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Breast Cancer Res. 2011 Sep 14;13(5):R88. doi: 10.1186/bcr2945.
It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs, however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal.
Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation.
Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment.
We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.
据推测，乳腺癌干细胞（bCSCs）介导疾病复发并驱动远处转移的形成——这是乳腺癌患者死亡的主要原因。然而，由于 bCSCs 的异质性和对现有治疗方法的耐药性，靶向 bCSCs 的治疗受到了阻碍。为了确定能够选择性地从乳腺癌中去除 bCSCs 的策略，而不论其临床亚型如何，我们寻找一种凋亡机制，该机制将靶向 bCSCs，但不会杀死正常细胞。细胞 FLICE 样抑制蛋白（c-FLIP）的抑制部分使乳腺癌细胞对抗癌剂肿瘤坏死因子相关凋亡诱导配体（TRAIL）敏感。在这里，我们在乳腺癌细胞系中证明，bCSCs 对这种促凋亡途径的去抑制非常敏感，导致实验性转移的显著减少和 bCSC 自我更新的丧失。
在四种乳腺癌细胞系中通过 siRNA（FLIPi）抑制 c-FLIP，并在鼠乳腺中通过条件性基因敲除抑制 c-FLIP。通过互补细胞凋亡测定法，包括一种新的异质细胞测定法，确定这些细胞对 TRAIL 的敏感性，而癌症干细胞亚群的肿瘤起始潜能则通过乳腺球体培养、醛氟测定和体内移植来确定。
c-FLIP 的遗传抑制导致 TRAIL 耐药癌细胞系对 TRAIL 的促凋亡作用部分敏感，而不论其细胞表型如何，但正常乳腺上皮细胞仍对杀伤有抗性。虽然在 TRAIL/FLIPi 处理后，10%至 30%的癌细胞群仍然存活，但随后的乳腺球体和醛氟测定表明，这种促凋亡刺激物选择性地靶向功能 bCSC 池，消除干细胞更新。这导致移植后原发性肿瘤减少 80%，转移减少 98%。残留肿瘤起始能力的复发与这样一种观察结果一致，即经处理的贴壁培养物在体外重新获得了 bCSC 样特性。然而，重要的是，在重复 TRAIL/FLIPi 治疗后，这种复发的 bCSC 活性被减弱。
我们描述了一种凋亡机制，该机制可选择性且反复地从乳腺癌细胞系中去除 bCSC 活性，并表明联合 TRAIL/FLIPi 治疗可能预防广泛的乳腺癌亚型的转移性疾病进展。
