Lutz Lisa, Fitzner Ingrid Coutiño, Ahrens Theresa, Geißler Anna-Lena, Makowiec Frank, Hopt Ulrich T, Bogatyreva Lioudmila, Hauschke Dieter, Werner Martin, Lassmann Silke
Department of Pathology, All University Medical Center Freiburg, Germany.
Department of Pathology, All University Medical CenterFreiburg, Germany; Faculty of Biology, University of FreiburgFreiburg, Germany.
Am J Cancer Res. 2016 Feb 15;6(3):664-76. eCollection 2016.
Little is known about histone modifiers and histone marks in colorectal cancers (CRC). The present study therefore addressed the role of histone acetylation and histone deacetylases (HDAC) in CRCs in situ and in vitro. Immunohistochemistry of primary CRCs (n=47) revealed that selected histone marks were frequently present (H3K4me3: 100%; H3K9me3: 77%; H3K9ac: 75%), partially displayed intratumoral heterogeneity (H3K9me3; H3K9ac) and were significantly linked to higher pT category (H3K9me3: p=0.023; H3K9ac: p=0.028). Furthermore, also HDAC1 (62%), HDAC2 (100%) and HDAC3 (72%) expression was frequent, revealing four CRC types: cases expressing 1) HDAC1, HDAC2 and HDAC3 (49%), 2) HDAC2 and HDAC3 (30%), 3) HDAC1 and HDAC2 (10.5%) and 4) exclusively HDAC2 (10.5%). Correlation to clinico-pathological parameters (pT, pN, G, MSI status) revealed that heterogeneous HDAC1 expression correlated with lymph node status (p=0.012). HDAC expression in situ was partially reflected by six CRC cell lines, with similar expression of all three HDACs (DLD1, LS174T), preferential HDAC2 and HDAC3 expression (SW480, Caco2) or lower HDAC2 and HDAC3 expression (HCT116, HT29). HDAC activity was variably higher in HCT116, HT29, DLD1 and SW480 compared to LS174T and Caco2 cells. Treatment with broad (SAHA) and specific (MS-275; FK228) HDAC inhibitors (HDACi) caused loss of cell viability in predominantly MSIpositive CRC cells (HCT116, LS174T, DLD1; SAHA, MS-275 and in part FK228). In contrast, MSI-negative CRC cells (Caco2, HT29, SW480) were resistant, except for high doses of FK228 (Caco2, HT29). Cell viability patterns were not linked to different efficacies of HDACi on reduction of HDAC activity or histone acetylation, p21 expression and/or induction of DNA damage (γH2A-X levels). In summary, this study reveals inter- and intra-tumoral heterogeneity of histone marks and HDAC expression in CRCs. This is reflected by diverse HDACi responses in vitro, which do not follow known modes of action. Together, this implies further exploitation of histone alterations in CRC for molecular classification and/or novel treatment options.
关于结直肠癌（CRC）中的组蛋白修饰因子和组蛋白标记，人们了解甚少。因此，本研究探讨了组蛋白乙酰化和组蛋白去乙酰化酶（HDAC）在CRC原位和体外的作用。对原发性CRC（n = 47）进行免疫组织化学分析发现，所选的组蛋白标记经常出现（H3K4me3：100%；H3K9me3：77%；H3K9ac：75%），部分表现出肿瘤内异质性（H3K9me3；H3K9ac），并且与较高的pT分期显著相关（H3K9me3：p = 0.023；H3K9ac：p = 0.028）。此外，HDAC1（62%）、HDAC2（100%）和HDAC3（72%）的表达也很常见，揭示了四种CRC类型：1）表达HDAC1、HDAC2和HDAC3的病例（49%），2）表达HDAC2和HDAC3的病例（30%），3）表达HDAC1和HDAC2的病例（10.5%），4）仅表达HDAC2的病例（10.5%）。与临床病理参数（pT、pN、G、微卫星不稳定性状态）的相关性分析表明，HDAC1的异质性表达与淋巴结状态相关（p = 0.012）。六种CRC细胞系部分反映了HDAC在原位的表达情况，其中所有三种HDAC的表达相似（DLD1、LS174T），HDAC2和HDAC3优先表达（SW480、Caco2），或者HDAC2和HDAC3表达较低（HCT116、HT29）。与LS174T和Caco2细胞相比，HCT116、HT29、DLD1和SW480细胞中的HDAC活性变化较大且更高。用广谱（SAHA）和特异性（MS - 275；FK228）HDAC抑制剂（HDACi）处理主要导致微卫星高度不稳定的CRC细胞（HCT116、LS174T、DLD1；SAHA、MS - 275以及部分FK228）的细胞活力丧失。相比之下，微卫星稳定的CRC细胞（Caco2、HT29、SW480）具有抗性，但高剂量的FK228对Caco2和HT29细胞有效。细胞活力模式与HDACi在降低HDAC活性或组蛋白乙酰化、p21表达和/或诱导DNA损伤（γH2A - X水平）方面的不同效果无关。总之，本研究揭示了CRC中组蛋白标记和HDAC表达的肿瘤间和肿瘤内异质性。这在体外表现为对不同HDACi的多样反应，且这些反应并不遵循已知的作用模式。综上所述，这意味着可进一步利用CRC中的组蛋白改变进行分子分类和/或开发新的治疗方案。
