鑑定抑制MDA-MB-231遷移與侵襲能力之分子標的

Abstract

癌症轉移在腫瘤發展的過程中是個難以治療與預測的現象，現有療法仍無法有效防止癌症轉移。目前對於抑制癌症轉移的標靶性療法仍有所不足。我們研究團隊先前發現一個擁有iodoacetamide基團的共價抑制劑I-Trp能夠對β-tubulin 354號位置的半胱胺酸進行烷基化，因此破壞β-tubulin和CCT-β間的交互作用，更進一步誘導CCT-β過度表現的癌細胞進行細胞凋亡，其中包含一株三陰性乳癌細胞株MDA-MB-231，而此株乳癌細胞株於本研究中用於測試I-Trp對其轉移能力之影響。本研究發現，低於半抑制濃度的I-Trp便能抑制MDA-MB-231細胞遷移和侵襲能力約7至8成。為了找出I-Trp於癌細胞中影響轉移能力的分子標的，本研究中利用帶有螢光基團的I-Trp探針，標記細胞中I-Trp的標靶蛋白質，再表現與癌細胞轉移有關的蛋白質進行烷基化位點研究。實驗發現被標記的其中一個蛋白為Rab5c，而Rab5c於先前報導顯示有調控癌細胞移動的能力。接著，將Rab5c的半胱胺酸位點進行突變，並表現於MDA-MB-231乳癌細胞中，測試I-Trp對其抑制遷徙和侵襲能力。實驗發現，Rab5c 20號以及64號位置的半胱胺酸會被I-Trp所烷基化，此外，若將這兩個位置的半胱胺酸突變為絲胺酸時，I-Trp便會失去抑制MDA-MB-231細胞遷徙和侵襲能力的藥效。總結而論，Rab5c以及先前研究發現的β-tubulin是I-Trp於細胞中的分子標的，而前者遭I-Trp所標的會影響癌症轉移能力，後者遭I-Trp標的則會誘導癌細胞凋亡。

Metastasis is a formidable process during tumor progression, often leading existing therapies to failure. As a result, there has been an unmet need for targeted therapies to effectively inhibit cancer metastasis. An iodoacetamide-based covalent inhibitor, I-Trp, was previously demonstrated to be capable of alkylating Cys354 of β-tubulin to disrupt the protein-protein interaction between β-tubulin and CCT-β, thus inducing apoptosis of CCT-β overexpressed cancer cells, including MDA-MB-231, a TNBC, which was used to test I-Trp on inhibiting its metastasis. In this study, a sub-IC50 concentration of I-Trp was found to decrease the migration and invasion of MDA-MB-231 by 70-80%. To identify the potential target for I-Trp inhibiting cell migration and invasion, total cellular proteins were treated with an I-Trp-derived fluorescent probe. The proteins labeled by I-Trp were further expressed in MDA-MB-231 to study their alkylating sites of I-Trp. One of the proteins, Rab5c, which has been reported to involve in metastasis, was mutated at the alkylating sites and transfected into MDA-MB-231 to test its role in I-Trp inhibiting cell migration and invasion. Cys20 and Cys64 of Rab5c were found to be alkylated by I-Trp and the alkylation resulted in suppression of MDA-MB-231 migration and invasion. In conclusion, this study combined with our previous findings show that I-Trp targets β-tubulin and Rab5c, thereby inducing cancer cell apoptosis and inhibiting cancer cell migration and invasion.