Arid3a is essential to execution of the first cell fate decision via direct embryonic and extraembryonic transcriptional regulation.

Despite their origin from the inner cell mass, embryonic stem (ES) cells undergo differentiation to the trophectoderm (TE) lineage by repression of the ES cell master regulator Oct4 or activation of the TE master regulator Caudal-type homeobox 2 (Cdx2). In contrast to the in-depth studies of ES cell self-renewal and pluripotency, few TE-specific regulators have been identified, thereby limiting our understanding of mechanisms underlying the first cell fate decision. Here we show that up-regulation and nuclear entry of AT-rich interactive domain 3a (Arid3a) drives TE-like transcriptional programs in ES cells, maintains trophoblast stem (TS) cell self-renewal, and promotes further trophoblastic differentiation both upstream and independent of Cdx2. Accordingly, Arid3a(-/-) mouse post-implantation placental development is severely impaired, resulting in early embryonic death. We provide evidence that Arid3a directly activates TE-specific and trophoblast lineage-specific genes while directly repressing pluripotency genes via differential regulation of epigenetic acetylation or deacetylation. Our results identify Arid3a as a critical regulator of TE and placental development through execution of the commitment and differentiation phases of the first cell fate decision.

Role of COX-2 in tumorospheres derived from a breast cancer cell line.

BACKGROUND: Cyclooxygenase-2 (COX-2) expression in primary breast cancer predicts tumor cell dissemination to bone marrow, which is a risk factor for recurrence and distant metastasis. "Stem-like" phenotype may be important in cancer metastasis. METHODS: To investigate the role of COX-2 protein in breast cancer stem-like cells, we analyzed it by co-immunofluorescence in tumorospheres derived from the MCF7 estrogen receptor-positive breast cancer cell line. To evaluate COX-2 function we utilized a COX-2 inhibitor in a clonogenicity assay performed with tumorospheres-derived cells. RESULTS: We detected rare cells in tumorospheres (one cell per tumorosphere) with very high COX-2 expression (COX-2(high)). COX-2 transfected MCF7 cells were able to generate long-term tumorospheres culture, even though transfection efficiency was only one in a million cells. We detected expression of OCT4 in some COX-2(high) cells, supporting the hypothesis that these cells could be cancer stem-like cells. It is important that COX-2(high) cells showed less expression of Ki-67 than did neighboring cells, indicating that COX-2(high) cells may be progenitors of tumorospheres. Celecoxib inhibited the growth of tumorosphere cultures and the ability of tumorosphere-derived cells to form colonies in vitro, indicating an active role of COX-2 in these processes. However, 2 µM celecoxib failed to eradicate tumorosphere-initiating cells. Finally, we detected rare COX-2(high) cells among SUM149 inflammatory breast cancer cells growing on plastic in serum-containing medium; the SUM149 cell line produces a very high level of COX-2 protein. CONCLUSION: Our results support a role for COX-2 in stem-like breast cancer cells and suggest a mechanism behind a role for COX-2 in disseminated tumor cells, which are known to exhibit characteristic biomarkers and functional properties of stem-like cells.

A CXCR4 antagonist CTCE-9908 inhibits primary tumor growth and metastasis of breast cancer.

BACKGROUND: CXCL12/CXCR4 signaling may be involved in tumor growth and angiogenesis, and homing of cancer cells to bone and other organs. Our purpose was to determine whether inhibition of CXCR4 with a peptide-based antagonist would reduce tumor growth and metastasis of breast cancer. METHODS: We used two mouse models of breast cancer. In the first model, 1 x 10(6) MDA-MB-231 breast cancer cells transfected with luciferase were implanted into the inguinal mammary fat pad to produce primary tumors. In the second model, 1 x 10(5) MDA-231-BSC12 cells were injected into the left cardiac ventricle to produce bone metastases. CTCE-9908, a peptide analog of CXCL12 that competitively binds to CXCR4, was used to test the effect of inhibiting CXCR4. Five mice from each mouse model were treated with CTCE-9908 (25 mg/kg, injected subcutaneously 5 d/wk). All mice were assessed weekly using bioluminescent imaging to quantify relative volumes of tumor burden. RESULTS: Bioluminescencent imaging showed that the mice treated with CTCE-9908 had significantly less primary tumor burden than the control mice. At 5 and 6 wk, the mice treated with CTCE-9908 had a 7-fold reduction and 5-fold reduction in primary tumor burden, respectively. Treatment with CTCE-9908 also significantly inhibited the rate of metastases compared with the control group. At 5 and 6 wk, the mice treated with CTCE-9908 demonstrated a 9-fold reduction and 20-fold reduction in metastatic tumor burden, respectively. CONCLUSION: Treatment with the CXCR4 antagonist CTCE-9908 significantly reduced metastasis as well as primary tumor growth in mouse models of breast cancer.

