Dai Meiou, Al-Odaini Amal A, Arakelian Ani, Rabbani Shafaat A, Ali Suhad, Lebrun Jean-Jacques
Breast Cancer Res. 2012 Sep 20;14(5):R127. doi: 10.1186/bcr3322.
Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.
A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.
We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.
Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.
肿瘤细胞迁移和侵袭是乳腺癌转移过程中的关键起始步骤，而乳腺癌转移是乳腺癌发病和死亡的主要原因。在此，我们研究了核心细胞周期机制成员p21Cip1（p21）在转化生长因子-β（TGFβ）介导的乳腺癌细胞迁移和侵袭中的作用。
使用乳腺脂肪垫异种移植小鼠模型评估乳腺肿瘤生长和局部侵袭。用5 ng/ml TGFβ处理三阴性人乳腺癌细胞系MDA-MB231及其亚子代SCP2和SCP25、SUM159PT、SUM149PT、SUM229PE和SUM1315MO2，通过蛋白质印迹法检测蛋白质表达水平。使用划痕/伤口愈合和Transwell实验检测细胞迁移和侵袭。使用荧光素酶实验通过TGFβ/Smad报告构建体（CAGA12-luc）测量TGFβ转录活性。q-PCR用于评估TGFβ下游靶基因。通过共免疫沉淀进行p21、p/CAF和Smad3之间的相互作用。此外，使用生物素化的Smad结合元件DNA探针通过DNA免疫沉淀测量Smad3对DNA的结合能力。最后，通过免疫组织化学在包含50个浸润性导管乳腺癌肿瘤的组织芯片中检测活性TGFβ/Smad信号传导、p21和p/CAF与淋巴结转移之间的关联，其中25个为淋巴结阳性。
我们发现p21表达与乳腺癌患者较差的总生存期和无远处转移生存期相关。此外，使用异种移植动物模型和体外研究，我们发现p21对肿瘤细胞侵袭至关重要。发现p21的侵袭作用与TGFβ下游的Smad3和p/CAF相互作用相关。p21和p/CAF通过控制Smad3乙酰化、DNA结合和转录活性来调节TGFβ介导的促转移基因转录。此外，我们发现使用乳腺癌患者的组织芯片，活性TGFβ/Smad信号传导与高p21和p/CAF表达水平以及淋巴结受累相关。
这些结果共同突出了p21和p/CAF在转录水平促进乳腺癌细胞迁移和侵袭中的重要作用，并可能为乳腺癌治疗开辟新途径。
