In the present study we investigated the mechanisms underlyi

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.
Material and methods
Results
Discussion The oncological significance of histone acetylation has been well characterized in previous studies [14]. With regard to cancer therapy, the beneficial effects of histone hyper-acetylation following HDAC inhibitors treatment has been fully identified, which is associated with reactivating tumor-suppressor gene transcription [33], [34], [35]. For example, the tumor-suppressor gene p21WAF1 may be re-activated to express by suberoylanilide hydroxamic Hydroxyfasudil or depsipeptide, two classical HDAC inhibitors, in cancer cells that have hypo-acetylated histone at p21WAF1 promoter regions [25], [33]. On the other hand, HDAC inhibitors could also help to reverse 5-FU resistance in CRC and enhance its cytotoxicity [15], [16], suggesting hyper-acetylation is adverse for cancer cell survival. Our finding revealed that CRC cells present hypo-acetylated in histone following 5-FU treatment, which might be beneficial for cancer cells under the stress of 5-FU. Firstly, global hypo-acetylation in H3 and H4 reduces global transcription level [11], [36], avoiding the interference of 5-FU to gene transcription and protein translation. Secondly, compacted chromatin induced by wide histone hypo-acetylation influences DNA replication process by reducing the accessibility of DNA to replication factors [37], therefore decreasing the probability of 5-FU incorporating into DNA. Thus, histone hypo-acetylation in CRC may be a protective response to 5-FU, notwithstanding long-term and extensive hypo-acetylation of histone is adverse for cell proliferation [11]. The critical regulators of histone acetylation responding to 5-FU are p300 and CBP, two homologous HATs and meanwhile strong transcription co-activators that are important for gene transcription [23], [38]. In this study, we cautiously demonstrated the importance of p300/CBP in regulating histone acetylation in CRC cells, where their reduction leads to widespread histone hypo-acetylation. Although the reduction of p300/CBP has been confirmed to be caused by lysosomal degradation, the mechanism how 5-FU affects the binding ability of p300/CBP to chromatin and their traffic to cytoplasm is still unclear. Actually, the degradation of p300/CBP involves both lysosome and proteasome pathways, depending on the specific stimulation or stressor [39], [40], [41]. This study suggests that the 5-FU induced degradation of p300/CBP is mediated by CMA, a more selective lysosome-dependent protein degradation pathway compared with other forms of autophagy [24]. CMA is a cytoplasmic protein degradation system containing the substrate recognition by hsc70 and recruitment of target proteins at lysosomes by LAMP2A [24], [42], but nuclear proteins could also be degraded by this pathway under some special conditions [43], [44]. For example, the transcription factor MEF2D, a critical modulator in the pathogenesis of Parkinson's disease, has been reported to undergo degradation via CMA under the oxidative stress [44]. Generally, autophagy is protective to cells involving removal of damaged or malfunctioning cellular components and proteins [20], [21]; and the 5-FU induced degradation of p300/CBP in this study is such a good example, since the accumulation of malfunctioning p300/CBP inside cells would break cellular homeostasis. On the other hand, downregulating the protein level of p300/CBP is helpful to decrease global transcription level, which is protective for cancer cells when exposing to 5-FU.