Evaluation of a CXCR4 antagonist in a xenograft mouse model of inflammatory breast cancer.

CXCL12/CXCR4 signaling, being important in the homing of cancer cells to lungs, bone and other organs, is a promising therapeutic target. Our purpose was to determine whether a peptide-based antagonist of CXCR4 would reduce primary tumor growth and/or metastasis in a preclinical mouse model of inflammatory breast cancer. We improved an existing model of inflammatory breast cancer for this study by luciferase transfection of SUM149 cells and the monitoring of such cells in mice by imaging and the luciferase assay. We implanted 2 x 10(6) SUM49-Luc cells along with matrigel into the left thoracic mammary fat pad of nude mice to produce tumors. Our mouse model exhibited important features of inflammatory breast cancer, namely, aggressive local disease, local metastases and distant metastases. To evaluate the efficacy of a CXCR4 antagonist CTCE-9908, by itself or in combination with paclitaxel, we treated groups of ten mice each with CTCE-9908 (25 mg/kg, injected subcutaneously 5 days/week), control peptide SC-9908, paclitaxel (10 mg/kg, injected subcutaneously twice a week), and CTCE-9908 plus paclitaxel concurrently. We assessed all mice weekly by whole-body luciferase imaging to quantify relative primary tumor burden and distant metastases. At the end of the experiment, we quantified primary tumors by weight and lung metastases by luciferase activity assay on tissue lysates. Paclitaxel, a known chemotherapeutic, inhibited primary tumor growth in our model (P < 0.05). CTCE-9908 did not significantly inhibit primary tumor growth or lung metastases as compared to control groups, without or with paclitaxel (P > 0.05). However, CTCE-9908 as a single therapy inhibited organ-specific metastasis to leg (P < 0.05 by chi-squared test and by two-sample t-test).

Cyclooxygenase-2 induces genomic instability, BCL2 expression, doxorubicin resistance, and altered cancer-initiating cell phenotype in MCF7 breast cancer cells.

INTRODUCTION: Cyclooxygenase (COX2) expression in primary breast cancer correlates with a worse prognosis. We reported previously that COX2 expression in MCF10A breast epithelial cells of basal subtype induces genomic instability. To understand the role of COX2 in estrogen receptor-positive (non-basal) breast cancer, we transfected the MCF7 cell line with COX2 and analyzed its chromosomal profile, BCL2 protein expression, and resistance to doxorubicin. We also analyzed cell cultures grown as mammospheres to determine whether COX2 expression affects the cancer-initiating ("stem") cell phenotype in MCF7 cells. METHODS: MCF7 Tet On cells (obtained from Clontech) were stably transfected with pTRE2pur-COX2 or pTRE2pur-COX2-GFP to produce COX2 or COX2-GFP protein, respectively. BCL2 protein was detected by Western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. Groups were compared using Chi-Square test. We analyzed the genomic instability phenotype by chromosome analysis of control and COX2 transfected metaphase-arrested MCF7 cells after Giemsa staining. We assessed the tumorigenic potential of cells grown as mammospheres with a clonogenic assay. RESULTS: Cytogenetic analysis of early passage COX2 transfected MCF7 cells demonstrated significant genomic instability as compared to parental MCF7 cells. COX2 overexpression was associated with a significant increase in chromosomal aberrations (chromatid breaks, chromosome fusions, C anaphase). COX2 transfected MCF7 cells produced a significantly higher level of the anti-apoptotic protein BCL2 than the parental cells. In a functional assay, we found that COX2 expression correlated with increased resistance to doxorubicin. In a complimentary approach to determine tumorigenic potential of cells, we found that COX2 increased the ability of MCF7 cells to grow as mammospheres in culture, which correlated with an increase in clonogenic efficiency. CONCLUSIONS: We found that COX2 expression in MCF7 breast cancer cells induced genomic instability, BCL2 expression, and doxorubicin resistance, thus making them significantly more tumorigenic. This data suggests that COX-2 may be an important target for breast cancer treatment.