAc peptides with anti- LysAc antibodies, and (iii) identification and quantification of the LysAc peptides by SILAC combined with 1D- HPLC/MS/MS or 2D-HPLC/MS/MS. This study is anticipated to identify a large number of non-nuclear LysAc proteins and to reveal their dynamics in cancer progression, therefore challenging the current concept that regulation of DNA-templated process is the major function of the modification. Given the difficulty in identifying new LysAc substrates, the novel, comprehensive LysAc datasets established in this study will address a critical barrier in the LysAc biology field. Specific aims of this research are: (1) To define the aberrant changes in lysine acetylation in cancer cells. LysAc will be quantified between two sets of matched normal and cancer cells, with an emphasis on quantifying LysAc with the highest possible sensitivity. (2) To identify downstream protein targets of suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved for clinical application. We will quantify LysAc, with or without SAHA, in both normal (SAHA-resistant cells) and matched cancer cells (SAHA-sensitive cells). (3) To identify the lysine-acetylation protein targets for p300 acetyltransferase. We will quantify LysAc proteins among transformed cell lines, with or without expression of the active p300 to generate an atlas of p300 substrates. And (4) to analyze and disseminate LysAc datasets to research community. The novel information from this study will be analyzed by bioinformatics tools and used to construct a LysAc database, providing an infrastructure to stimulate drug development and LysAc-biology research. Lysine acetylation, a post-translational modification in proteins, is dysregulated in cancer development. The proposed study aims to elucidate the dynamic changes of lysine acetylation in cancer progression. The resulting knowledge can be used to understand the biological functions of the modification, to assist development of novel biomarkers for diagnosis of cancer and development of novel anti-tumor drug with high potency and better safety profile.

R01CA126832

2008-06-01

2013-04-30