Triple-negative breast cancer (TNBC) is a highly heterogeneous and recurrent subtype of breast cancer that lacks an effective targeted therapy. To identify candidate therapeutic targets, we profiled global gene expression in TNBC and breast tumor-initiating cells with a patient survival dataset. Eight TNBC-related kinases were found to be overexpressed in TNBC cells with stem-like properties. Among them, expression of PKC-α, MET, and CDK6 correlated with poorer survival outcomes. In cases coexpressing two of these three kinases, survival rates were lower than in cases where only one of these kinases was expressed. In functional tests, two-drug combinations targeting these three kinases inhibited TNBC cell proliferation and tumorigenic potential in a cooperative manner. A combination of PKC-α-MET inhibitors also attenuated tumor growth in a cooperative manner in vivo. Our findings define three kinases critical for TNBC growth and offer a preclinical rationale for their candidacy as effective therapeutic targets in treating TNBC. Cancer Res; 74(17); 1–14. ©2014 AACR.

The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are coexpressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed-forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development. Cancer Res; 74(17); 1–11. ©2014 AACR.

Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer. Cancer Res; 74(17); 1–10. ©2014 AACR.

Despite its aggressive nature, triple-negative breast cancer (TNBC) often exhibits leucocyte infiltrations that correlate with favorable prognosis. In this study, we offer an explanation for this apparent conundrum by defining TNBC cell subsets that overexpress the IL15 immune receptor IL15RA. This receptor usually forms a heterotrimer with the IL2 receptors IL2RB and IL2RG, which regulates the proliferation and differentiation of cytotoxic T cells and NK cells. However, unlike IL15RA, the IL2RB and IL2RG receptors are not upregulated in basal-like TNBC breast cancer cells that express IL15RA. Mechanistic investigations indicated that IL15RA signaling activated JAK1, STAT1, STAT2, AKT, PRAS40, and ERK1/2 in the absence of IL2RB and IL2RG, whereas neither STAT5 nor JAK2 were activated. RNAi-mediated attenuation of IL15RA established its role in cell growth, apoptosis, and migration, whereas expression of the IL15 cytokine in IL15RA-expressing cells stimulated an autocrine signaling cascade that promoted cell proliferation and migration and blocked apoptosis. Notably, coexpression of IL15RA and IL15 was also sufficient to activate peripheral blood mononuclear cells upon coculture in a paracrine signaling manner. Overall, our findings offer a mechanistic explanation for the paradoxical association of some high-grade breast tumors with better survival outcomes, due to engagement of the immune stroma. Cancer Res; 74(17); 1–14. ©2014 AACR.

A central confounding factor in the development of targeted therapies is tumor cell heterogeneity, particularly in tumor-initiating cells (TIC), within clinically identical tumors. Here, we show how activation of the Sonic Hedgehog (SHH) pathway in neural stem and progenitor cells creates a foundation for tumor cell evolution to heterogeneous states that are histologically indistinguishable but molecularly distinct. In spontaneous medulloblastomas that arise in Patched (Ptch)+/− mice, we identified three distinct tumor subtypes. Through cell type–specific activation of the SHH pathway in vivo, we determined that different cells of origin evolved in unique ways to generate these subtypes. Moreover, TICs in each subtype had distinct molecular and cellular phenotypes. At the bulk tumor level, the three tumor subtypes could be distinguished by a 465-gene signature and by differential activation levels of the ERK and AKT pathways. Notably, TICs from different subtypes were differentially sensitive to SHH or AKT pathway inhibitors, highlighting new mechanisms of resistance to targeted therapies. In summary, our results show how evolutionary processes act on distinct cells of origin to contribute to tumoral heterogeneity, at both bulk tumor and TIC levels. Cancer Res; 74(17); 1–11. ©2014 AACR.