Research Area, Instituto de Oncología "Angel H. Roffo", Ciudad de Buenos Aires, Argentina.
Members of the Research Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad de Buenos Aires, Argentina.
Cancer Res Treat. 2017 Oct;49(4):869-879. doi: 10.4143/crt.2016.378. Epub 2016 Dec 6.
We investigated the effects of laminin on the fraction of cells with self-renewing capacity in the estrogen-dependent, tamoxifen-sensitive LM05-E breast cancer cell line. We also determined whether laminin affected the response to tamoxifen.
The LM05-E breast cancer cell line was used as a model for all experiments. Aldehyde dehydrogenase (ALDH) activity, clonogenic and mammosphere assays were performed to measure the effects of laminin on modulation of the stem cell subpopulation. Pluripotent gene expression was analyzed by reverse transcriptase-polymerase chain reaction. The involvement of the mitogen-activated protein kinase (MAPK)/ERK pathway was determined using specific inhibitors. The effects of laminin on the response to tamoxifenwere determined and the involvement of α6 integrin was investigated.
We found that pretreatment with laminin leads to a decrease in cells with the ability to form mammospheres that was accompanied by a decrease in ALDH activity. Moreover, exposure of mammospheres to laminin reduced the capacity to form secondary mammospheres and decreased the expression of Sox-2, Nanog, and Oct-4. We previously reported that 4-OH-tamoxifen leads to an increase in the expression of these genes in LM05-E cells. Treatment with signaling pathway inhibitors revealed that the MAPK/ERK pathway mediates the effects of laminin. Finally, laminin induced tamoxifen resistance in LM05-E cells through α6 integrin.
Our results suggest that the final number of cells with self-renewing capacity in estrogen-dependent breast tumors may result from the combined effects of endocrine treatment and microenvironmental cues.
我们研究了层粘连蛋白对依赖雌激素的他莫昔芬敏感的 LM05-E 乳腺癌细胞系中具有自我更新能力的细胞比例的影响。我们还确定了层粘连蛋白是否影响他莫昔芬的反应。
LM05-E 乳腺癌细胞系被用作所有实验的模型。醛脱氢酶（ALDH）活性、集落形成和乳腺球体测定用于测量层粘连蛋白对调节干细胞亚群的影响。多能基因表达通过逆转录-聚合酶链反应进行分析。使用特定的抑制剂确定丝裂原活化蛋白激酶（MAPK）/ERK 途径的参与。确定层粘连蛋白对他莫昔芬反应的影响，并研究α6 整合素的参与。
我们发现，层粘连蛋白预处理会导致形成乳腺球体的能力下降，伴随 ALDH 活性下降。此外，暴露于层粘连蛋白的乳腺球体减少了形成二级乳腺球体的能力，并降低了 Sox-2、Nanog 和 Oct-4 的表达。我们之前报道过，4-OH-他莫昔芬会导致 LM05-E 细胞中这些基因的表达增加。信号通路抑制剂的处理表明，MAPK/ERK 途径介导了层粘连蛋白的作用。最后，层粘连蛋白通过α6 整合素诱导 LM05-E 细胞对他莫昔芬产生耐药性。
我们的结果表明，雌激素依赖性乳腺癌肿瘤中具有自我更新能力的细胞总数可能是内分泌治疗和微环境线索综合作用的结果。
