Identification and characterization of CXCR4 as a modulator of progression in the MCF10AT model of proliferative breast disease

The MCF10AT series of cell lines and microarray analysis was used to identify those genes that are important to the transition from premalignant to malignant breast cancer. Specifically, microarray analysis was used to identify genes whose expression levels were altered between premalignant (MCF10AT1k.cl2) and malignant cell lines (MCF10CA1a.cl1, MCF10CA1d, MCF10DCIS.com, and MCF10CA2) and genes that were affected by estrogen treatment in premalignant cells (MCF10AT1). Gene expression changes that are common to both may be crucial for the progression of breast cancer to malignancy. Agglomerative clustering showed that premalignant cells treated with estrogen demonstrate a gene expression pattern more similar to the malignant cell lines than the premalignant cell lines. The chemokine (C-X-C) receptor 4 (CXCR4) was over-expressed in the malignant cell lines and up-regulated by estrogen in the premalignant MCF10AT1 cells. Lentiviral-mediated shRNA expression was used to knockdown expression of CXCR4 in the malignant MCF10DCIS.com and MCF10CA1a.cl1 cell lines. Knockdown was either constitutive or inducible by tetracycline treatment. In vitro, constitutive knockdown of CXCR4 in MCF10DCIS.com resulted in slower proliferation, reduced migration, and morphology on Matrigel that was more similar to premalignant cells. In vivo, xenograft of MCF10DCIS.com with constitutive knockdown of CXCR4 produced tumors that were more invasive than normal MCF10DCIS.com xenografts. Xenograft of MCF10CA1a.cl1 with constitutive CXCR4 knockdown, however, produced tumors with more differentiation compared to normal MCF10CA1a.cl1 xenografts. A MCF10DCIS.com cell line was then developed that demonstrates tetracycline inducible expression of CXCR4 shRNA. To develop an inducible CXCR4 shRNA knockdown system, MCF10DCIS.com cells were first transduced with lentivirus to produce cells that express the Tet repressor (TetR) protein. These TetR-expressing cells were used in xenografts to determine whether MCF10DCIS.com tumor development was affected by lentiviral transduction. Xenografts of 6 TetR-expressing MCF10DCIS.com clones resulted in only 1 that produced a tumor similar to the non-transduced MCF10DCIS.com. This clone was then transduced to generate a cell line with inducible CXCR4 shRNA expression. This CXCR4 shRNA inducible MCF10DCIS.com cell line can be used to further investigate the role of CXCR4 in vivo and will provide a system to investigate the importance of CXCR4 at different stages of tumor development.