靶向 CBP 逆转 CDX2/REG4 双阳性胃癌对 5-FU 的化疗耐药性

IF 7.9 1区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL Clinical and Translational Medicine Pub Date : 2024-10-25 DOI:10.1002/ctm2.70069
Zhiyuan Fan, Fangyuan Li, Xiao Jiang, Tao Pan, Mingde Zang, Jianfang Li, Beiqin Yu, Qingqing Sang, Wentao Liu, Liping Su, Chen Li, Zhenggang Zhu, Min Yan, Chao Yan, Fei Yuan, Bingya Liu
{"title":"靶向 CBP 逆转 CDX2/REG4 双阳性胃癌对 5-FU 的化疗耐药性","authors":"Zhiyuan Fan,&nbsp;Fangyuan Li,&nbsp;Xiao Jiang,&nbsp;Tao Pan,&nbsp;Mingde Zang,&nbsp;Jianfang Li,&nbsp;Beiqin Yu,&nbsp;Qingqing Sang,&nbsp;Wentao Liu,&nbsp;Liping Su,&nbsp;Chen Li,&nbsp;Zhenggang Zhu,&nbsp;Min Yan,&nbsp;Chao Yan,&nbsp;Fei Yuan,&nbsp;Bingya Liu","doi":"10.1002/ctm2.70069","DOIUrl":null,"url":null,"abstract":"<p>Dear Editor,</p><p>We conducted a study exploring the potential of cyclic-AMP response element binding protein (CBP) inhibitors in overcoming the chemoresistance of CDX2/REG4 double-positive gastric cancer (GC) to 5-FU chemotherapy.</p><p>CDX2 is a classical transcription factor belonging to the caudal-related homeobox gene family, which determines the development and maintenance of intestinal differentiation in the gut and is overexpressed  in part of GC.<span><sup>1</sup></span> Our study aims to investigate the heterogeneity of CDX2+ GC, which accounts for approximately 50% of all GC,<span><sup>1</sup></span> and discover potential therapies. Using 111 GC samples, molecular classification based on CDX2 expression revealed that CDX2+ GC could be further divided into two subtypes: REG4<sup>hi</sup> and REG4<sup>lo</sup> (Figure 1A). REG4 is a direct target of CDX2 and has been implicated in the progression and chemoresistance of GC.<span><sup>2</sup></span> The REG4<sup>hi</sup> subtype showed significantly shorter overall survival (OS, Figure 1B, hazard ratio: CDX2<sup>hi</sup> REG4<sup>hi</sup> vs. CDX2<sup>lo</sup> .99, 95% CI .60–1.64, <i>p</i> = .973; CDX2<sup>hi</sup> REG4<sup>lo</sup> vs. CDX2<sup>lo</sup> .11, 95% CI .04–.30, <i>p</i> &lt; .001; CDX2<sup>hi</sup> REG4<sup>lo</sup> vs. CDX2<sup>hi</sup> REG4<sup>hi</sup> .12, 95% CI .04–.30, <i>p</i> &lt; .001) and poorer differentiation compared to REG4<sup>lo</sup> (Figure 1C, Tables S1 and S2). REG4 positive expression was significantly associated with CDX2+ cases (Figure 1D). Additionally, CDX2+ REG4<sup>hi</sup> GC patients were more resistant to 5-FU-based chemotherapy (Figure 1E). We identified CDX2+ GC cell lines (Figure 1F) with high or low REG4 expression (Figure 1G) using the CCLE database. The IC<sub>50</sub> of CDX2+ REG4<sup>hi</sup> GC cells to 5-FU were much higher than those of CDX2+ REG4<sup>lo</sup> GC cells (Figure 1H–J).</p><p>We selected GC cell lines which showed consistent expression patterns of CDX2 and REG4 with specific GC types suggested by CCLE database and confirmed by immunoblotting (Figure 2A). A screen of 17 small molecule inhibitors targeting epigenetic regulators (Table S3) identified CPI-637, a CBP/p300 inhibitor, as particularly effective against CDX2+ REG4<sup>hi</sup> GC cells (Figure 2A and B). These cells showed significant growth inhibition (Figure 2C) and lower IC50 values (Figure 2D) with CPI-637 compared to CDX2+ REG4<sup>lo</sup> cells. In vivo experiments demonstrated that CPI-637 significantly inhibited tumour growth in CDX2+ REG4<sup>hi</sup> cell derived xenograft CDX (Figure 2E), resulting in smaller tumour volumes (Figure 2F), less tumour weight (Figure 2G) and higher tumour growth inhibition rates (Figure 2H).</p><p>CPI-637 is a selective inhibitor targeting both CBP and p300<span><sup>3</sup></span> and its role have been investigated in tumour treatment.