Robertson Fredika M, Simeone Ann-Marie, Mazumdar Abhijit, Shah Ashish H, McMurray John S, Ghosh Sukhen, Cristofanilli Massimo
Department of Experimental Therapeutics, 1515 Holcombe Blvd, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
J Exp Ther Oncol. 2008;7(4):299-312.
Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC) characterized by rapid growth and aggressive invasion with no selective therapies developed to treat IBC. Cyclooxygenase-2 (Cox-2), which produces prostaglandin E2 (PGE2) is known to be upregulated in primary IBC tumors and metastatic lesions, however the use of selective Cox-2 inhibitors has diminished due to cardiovascular side effects. One alternative approach to targeting Cox-2 enzyme activity is to block binding of the PGE2 ligand to its prostanoid (EP) receptors, which are designated as EP1, EP2, EP3, and EP4 and are members of a subfamily of G protein coupled receptors (GPCRs). While SUM149 IBC tumor cells and MCF-7 non-IBC breast tumor cells produce both EP2 and EP4 receptors, the invasive MDA-MB-231 non-IBC breast tumor cells produced low but detectable levels of these receptors. PGE2 and the EP4 agonist, PGE2 alcohol, stimulated significantly increased (p < 0.05) levels of proliferation and invasion by SUM149 IBC tumor cells, with no effect on proliferation of either of the two non-IBC breast tumor cell lines. In contrast, the EP2 agonist butaprost had no effect on proliferation or invasion of any cell line examined. The selective EP4 antagonist, GW627368X, induced inhibition of proliferation and invasion of human SUM149 IBC tumor cells beginning at 0.1 microM, with inhibition of proliferation and invasion by MDA-MB-231 non-IBC cells at higher concentrations of GW627368X. Molecular knockdown of the EP4 receptor was accomplished by stable transfection of an EP4 short hairpin RNA (shRNA) construct, with a clonally derived cell line designated as SUM149/Clone 1 exhibiting significantly slowed proliferation and diminished invasion compared to SUM149/Vector 5 which contained a scrambled shRNA control vector. This is the first report using both a selective pharmacologic inhibitor and a molecular shRNA knockdown approach to demonstrate that EP4 is directly involved in regulation of proliferation and invasion of IBC cells.
炎性乳腺癌（IBC）是局部晚期乳腺癌（LABC）中最具侵袭性的形式，其特征是生长迅速且侵袭性强，目前尚无针对IBC的特异性治疗方法。已知可产生前列腺素E2（PGE2）的环氧合酶-2（Cox-2）在原发性IBC肿瘤和转移病灶中上调，然而，由于心血管副作用，选择性Cox-2抑制剂的使用已减少。靶向Cox-2酶活性的另一种方法是阻断PGE2配体与其前列腺素（EP）受体的结合，这些受体被命名为EP1、EP2、EP3和EP4，属于G蛋白偶联受体（GPCR）亚家族。虽然SUM149 IBC肿瘤细胞和MCF-7非IBC乳腺肿瘤细胞均产生EP2和EP4受体，但侵袭性MDA-MB-231非IBC乳腺肿瘤细胞产生的这些受体水平较低但可检测到。PGE2和EP4激动剂PGE2醇可显著刺激SUM149 IBC肿瘤细胞的增殖和侵袭水平增加（p < 0.05），对两种非IBC乳腺肿瘤细胞系的增殖均无影响。相比之下，EP2激动剂布他前列素对所检测的任何细胞系的增殖或侵袭均无影响。选择性EP4拮抗剂GW627368X从0.1 microM开始诱导抑制人SUM149 IBC肿瘤细胞的增殖和侵袭，在更高浓度的GW627368X下可抑制MDA-MB-231非IBC细胞的增殖和侵袭。通过稳定转染EP4短发夹RNA（shRNA）构建体实现EP4受体的分子敲低，与含有乱序shRNA对照载体的SUM149/载体5相比，克隆衍生的细胞系SUM149/克隆1表现出增殖明显减慢和侵袭减少。这是首次使用选择性药理抑制剂和分子shRNA敲低方法证明EP4直接参与IBC细胞增殖和侵袭调控的报告。
