5-Fluorouracil targets histone acetyltransferases p300/CBP in the treatment of colorectal cancer

Highlights

• A novel pharmacological effect of 5-FU involving global histone de-acetylation in colorectal cancer is illuminated.

• The histone de-acetylation induced by 5-FU results from the degradation of histone acetyltransferases p300/CBP via lysosome.

• Low-expression of p300/CBP in colorectal cancer tissue is closely associated with poor clinical response to 5-FU treatment.

Abstract

Although 5-fluorouracil (5-FU) is known to interfere with the synthesis of ribonucleic acid and deoxyribonucleic acid, the mechanism underlying its therapeutic efficacy in colorectal cancer (CRC) has not been fully elucidated. We aimed to investigate the influence of 5-FU on histone acetylation, a well-established anti-cancer target, to reveal novel pharmacological effects of 5-FU and their significance for CRC therapy. Results demonstrated that 5-FU induces global histone de-acetylation in multiple CRC cell lines. We identified that 5-FU reduces the binding ability of histone acetyltransferases p300 and CBP to chromatin, and induces their degradation through lysosome. Further work revealed that the degradation of p300/CBP induced by 5-FU was dependent on chaperone-mediated autophagy, mediated by heat-shock cognate protein 70 kDa (hsc70) and lysosomal-associated membrane protein 2A (LAMP2A). Moreover, the degradation of p300/CBP is relevant to cellular resistance to 5-FU, since blocking the degradation enhances 5-FU's cytotoxicity in CRC cells. From clinical data, we demonstrated that low expression of p300/CBP in CRC tissue was closely associated with poor clinical response to 5-FU based-chemotherapy, based on the analysis of 262 colorectal samples from the patients receiving 5-FU treatment: compared to cases with high expression of p300/CBP, those with low expression had lower long-term disease-free survival rate and increased early-progression. These results elucidate a novel pharmacological effect of 5-FU involving global histone de-acetylation by promoting the degradation of p300/CBP, and highlights p300 and CBP as promising predictors of chemo-sensitivity to 5-FU treatment.

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.