<span><sup>4, 5</sup></span> CBP/p300 activates gene expression using its protein lysine acetyltransferase (KAT) domain to catalyse the histone H3 lysine 18 or lysine 27 acetylation (H3K18Ac or H3K27Ac) and its bromodomain to recognise acetyl-lysine residues in histone tails.<span><sup>6</sup></span> AGS and MKN28 had similarly high levels of CBP and silenced expression of p300 (Figure S1A) probably due to EP300 nonsense mutations (Figure S1B). p300 showed relatively low expression in MKN45 and NCI-N87 cells with no mutations (Figure S1A). We further found that H3K18Ac/H3K27Ac decreased upon CPI-637 treatment in AGS and NCI-N87 cells was in a dose-dependent manner (Figure S1C and D). Significant reductions in H3K18Ac/H3K27Ac levels with 5 µM CPI-637 were observed in GC cell lines (Figure S1C–E). CPI-637 impaired REG4 expression in CDX2+ REG4<sup>hi</sup> GC cells but did not affect REG4 expression in CDX2+ REG4<sup>lo</sup> ones (Figure S1E and F).</p><p>REG4 was identified as a direct target of CDX2 in a previous study.<span><sup>2, 7</sup></span> Two previously reported CDX2-binding sites on the REG4 promoter located near the transcriptional start site (TSS) (Figure 3A). ChIP analyses revealed that H3K27Ac was abundant on the REG4 promoter in control-treated CDX2+ REG4<sup>hi</sup> AGS cells (Figure 3B), while CPI-637 treatment induced a transition to H3K27Me3 (Figure S2A). Conversely, in CDX2<sup>+</sup> REG4<sup>lo</sup> NCI-N87 cells, H3K27Me3, rather than H3K27Ac or H3K18Ac, was specifically enriched on the <i>REG4</i> promoter, which was not affected by CPI-637 treatment (Figure S2B). Co-IP experiments showed that CDX2 physically interacted with CBP (Figure 3C), and this interaction was disrupted by CPI-637 treatment or CDX2 silencing (Figure 3D and E), indicating that the regulation of REG4 by CBP was CDX2-dependent.</p><p>GSEA in our GSE54129 cohort revealed GATA4, GATA6 and ecotropic viral integration site-1 (EVI1) were most significantly enriched in CDX2<sup>+</sup> REG4<sup>hi</sup> GC (Figure S2C, all FDR <i>q</i> value &lt; .001). A total of 168 differentially expressed genes (DEGs) between two subtypes of CDX2<sup>+</sup> GC were identified among GSE54129 and two other independent GC cell line cohorts (GSE15455 and GSE22183, Figure S2D, Table S4). EVI1 was found to co-occupy the REG4 promoter with CDX2 and CBP (Figure 3F and G). EVI1 silencing abrogated REG4 expression (Figure 3H and I) and blocked the interaction between CDX2 and CBP (Figure 3J–L), indicating that EVI1 was crucial for the recruitment of CBP to the REG4 promoter by CDX2. GATA4 and GATA6 were not significantly differentially expressed between the two GC cell subtypes (Figure S2E). Sequential ChIP analyses showed none of the three transcriptional factors (TFs) occupied the REG4 promoter in MKN28 and NCI-N87 cells (Figure S2F).</p><p>CBP can catalyse lysine acetylation via its KAT activity.<span><sup>8, 9</sup></span> We found that the EVI1 acetylation level was high in CDX2+ REG4<sup>hi</sup> AGS cells (Figure S2G) and CBP silencing by siRNA nearly abrogated EVI1 acetylation (Figure S2G). The region between amino acids 283 and 514 of the EVI1 protein was reported to contribute to the interaction between EVI1 and CBP.<span><sup>10</sup></span> We identified three candidate CBP KAT-specific lysine acetylation sites (lysine 359, 421 and 425) in this EVI1 region using the GPS-PAIL algorithm<span><sup>9</sup></span> (Figure S2H and I). AGS cells with silenced endogenous EVI1 were reintroduced with the wild-type EVI1 (EVI1 WT) or three EVI1 mutants (EVI1 K359R, EVI1 K421R, or EVI1 K425R) (Figure S2J). Co-IP analyses showed that the mutation of K421 rather than K359 and K425 significantly attenuated CBP-induced EVI1 acetylation (Figure S2K). The mutation of EVI1 K421 to arginine abrogated the physical binding of EVI1 to CBP or CDX2 and promoted the recruitment of the corepressor