The cell-biological program termed the epithelial-mesenchymal transition (EMT) confers on cancer cells mesenchymal traits and an ability to enter the cancer stem cell (CSC) state. However, the interactions between CSCs and their surrounding microenvironment are poorly understood. Here we show that tumor-associated monocytes and macrophages (TAMs) create a CSC-niche via juxtacrine signaling with CSCs. We performed quantitative proteomic profiling and found that the EMT program upregulates the expression of CD90/Thy1 and EphA4, which mediate the physical interactions of CSCs with TAMs by directly binding with their respective counter-receptors on these cells. In response, the EphA4 receptor on the carcinoma cells activates Src and NF-κB, the latter results in the secretion of a variety of cytokines by the CSCs; these cytokines serve to sustain the stem-cell state. Indeed, admixed macrophages enhance the CSC activities of carcinoma cells. These findings underscore the significance of TAMs as important components of the CSC niche.

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.

SUMMARY We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40–70% of breakpoints at proB/pre-B stage translocation regions – specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage-specificity, lineage-specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.

SUMMARY The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs, but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling, and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs.