Taussig Cancer Institute, Translational Hematology & Oncology Research, Cleveland Clinic, Cleveland, OH 44195, USA.
Matrix Biol. 2013 Mar 11;32(2):123-32. doi: 10.1016/j.matbio.2012.11.010. Epub 2012 Nov 30.
Reversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated.
5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of methylation patterns in the promoter of TSP1 and comparison of aberrantly methylated TSP1 in melanoma with non-malignant nevi identified a significantly higher frequency of promoter methylation in tumor samples from melanoma patients.
Pharmacological reversal of methylation silenced TSP1 had functional biological consequences in enhancing angiogenesis inhibition and inducing antitumor effects to decrease murine melanoma growth. Angiogenesis inhibition is an additional mechanism by which epigenetic modulators can have antitumor effects.
通过药理学手段逆转异常甲基化是通过表观遗传重编程实现治疗的一个有吸引力的目标。为了确定药物逆转甲基化可以导致血管生成的功能抑制，我们对血小板反应蛋白 1（TSP1）进行了体外和体内研究，TSP1 是一种已知的血管生成抑制剂。TSP1 在几种恶性肿瘤中被甲基化，并能抑制黑色素瘤异种移植物中的血管生成。我们分析了 5-氮杂-2′-脱氧胞苷（5-Aza-dC）对体外黑色素瘤细胞的影响，以确认启动子超甲基化的逆转和 TSP1 表达的恢复。然后我们研究了 TSP1 表达对体内新血管形成和肿瘤生长的影响。最后，为了确定潜在的临床转化，我们研究了痣和黑色素瘤组织中 TSP1 启动子区域的甲基化状态。
5-Aza-dC 降低了 DNA（胞嘧啶-5）-甲基转移酶 1（DNMT1）蛋白，逆转了启动子超甲基化，并恢复了五种黑色素瘤细胞系中的 TSP1 表达，而对正常人黑素细胞中的 TSP1 蛋白水平没有影响。在体内新血管生成研究中，将黑色素瘤细胞（A375）植入未经处理或用 5Aza-dC 处理的小鼠中。观察者对处理情况不知情，通过对肿瘤部位的血管进行计数，发现预处理肿瘤部位的肿瘤血管数量明显减少。这种差异发生在肿瘤体积出现明显差异之前，但在进一步的研究中，用 5-Aza-dC 体内处理的小鼠的平均肿瘤体积减少了 55%，与未处理的对照组相比。用 shRNA 敲低 TSP1 表达可使肿瘤诱导的血管生成增加 68%。对未处理和处理小鼠来源的肿瘤中 TSP1 启动子甲基化状态的分析表明，未处理组的 67%肿瘤和处理组的 17%肿瘤存在部分甲基化，与 A375 细胞的甲基化特异性 PCR 分析一致。对 TSP1 启动子的甲基化模式进行检查，并比较黑色素瘤中异常甲基化的 TSP1 与非恶性痣，发现黑色素瘤患者肿瘤样本中启动子甲基化的频率明显更高。
药物逆转甲基化沉默的 TSP1 在增强血管生成抑制和诱导抗肿瘤作用以减少小鼠黑色素瘤生长方面具有功能生物学意义。血管生成抑制是表观遗传调节剂具有抗肿瘤作用的另一种机制。