C-terminal binding protein 1 (CtBP1) to EVI1 (Figure S2L). S2</p><p>We also analysed whether the application of the CBP inhibitor could overcome the 5-FU resistance of CDX2<sup>+</sup> REG4<sup>hi</sup> GC cells. CPI-637 and 5-FU combination treatment significantly improved the efficacy of 5-FU or CPI-637 alone in AGS and MKN45 cells (Figure 4A and B). The calculation of combination index (CI) values showed that CPI-637 and 5-FU combination had a synergistic effect in CDX2<sup>+</sup> REG4<sup>hi</sup> AGS and MKN45 cells (Figure 4C), but the addition of CPI-637 didn't improve the effect of 5-FU in CDX2<sup>+</sup> REG4<sup>lo</sup> MKN28 and NCI-N87 cells (Figure 4D).</p><p>In both CDX2<sup>+</sup> REG4<sup>hi</sup> CDX and patient derived xenograft (PDX) in vivo, treatment with CPI-637 and particularly with the CPI-637 and 5-FU combination significantly inhibited tumour growth (Figure 4E and F). Furthermore, CPI-637 and 5-FU combination markedly improved mouse survival compared to 5-FU or CPI-637 alone (Figure 4E and F). We compared the tumor inhibition rate and found that 5-FU was more effective in CDX2+ REG4lo GC while CPI-637 was more effective in CDX2+ REG4hi GC (Figure 2G). The combination did not result in more systemic toxicity or weight loss than 5-FU alone (Figure 4H and I). Collectively, these data indicate that targeting CBP can overcome 5-FU resistance and thus provide a therapeutic option for efficacy improvement of 5-FU-based chemotherapy in CDX2<sup>+</sup> REG4<sup>hi</sup> GC (Figure 4J). This study provides crucial insights into the molecular mechanisms driving chemoresistance and offers potential for clinical translation.</p>","PeriodicalId":10189,"journal":{"name":"Clinical and Translational Medicine","volume":null,"pages":null},"PeriodicalIF":7.9000,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511671/pdf/","citationCount":"0","resultStr":"{\"title\":\"Targeting CBP revers chemoresistance to 5-FU of CDX2/REG4 double-positive gastric cancer\",\"authors\":\"Zhiyuan Fan,&nbsp;Fangyuan Li,&nbsp;Xiao Jiang,&nbsp;Tao Pan,&nbsp;Mingde Zang,&nbsp;Jianfang Li,&nbsp;Beiqin Yu,&nbsp;Qingqing Sang,&nbsp;Wentao Liu,&nbsp;Liping Su,&nbsp;Chen Li,&nbsp;Zhenggang Zhu,&nbsp;Min Yan,&nbsp;Chao Yan,&nbsp;Fei Yuan,&nbsp;Bingya Liu\",\"doi\":\"10.1002/ctm2.70069\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Dear Editor,</p><p>We conducted a study exploring the potential of cyclic-AMP response element binding protein (CBP) inhibitors in overcoming the chemoresistance of CDX2/REG4 double-positive gastric cancer (GC) to 5-FU chemotherapy.</p><p>CDX2 is a classical transcription factor belonging to the caudal-related homeobox gene family, which determines the development and maintenance of intestinal differentiation in the gut and is overexpressed  in part of GC.<span><sup>1</sup></span> Our study aims to investigate the heterogeneity of CDX2+ GC, which accounts for approximately 50% of all GC,<span><sup>1</sup></span> and discover potential therapies. Using 111 GC samples, molecular classification based on CDX2 expression revealed that CDX2+ GC could be further divided into two subtypes: REG4<sup>hi</sup> and REG4<sup>lo</sup> (Figure 1A). REG4 is a direct target of CDX2 and has been implicated in the progression and chemoresistance of GC.