Regulators of differentiated cell fate can offer targets for managing cancer development and progression. Here, we identify Runx2 as a new regulator of epithelial cell fate in mammary gland development and breast cancer. Runx2 is expressed in the epithelium of pregnant mice in a strict temporally and hormonally regulated manner. During pregnancy, Runx2 genetic deletion impaired alveolar differentiation in a manner that disrupted alveolar progenitor cell populations. Conversely, exogenous transgenic expression of Runx2 in mammary epithelial cells blocked milk production, suggesting that the decrease in endogenous Runx2 observed late in pregnancy is necessary for full differentiation. In addition, overexpression of Runx2 drove epithelial-to-mesenchymal transition–like changes in normal mammary epithelial cells, whereas Runx2 deletion in basal breast cancer cells inhibited cellular phenotypes associated with tumorigenesis. Notably, loss of Runx2 expression increased tumor latency and enhanced overall survival in a mouse model of breast cancer, with Runx2-deficient tumors exhibiting reduced cell proliferation. Together, our results establish a previously unreported function for Runx2 in breast cancer that may offer a novel generalized route for therapeutic interventions. Cancer Res; 74(18); 1–10. ©2014 AACR.

Understanding supports for cancer stem–like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem–like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG–siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reduced GSC along with glioma growth. Our findings identify TLR9 as a functional marker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 1–11. ©2014 AACR.

Drug-induced gene expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug-repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer (CRC), for which there is a pressing need to find novel anti-metastatic compounds. We computationally predicted three novel and still unknown compounds against CRC; citalopram (an anti-depressant), troglitazone (an anti-diabetic) and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation and migration and in a subcutaneous mouse model. We found evidence that the mode-of-action of these compounds may be through inhibition of TGF-β signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of CRC. This study proposes citalopram as a potential therapeutic option for CRC patients illustrating the potential of systems pharmacology.

Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions which are inhibited by the prototypic Th-2 cytokine IL-4 and the TGF-βsuperfamily members TGF-β1 and activin-A. Interestingly, the largest subgroup of the TGF-βsuperfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling to NK cell effector functions have not been evaluated. Here we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8 which mediate BMP family member signaling. In opposition to the inhibitory effects of TGF-β1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L activated NK cells revealed that BMP signaling optimized IFN-γ and global cytokine and chemokine production; phenotypic activation and proliferation; autologous DC activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one which might be therapeutically manipulated to help eradicate tumors.

Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR–bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 1–11. ©2014 AACR.