Original Article5-Fluorouracil targets histone acetyltransferases p300/CBP in the treatment of colorectal cancer
Introduction
Colorectal cancer (CRC) is the third more common malignant neoplasm worldwide and the second leading cause of cancer deaths in developed countries [1]. Although adjuvant or palliative chemotherapy based on 5-Fluorouracil (5-FU) is an essential treatment for the majority of CRC patients [2], [3], the response rate of current treatment regimens remains discouragingly low (10–15% with 5-FU alone and less than 50% when 5-FU is combined with other cytotoxic drugs) [4], [5]. An underlying resistance to 5-FU chemotherapy, including intrinsic and acquired resistance, remains a leading cause for treatment failure [6]. Moreover, current biomarkers for predicting therapeutic efficacy following 5-FU treatment possess their own limitations in clinical practice [7], [8]. An improved understanding of the mechanisms underlying the anti-neoplastic properties of 5-FU is required to better predict and improve the clinical response to 5-FU.
5-FU is known to be metabolized like uracil to interfere with the metabolism of nucleic acids [4]. The metabolite fluorodeoxyuridine monophosphate also inhibits the activity of thymidylate synthase, a critical enzyme of nucleotide synthesis, to shut off DNA synthesis [9]. However, these findings cannot fully explain the complex anti-neoplastic effects of 5-FU, or allow us to accurately predict the clinical response to 5-FU treatment.
In addition to blocking nucleic acid metabolism, 5-FU possesses a myriad of biological activities, including the ability to recode histone modifications [10]. Histone modification, especially histone acetylation, is important in establishing chromatin environments and regulating gene expression [11]. Moreover, histone modification is closely associated with therapeutic sensitivity of tumors [12], [13], which makes it a well-established anti-cancer target [14]. For example, many studies have demonstrated over-increasing histone acetylation may reverse 5-FU resistance and potentially improve therapeutic outcome in CRC [15], [16]. Therefore, a better understanding of the 5-FU histone-modifying effects is helpful to reveal the mechanism of 5-FU resistance. Many factors such as oxidative stress, DNA damage or some artificial compounds may influence histone acetylation in cancer cells by working on histone acetyltransferases (HATs) or histone deacetylases (HDACs) [17], [18]. It therefore is possible that 5-FU affects histone acetylation either by altering the amount of histone-modifying enzymes or by influencing their catalytic activity. Besides the possibility that 5-FU may inhibit the synthesis of histone-modifying enzymes, it remains possible that 5-FU promotes their degradation, since 5-FU has been shown to induce extensive protein degradation by activating autophagy in cancer cells [19]. Autophagy is a beneficial protein-degradation system for cells to maintain cellular metabolism under starvation or stress, which is also relevant to cellular resistance to cytotoxic drugs in cancer [20], [21].
In the present study, we investigated the mechanisms underlying the ability of 5-FU to modify histone acetylation. Our results suggested that 5-FU induces global histone de-acetylation in CRC by promoting the degradation of p300 and CBP, two important homologous HATs catalyzing acetylation at multiple sites of lysine on histone [22], [23]. We further demonstrated that this degradation is dependent on chaperone-mediated autophagy (CMA), a selective protein degradation pathway mediated by heat-shock cognate protein 70 kDa (Hsc70) and lysosomal-associated membrane protein 2A (LAMP2A) [24]. Finally, we demonstrated that the degradation of p300/CBP is associated with cellular resistance to 5-FU, and found low-expression of p300/CBP in CRC samples is closely associated with poor clinical response to 5-FU treatment, suggesting that they may serve as biomarkers to predict therapeutic outcome.
Section snippets
Cell culture
Cells were cultured at 37 °C using 5% CO2 in mediums supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (HCT116 in McCoy's 5A medium, HCT-8 in RPMI 1640 medium, SW480 and SW620 in L-15medium, MEF in DMEM). Cells in the exponential growth phase were plated at a density of 1 × 105 cells on 6-cm culture dishes.
Protein extraction
Cells were lysed using the lysis-buffer with 1% Triton X-100, and protein was preserved in loading-buffer with 10% SDS. Histones were isolated using modified acid
5-FU induces H3 and H4 global de-acetylation
To investigate whether 5-FU affects histone acetylation in CRC cells, HCT 116 cells were exposed to gradient concentrations of 5-FU for varying periods of time. A dose- and time-dependent global reduction of H3 and H4 acetylation was observed (Fig. 1A). In addition, multiple lysine residues in H3 and H4, including H3K9, H3K14, H3K18, H3K27, H3K56, H4K5, H4K8, and H4K12, were found significantly de-acetylated following 5-FU treatment (Fig. 1B). Given that histone acetylation is associated with
Discussion
In the present study, we identified a novel mechanism of 5-FU involving the induction of global histone hypo-acetylation. We demonstrated that 5-FU promotes the degradation of p300/CBP via chaperone-mediated autophagy, which is relevant to chemo-resistance of 5-FU. We further confirmed low-expression of p300/CBP is predictive to clinical resistance of 5-FU treatment.
The oncological significance of histone acetylation has been well characterized in previous studies [14]. With regard to cancer
Funding
This work was supported by National Natural Science Foundation of China grants NSFC91319302; Beijing Municipal Administration of Hospitals' Youth Program QML20161105; Discipline Construction Funding of Shenzhen (2016) and Shenzhen Municipal Commission of Science and Technology Innovation grants JCYJ20160427104855100.
Author contributions
Study design: Zhu W-G and Gu J. Acquisition and interpretation of data: Du C, Huang D, Peng Y, Yao Y, Zhao Y, Yang Y, Wang H, Cao L. Drafting of the manuscript: Du C, Huang D, Peng Y, they do equal contribution. Critical revision of the manuscript: Zhu W-G and Gu J, they do equal contribution.
Acknowledgements
We greatly appreciate the following staff members who contributed to this work:
Prof. Aiwen Wu, Prof. Zhiqian Zhang, Mrs. Jinying Jia, and Mr. Zhaowei Li in Peking University Cancer Hospital; Mrs. Lina Wang in Peking University Health Science Center.
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2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Similar confusion arises regarding the impact of macroautophagy in cancer treatment [8]. However, recent studies have made great progress in sorting out this controversy by focusing on CMA, another equally important but less-well-studied form of autophagy [4,8,21–24]. It was shown that there is cross-talk between these autophagic pathways that allows one pathway to compensate for the other [25], thus it may not be sufficient to only study macroautophagy.