<span><sup>2</sup></span> The REG4<sup>hi</sup> subtype showed significantly shorter overall survival (OS, Figure 1B, hazard ratio: CDX2<sup>hi</sup> REG4<sup>hi</sup> vs. CDX2<sup>lo</sup> .99, 95% CI .60–1.64, <i>p</i> = .973; CDX2<sup>hi</sup> REG4<sup>lo</sup> vs. CDX2<sup>lo</sup> .11, 95% CI .04–.30, <i>p</i> &lt; .001; CDX2<sup>hi</sup> REG4<sup>lo</sup> vs. CDX2<sup>hi</sup> REG4<sup>hi</sup> .12, 95% CI .04–.30, <i>p</i> &lt; .001) and poorer differentiation compared to REG4<sup>lo</sup> (Figure 1C, Tables S1 and S2). REG4 positive expression was significantly associated with CDX2+ cases (Figure 1D). Additionally, CDX2+ REG4<sup>hi</sup> GC patients were more resistant to 5-FU-based chemotherapy (Figure 1E). We identified CDX2+ GC cell lines (Figure 1F) with high or low REG4 expression (Figure 1G) using the CCLE database. The IC<sub>50</sub> of CDX2+ REG4<sup>hi</sup> GC cells to 5-FU were much higher than those of CDX2+ REG4<sup>lo</sup> GC cells (Figure 1H–J).</p><p>We selected GC cell lines which showed consistent expression patterns of CDX2 and REG4 with specific GC types suggested by CCLE database and confirmed by immunoblotting (Figure 2A). A screen of 17 small molecule inhibitors targeting epigenetic regulators (Table S3) identified CPI-637, a CBP/p300 inhibitor, as particularly effective against CDX2+ REG4<sup>hi</sup> GC cells (Figure 2A and B). These cells showed significant growth inhibition (Figure 2C) and lower IC50 values (Figure 2D) with CPI-637 compared to CDX2+ REG4<sup>lo</sup> cells. In vivo experiments demonstrated that CPI-637 significantly inhibited tumour growth in CDX2+ REG4<sup>hi</sup> cell derived xenograft CDX (Figure 2E), resulting in smaller tumour volumes (Figure 2F), less tumour weight (Figure 2G) and higher tumour growth inhibition rates (Figure 2H).</p><p>CPI-637 is a selective inhibitor targeting both CBP and p300<span><sup>3</sup></span> and its role have been investigated in tumour treatment.<span><sup>4, 5</sup></span> CBP/p300 activates gene expression using its protein lysine acetyltransferase (KAT) domain to catalyse the histone H3 lysine 18 or lysine 27 acetylation (H3K18Ac or H3K27Ac) and its bromodomain to recognise acetyl-lysine residues in histone tails.<span><sup>6</sup></span> AGS and MKN28 had similarly high levels of CBP and silenced expression of p300 (Figure S1A) probably due to EP300 nonsense mutations (Figure S1B). p300 showed relatively low expression in MKN45 and NCI-N87 cells with no mutations (Figure S1A). We further found that H3K18Ac/H3K27Ac decreased upon CPI-637 treatment in AGS and NCI-N87 cells was in a dose-dependent manner (Figure S1C and D). Significant reductions in H3K18Ac/H3K27Ac levels with 5 µM CPI-637 were observed in GC cell lines (Figure S1C–E). CPI-637 impaired REG4 expression in CDX2+ REG4<sup>hi</sup> GC cells but did not affect REG4 expression in CDX2+ REG4<sup>lo</sup> ones (Figure S1E and F).</p><p>REG4 was identified as a direct target of CDX2 in a previous study.<span><sup>2, 7</sup></span> Two previously reported CDX2-binding sites on the REG4 promoter located near the transcriptional start site (TSS) (Figure 3A). ChIP analyses revealed that H3K27Ac was abundant on the REG4 promoter in control-treated CDX2+ REG4<sup>hi</sup> AGS cells (Figure 3B), while CPI-637 treatment induced a transition to H3K27Me3 (Figure S2A). Conversely, in CDX2<sup>+</sup> REG4<sup>lo</sup> NCI-N87 cells, H3K27Me3, rather than H3K27Ac or H3K18Ac, was specifically enriched on the <i>REG4</i> promoter, which was not affected by CPI-637 treatment (Figure S2B). Co-IP experiments showed that CDX2 physically interacted with CBP (Figure 3C), and this interaction was disrupted by CPI-637 treatment or CDX2 silencing (Figure 3D and E), indicating that the regulation of REG4 by CBP was CDX2-dependent.