Whipple Rebecca A, Vitolo Michele I, Boggs Amanda E, Charpentier Monica S, Thompson Keyata, Martin Stuart S
Breast Cancer Res. 2013;15(5):R83. doi: 10.1186/bcr3477.
Detyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-κB) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments.
Using metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-κB inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-κB inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-κB inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study.
Sesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-κB. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network.
Our data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-κB inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.
去酪氨酸化微管蛋白是α-微管蛋白的一种翻译后修饰，也是稳定微管的一个标志，鉴于其与肿瘤进展、侵袭性和化疗耐药性相关，最近受到了关注。我们最近还报道，上皮-间质转化（EMT）通过抑制微管蛋白酪氨酸连接酶（TTL）促进微管蛋白去酪氨酸化。此外，富含去酪氨酸化微管蛋白的膜突起，即微触须（McTN），有助于肿瘤细胞重新附着于内皮细胞层。鉴于EMT与炎症和癌症进展相关，我们在一组人乳腺癌细胞上测试了抗炎核因子-κB（NF-κB）抑制剂，以研究它们对去酪氨酸化微管蛋白的影响，从而确定更具特异性的针对微管蛋白的抗癌治疗方法。
我们使用转移性人乳腺癌细胞MDA-MB-157、MDA-MB-436和Bt-549，通过蛋白质表达分析和免疫荧光测量NF-κB抑制剂小白菊内酯、木香烯内酯和白藜芦醇对去酪氨酸化微管蛋白的影响。进行荧光素酶报告基因检测和活力筛选，以确定这些作用是否与其NF-κB抑制特性相关，或是凋亡的结果。利用微电子传感器阵列上的电阻抗实时监测细胞与基质的附着情况。在整个研究过程中，我们将NF-κB抑制剂特异性靶向去酪氨酸化微管蛋白的选择性与传统的靶向微管蛋白的治疗药物紫杉醇和秋水仙碱进行了比较。
倍半萜内酯类化合物小白菊内酯和木香烯内酯选择性降低去酪氨酸化微管蛋白，而与它们对NF-κB的抑制作用无关。对用小白菊内酯和木香烯内酯处理的悬浮细胞进行活细胞评分，结果显示微触须的频率降低且重新附着受到抑制。结构分析表明，小白菊内酯和木香烯内酯可以减少去酪氨酸化微管，而不会显著破坏整体微管网络或细胞活力。紫杉醇和秋水仙碱对微管网络有不加区分的破坏作用。
我们的数据表明，用小白菊内酯和木香烯内酯选择性靶向去酪氨酸化微管蛋白可以降低微触须频率并抑制肿瘤细胞重新附着。这些作用与其对NF-κB的抑制作用无关，呈现出一种新的抗癌特性和治疗机会，即选择性靶向循环肿瘤细胞中稳定的微管子集，以降低乳腺癌患者的转移潜能，同时毒性更小。