</p><p>GSEA in our GSE54129 cohort revealed GATA4, GATA6 and ecotropic viral integration site-1 (EVI1) were most significantly enriched in CDX2<sup>+</sup> REG4<sup>hi</sup> GC (Figure S2C, all FDR <i>q</i> value &lt; .001). A total of 168 differentially expressed genes (DEGs) between two subtypes of CDX2<sup>+</sup> GC were identified among GSE54129 and two other independent GC cell line cohorts (GSE15455 and GSE22183, Figure S2D, Table S4). EVI1 was found to co-occupy the REG4 promoter with CDX2 and CBP (Figure 3F and G). EVI1 silencing abrogated REG4 expression (Figure 3H and I) and blocked the interaction between CDX2 and CBP (Figure 3J–L), indicating that EVI1 was crucial for the recruitment of CBP to the REG4 promoter by CDX2. GATA4 and GATA6 were not significantly differentially expressed between the two GC cell subtypes (Figure S2E). Sequential ChIP analyses showed none of the three transcriptional factors (TFs) occupied the REG4 promoter in MKN28 and NCI-N87 cells (Figure S2F).</p><p>CBP can catalyse lysine acetylation via its KAT activity.<span><sup>8, 9</sup></span> We found that the EVI1 acetylation level was high in CDX2+ REG4<sup>hi</sup> AGS cells (Figure S2G) and CBP silencing by siRNA nearly abrogated EVI1 acetylation (Figure S2G). The region between amino acids 283 and 514 of the EVI1 protein was reported to contribute to the interaction between EVI1 and CBP.<span><sup>10</sup></span> We identified three candidate CBP KAT-specific lysine acetylation sites (lysine 359, 421 and 425) in this EVI1 region using the GPS-PAIL algorithm<span><sup>9</sup></span> (Figure S2H and I). AGS cells with silenced endogenous EVI1 were reintroduced with the wild-type EVI1 (EVI1 WT) or three EVI1 mutants (EVI1 K359R, EVI1 K421R, or EVI1 K425R) (Figure S2J). Co-IP analyses showed that the mutation of K421 rather than K359 and K425 significantly attenuated CBP-induced EVI1 acetylation (Figure S2K). The mutation of EVI1 K421 to arginine abrogated the physical binding of EVI1 to CBP or CDX2 and promoted the recruitment of the corepressor C-terminal binding protein 1 (CtBP1) to EVI1 (Figure S2L). S2</p><p>We also analysed whether the application of the CBP inhibitor could overcome the 5-FU resistance of CDX2<sup>+</sup> REG4<sup>hi</sup> GC cells. CPI-637 and 5-FU combination treatment significantly improved the efficacy of 5-FU or CPI-637 alone in AGS and MKN45 cells (Figure 4A and B). The calculation of combination index (CI) values showed that CPI-637 and 5-FU combination had a synergistic effect in CDX2<sup>+</sup> REG4<sup>hi</sup> AGS and MKN45 cells (Figure 4C), but the addition of CPI-637 didn't improve the effect of 5-FU in CDX2<sup>+</sup> REG4<sup>lo</sup> MKN28 and NCI-N87 cells (Figure 4D).</p><p>In both CDX2<sup>+</sup> REG4<sup>hi</sup> CDX and patient derived xenograft (PDX) in vivo, treatment with CPI-637 and particularly with the CPI-637 and 5-FU combination significantly inhibited tumour growth (Figure 4E and F). Furthermore, CPI-637 and 5-FU combination markedly improved mouse survival compared to 5-FU or CPI-637 alone (Figure 4E and F). We compared the tumor inhibition rate and found that 5-FU was more effective in CDX2+ REG4lo GC while CPI-637 was more effective in CDX2+ REG4hi GC (Figure 2G). The combination did not result in more systemic toxicity or weight loss than 5-FU alone (Figure 4H and I). Collectively, these data indicate that targeting CBP can overcome 5-FU resistance and thus provide a therapeutic option for efficacy improvement of 5-FU-based chemotherapy in CDX2<sup>+</sup> REG4<sup>hi</sup> GC (Figure 4J). This study provides crucial insights into the molecular mechanisms driving chemoresistance and offers potential for clinical translation.</p>\",\"PeriodicalId\":10189,\"journal\":{\"name\":\"Clinical and Translational Medicine\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":7.9000,\"publicationDate\":\"2024-10-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11511671/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Clinical and Translational Medicine\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/ctm2.70069\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and Translational Medicine","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/ctm2.70069","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
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摘要

亲爱的编辑,我们开展了一项研究,探索环-AMP反应元件结合蛋白(CBP)抑制剂在克服CDX2/REG4双阳性胃癌(GC)对5-FU化疗的化疗耐药性方面的潜力。CDX2是属于尾部相关同工酶基因家族的经典转录因子,它决定着肠道分化的发育和维持,并在部分GC中过表达。我们的研究旨在调查 CDX2+ GC(约占所有 GC 的 50%1 )的异质性,并发现潜在的治疗方法。1 我们的研究旨在调查 CDX2+ GC 的异质性,CDX2+ GC 约占所有 GC 的 50%,1 并发现潜在的治疗方法。使用 111 个 GC 样本,基于 CDX2 表达的分子分类显示 CDX2+ GC 可进一步分为两个亚型:REG4hi和REG4lo(图1A)。REG4 是 CDX2 的直接靶点,与 GC 的进展和化疗耐药有关。2 REG4hi 亚型的总生存期(OS,图 1B,危险比:CDX2hi REG4hi)明显较短:2 与REG4lo相比,REG4hi亚型的总生存期(OS,图1B,危险比:CDX2hi REG4hi vs. CDX2lo .99,95% CI .60-1.64,p = .973;CDX2hi REG4lo vs. CDX2lo .11,95% CI .04-.30,p &lt;.001;CDX2hi REG4lo vs. CDX2hi REG4hi .12,95% CI .04-.30,p &lt;.001)明显更短,分化能力更差(图1C,表S1和S2)。REG4 阳性表达与 CDX2+ 病例明显相关(图 1D)。此外,CDX2+ REG4hi GC 患者对基于 5-FU 的化疗更耐受(图 1E)。我们利用CCLE数据库鉴定了REG4高表达或低表达的CDX2+ GC细胞系(图1F)(图1G)。CDX2+ REG4hi GC细胞对5-FU的IC50远高于CDX2+ REG4lo GC细胞(图1H-J)。我们选择了CDX2和REG4表达模式与CCLE数据库提示的特定GC类型一致并经免疫印迹证实的GC细胞系(图2A)。通过筛选 17 种针对表观遗传调节因子的小分子抑制剂(表 S3),发现 CBP/p300 抑制剂 CPI-637 对 CDX2+ REG4hi GC 细胞特别有效(图 2A 和 B)。与 CDX2+ REG4lo 细胞相比,CPI-637 能明显抑制这些细胞的生长(图 2C),且 IC50 值更低(图 2D)。体内实验表明,CPI-637 能显著抑制 CDX2+ REG4hi 细胞衍生的异种 CDX 的肿瘤生长(图 2E),使肿瘤体积更小(图 2F)、肿瘤重量更轻(图 2G)、肿瘤生长抑制率更高(图 2H)、5 CBP/p300 利用其蛋白赖氨酸乙酰转移酶(KAT)结构域催化组蛋白 H3 赖氨酸 18 或赖氨酸 27 乙酰化(H3K18Ac 或 H3K27Ac),并利用其溴结构域识别组蛋白尾部的乙酰赖氨酸残基,从而激活基因表达。AGS 和 MKN28 中的 CBP 水平同样很高,而 p300 的表达却很沉默(图 S1A),这可能是由于 EP300 的无义突变所致(图 S1B)。我们进一步发现,CPI-637处理AGS和NCI-N87细胞后,H3K18Ac/H3K27Ac的减少呈剂量依赖性(图S1C和D)。在 GC 细胞系中,5 µM CPI-637 可显著降低 H3K18Ac/H3K27Ac 水平(图 S1C-E)。CPI-637 影响了 CDX2+ REG4hi GC 细胞中 REG4 的表达,但不影响 CDX2+ REG4lo 细胞中 REG4 的表达(图 S1E 和 F)。ChIP 分析显示,在对照组处理的 CDX2+ REG4hi AGS 细胞中,REG4 启动子上的 H3K27Ac 含量丰富(图 3B),而 CPI-637 处理会诱导 H3K27Me3 的转变(图 S2A)。相反,在 CDX2+ REG4lo NCI-N87 细胞中,H3K27Me3 而不是 H3K27Ac 或 H3K18Ac 特异性地富集在 REG4 启动子上,而 CPI-637 处理不会影响该启动子(图 S2B)。Co-IP实验表明,CDX2与CBP有物理相互作用(图3C),CPI-637处理或CDX2沉默会破坏这种相互作用(图3D和E),表明CBP对REG4的调控是CDX2依赖性的。GSE54129队列中的GSEA显示,GATA4、GATA6和生态病毒整合位点-1(EVI1)在CDX2+ REG4hi GC中的富集最为显著(图S2C,所有FDR q值均为0.001)。在 GSE54129 和另外两个独立的 GC 细胞系队列(GSE15455 和 GSE22183,图 S2D,表 S4)中,CDX2+ GC 两个亚型之间共鉴定出 168 个差异表达基因(DEGs)。发现 EVI1 与 CDX2 和 CBP 共同占据 REG4 启动子(图 3F 和 G)。沉默 EVI1 可抑制 REG4 的表达(图 3H 和 I)并阻断 CDX2 与 CBP 之间的相互作用(图 3J-L),这表明 EVI1 对 CDX2 将 CBP 招募到 REG4 启动子至关重要。 GATA4和GATA6在两种GC细胞亚型中的表达没有明显差异(图S2E)。我们发现 CDX2+ REG4hi AGS 细胞中 EVI1 乙酰化水平很高(图 S2G),而且通过 siRNA 沉默 CBP 几乎可以消减 EVI1 乙酰化(图 S2G)。据报道,EVI1 蛋白的 283 和 514 氨基酸之间的区域有助于 EVI1 和 CBP 之间的相互作用10 。我们使用 GPS-PAIL 算法9 在 EVI1 的这一区域鉴定了三个候选 CBP KAT 特异性赖氨酸乙酰化位点(赖氨酸 359、421 和 425)(图 S2H 和 I)。用野生型 EVI1(EVI1 WT)或三种 EVI1 突变体(EVI1 K359R、EVI1 K421R 或 EVI1 K425R)重新诱导内源性 EVI1 沉默的 AGS 细胞(图 S2J)。Co-IP 分析表明,K421 而不是 K359 和 K425 的突变显著减弱了 CBP 诱导的 EVI1 乙酰化(图 S2K)。将 EVI1 的 K421 突变为精氨酸后,EVI1 与 CBP 或 CDX2 的物理结合失效,并促进了核心抑制因子 C 端结合蛋白 1(CtBP1)与 EVI1 的招募(图 S2L)。S2我们还分析了应用CBP抑制剂能否克服CDX2+ REG4hi GC细胞对5-FU的耐药性。CPI-637和5-FU联合治疗明显提高了5-FU或CPI-637单独治疗对AGS和MKN45细胞的疗效(图4A和B)。联合指数(CI)值的计算显示,CPI-637和5-FU联合治疗在CDX2+ REG4hi AGS和MKN45细胞中具有协同作用(图4C),但在CDX2+ REG4lo MKN28和NCI-N87细胞中,加入CPI-637并不能改善5-FU的疗效(图4D)。在体内CDX2+ REG4hi CDX和患者衍生异种移植(PDX)中,使用CPI-637,特别是CPI-637和5-FU联合治疗可显著抑制肿瘤生长(图4E和F)。此外,与单独使用 5-FU 或 CPI-637 相比,CPI-637 和 5-FU 联合疗法明显提高了小鼠的存活率(图 4E 和 F)。我们比较了肿瘤抑制率,发现5-FU对CDX2+ REG4lo GC更有效,而CPI-637对CDX2+ REG4hi GC更有效(图2G)。与单用 5-FU 相比,联合用药不会导致更多的全身毒性或体重减轻(图 4H 和 I)。总之,这些数据表明,靶向 CBP 可以克服 5-FU 耐药性,从而为 CDX2+ REG4hi GC 基于 5-FU 的化疗疗效改善提供了一种治疗选择(图 4J)。这项研究为了解驱动化疗耐药的分子机制提供了重要见解,并为临床转化提供了潜力。
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Targeting CBP revers chemoresistance to 5-FU of CDX2/REG4 double-positive gastric cancer

Dear Editor,

We conducted a study exploring the potential of cyclic-AMP response element binding protein (CBP) inhibitors in overcoming the chemoresistance of CDX2/REG4 double-positive gastric cancer (GC) to 5-FU chemotherapy.

CDX2 is a classical transcription factor belonging to the caudal-related homeobox gene family, which determines the development and maintenance of intestinal differentiation in the gut and is overexpressed  in part of GC.1 Our study aims to investigate the heterogeneity of CDX2+ GC, which accounts for approximately 50% of all GC,1 and discover potential therapies. Using 111 GC samples, molecular classification based on CDX2 expression revealed that CDX2+ GC could be further divided into two subtypes: REG4hi and REG4lo (Figure 1A). REG4 is a direct target of CDX2 and has been implicated in the progression and chemoresistance of GC.2 The REG4hi subtype showed significantly shorter overall survival (OS, Figure 1B, hazard ratio: CDX2hi REG4hi vs. CDX2lo .99, 95% CI .60–1.64, p = .973; CDX2hi REG4lo vs. CDX2lo .11, 95% CI .04–.30, p < .001; CDX2hi REG4lo vs. CDX2hi REG4hi .12, 95% CI .04–.30, p < .001) and poorer differentiation compared to REG4lo (Figure 1C, Tables S1 and S2). REG4 positive expression was significantly associated with CDX2+ cases (Figure 1D). Additionally, CDX2+ REG4hi GC patients were more resistant to 5-FU-based chemotherapy (Figure 1E). We identified CDX2+ GC cell lines (Figure 1F) with high or low REG4 expression (Figure 1G) using the CCLE database. The IC50 of CDX2+ REG4hi GC cells to 5-FU were much higher than those of CDX2+ REG4lo GC cells (Figure 1H–J).

We selected GC cell lines which showed consistent expression patterns of CDX2 and REG4 with specific GC types suggested by CCLE database and confirmed by immunoblotting (Figure 2A). A screen of 17 small molecule inhibitors targeting epigenetic regulators (Table S3) identified CPI-637, a CBP/p300 inhibitor, as particularly effective against CDX2+ REG4hi GC cells (Figure 2A and B). These cells showed significant growth inhibition (Figure 2C) and lower IC50 values (Figure 2D) with CPI-637 compared to CDX2+ REG4lo cells. In vivo experiments demonstrated that CPI-637 significantly inhibited tumour growth in CDX2+ REG4hi cell derived xenograft CDX (Figure 2E), resulting in smaller tumour volumes (Figure 2F), less tumour weight (Figure 2G) and higher tumour growth inhibition rates (Figure 2H).

CPI-637 is a selective inhibitor targeting both CBP and p3003 and its role have been investigated in tumour treatment.4, 5 CBP/p300 activates gene expression using its protein lysine acetyltransferase (KAT) domain to catalyse the histone H3 lysine 18 or lysine 27 acetylation (H3K18Ac or H3K27Ac) and its bromodomain to recognise acetyl-lysine residues in histone tails.6 AGS and MKN28 had similarly high levels of CBP and silenced expression of p300 (Figure S1A) probably due to EP300 nonsense mutations (Figure S1B). p300 showed relatively low expression in MKN45 and NCI-N87 cells with no mutations (Figure S1A). We further found that H3K18Ac/H3K27Ac decreased upon CPI-637 treatment in AGS and NCI-N87 cells was in a dose-dependent manner (Figure S1C and D). Significant reductions in H3K18Ac/H3K27Ac levels with 5 µM CPI-637 were observed in GC cell lines (Figure S1C–E). CPI-637 impaired REG4 expression in CDX2+ REG4hi GC cells but did not affect REG4 expression in CDX2+ REG4lo ones (Figure S1E and F).

REG4 was identified as a direct target of CDX2 in a previous study.2, 7 Two previously reported CDX2-binding sites on the REG4 promoter located near the transcriptional start site (TSS) (Figure 3A). ChIP analyses revealed that H3K27Ac was abundant on the REG4 promoter in control-treated CDX2+ REG4hi AGS cells (Figure 3B), while CPI-637 treatment induced a transition to H3K27Me3 (Figure S2A). Conversely, in CDX2+ REG4lo NCI-N87 cells, H3K27Me3, rather than H3K27Ac or H3K18Ac, was specifically enriched on the REG4 promoter, which was not affected by CPI-637 treatment (Figure S2B). Co-IP experiments showed that CDX2 physically interacted with CBP (Figure 3C), and this interaction was disrupted by CPI-637 treatment or CDX2 silencing (Figure 3D and E), indicating that the regulation of REG4 by CBP was CDX2-dependent.

GSEA in our GSE54129 cohort revealed GATA4, GATA6 and ecotropic viral integration site-1 (EVI1) were most significantly enriched in CDX2+ REG4hi GC (Figure S2C, all FDR q value < .001). A total of 168 differentially expressed genes (DEGs) between two subtypes of CDX2+ GC were identified among GSE54129 and two other independent GC cell line cohorts (GSE15455 and GSE22183, Figure S2D, Table S4). EVI1 was found to co-occupy the REG4 promoter with CDX2 and CBP (Figure 3F and G). EVI1 silencing abrogated REG4 expression (Figure 3H and I) and blocked the interaction between CDX2 and CBP (Figure 3J–L), indicating that EVI1 was crucial for the recruitment of CBP to the REG4 promoter by CDX2. GATA4 and GATA6 were not significantly differentially expressed between the two GC cell subtypes (Figure S2E). Sequential ChIP analyses showed none of the three transcriptional factors (TFs) occupied the REG4 promoter in MKN28 and NCI-N87 cells (Figure S2F).

CBP can catalyse lysine acetylation via its KAT activity.8, 9 We found that the EVI1 acetylation level was high in CDX2+ REG4hi AGS cells (Figure S2G) and CBP silencing by siRNA nearly abrogated EVI1 acetylation (Figure S2G). The region between amino acids 283 and 514 of the EVI1 protein was reported to contribute to the interaction between EVI1 and CBP.10 We identified three candidate CBP KAT-specific lysine acetylation sites (lysine 359, 421 and 425) in this EVI1 region using the GPS-PAIL algorithm9 (Figure S2H and I). AGS cells with silenced endogenous EVI1 were reintroduced with the wild-type EVI1 (EVI1 WT) or three EVI1 mutants (EVI1 K359R, EVI1 K421R, or EVI1 K425R) (Figure S2J). Co-IP analyses showed that the mutation of K421 rather than K359 and K425 significantly attenuated CBP-induced EVI1 acetylation (Figure S2K). The mutation of EVI1 K421 to arginine abrogated the physical binding of EVI1 to CBP or CDX2 and promoted the recruitment of the corepressor C-terminal binding protein 1 (CtBP1) to EVI1 (Figure S2L). S2

We also analysed whether the application of the CBP inhibitor could overcome the 5-FU resistance of CDX2+ REG4hi GC cells. CPI-637 and 5-FU combination treatment significantly improved the efficacy of 5-FU or CPI-637 alone in AGS and MKN45 cells (Figure 4A and B). The calculation of combination index (CI) values showed that CPI-637 and 5-FU combination had a synergistic effect in CDX2+ REG4hi AGS and MKN45 cells (Figure 4C), but the addition of CPI-637 didn't improve the effect of 5-FU in CDX2+ REG4lo MKN28 and NCI-N87 cells (Figure 4D).

In both CDX2+ REG4hi CDX and patient derived xenograft (PDX) in vivo, treatment with CPI-637 and particularly with the CPI-637 and 5-FU combination significantly inhibited tumour growth (Figure 4E and F). Furthermore, CPI-637 and 5-FU combination markedly improved mouse survival compared to 5-FU or CPI-637 alone (Figure 4E and F). We compared the tumor inhibition rate and found that 5-FU was more effective in CDX2+ REG4lo GC while CPI-637 was more effective in CDX2+ REG4hi GC (Figure 2G). The combination did not result in more systemic toxicity or weight loss than 5-FU alone (Figure 4H and I). Collectively, these data indicate that targeting CBP can overcome 5-FU resistance and thus provide a therapeutic option for efficacy improvement of 5-FU-based chemotherapy in CDX2+ REG4hi GC (Figure 4J). This study provides crucial insights into the molecular mechanisms driving chemoresistance and offers potential for clinical translation.

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来源期刊
CiteScore
15.90
自引率
1.90%
发文量
450
审稿时长
4 weeks
期刊介绍: Clinical and Translational Medicine (CTM) is an international, peer-reviewed, open-access journal dedicated to accelerating the translation of preclinical research into clinical applications and fostering communication between basic and clinical scientists. It highlights the clinical potential and application of various fields including biotechnologies, biomaterials, bioengineering, biomarkers, molecular medicine, omics science, bioinformatics, immunology, molecular imaging, drug discovery, regulation, and health policy. With a focus on the bench-to-bedside approach, CTM prioritizes studies and clinical observations that generate hypotheses relevant to patients and diseases, guiding investigations in cellular and molecular medicine. The journal encourages submissions from clinicians, researchers, policymakers, and industry professionals.
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