Notch signaling through Jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells but must be switched off to complete chondrogenesis.

We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three-dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14-day chondrogenic cultures showed that the Notch ligand Jagged-1 (Jag-1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY-1, was also expressed, with a temporal profile that closely followed the expression of Jag-1, and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut-down in Notch signaling was critical for full chondrogenesis, as adenoviral human Jag-1 transduction of hMSC, which caused continuous elevated expression of Jag-1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures, there was inhibited production of extracellular matrix, and the gene expression of aggrecan and type II collagen were strongly suppressed; this may reflect the retention of a prechondrogenic state. The JAG-1-mediated Notch signaling was also shown to be necessary for chondrogenesis, as N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) added to cultures on days 0-14 or just days 0-5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag-1-mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed.

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin.

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.

Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-α status.

Tumor hypoxia is often linked to decreased survival in patients with breast cancer and current therapeutic strategies aim to target the hypoxic response. One way in which this is done is by blocking hypoxia-induced angiogenesis. Antiangiogenic therapies show some therapeutic potential with increased disease-free survival, but these initial promising results are short lived and followed by tumor progression. We hypothesized that this may be due to altered cancer stem cell (CSC) activity resulting from increased tumor hypoxia. We studied the effects of hypoxia on CSC activity, using in vitro mammosphere and holoclone assays as well as in vivo limiting dilution experiments, in 13 patient-derived samples and four cell lines. There was a HIF-1α-dependent CSC increase in ER-α-positive cancers following hypoxic exposure, which was blocked by inhibition of estrogen and Notch signaling. A contrasting decrease in CSC was seen in ER-α-negative cancers. We next developed a xenograft model of cell lines and patient-derived samples to assess the hypoxic CSC response. Varying sizes of xenografts were collected and analyzed for HIF1-α expression and CSC. The same ER-α-dependent contrasting hypoxic-CSC response was seen validating the initial observation. These data suggest that ER-α-positive and negative breast cancer subtypes respond differently to hypoxia and, as a consequence, antiangiogenic therapies will not be suitable for both subgroups.

Breast cancer stem cells: something out of notching?

We and others have established that the developmental Notch receptor signaling pathway is active in breast cancer cell lines, as well as in preinvasive and invasive primary samples. Recently, a role for Notch in regulating the hierarchy of stem and progenitor cells in both normal and cancer epithelium has been elucidated. Because inhibiting the Notch receptor signaling pathway is a possible future breast cancer therapy, here, we review the expression and activity of the different ligands and receptors and summarize the various ways in which the pathway's activity can be inhibited, and the likely effects of inhibition on different tumor cell subpopulations.

Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor.

Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.

Wnt proteins in mammary development and cancer.

Secreted proteins of the Wnt family play widespread roles in the regulation of embryonic development, and aberrant activation of the canonical Wnt/beta-catenin pathway is one of the most frequent signaling abnormalities known in human cancer. While the consequences of Wnt signaling in development are diverse at the cellular level, they are often concerned with cell fate determination. Recent data also indicate that Wnt proteins influence the self-renewal of stem cells in certain tissues. In the mammary gland, Wnt signals are strongly implicated in initial development of the mammary rudiments, and in the ductal branching and alveolar morphogenesis that occurs during pregnancy. Transgenic expression of Wnt1 or Wnt10b in the mouse mammary gland leads to lobuloalveolar hyperplasia with a major risk of progression to carcinoma. Recent evidence suggests that this phenotype is associated with expansion of a multipotent progenitor cell population. In human breast cancer, evidence of beta-catenin accumulation implies that the canonical Wnt signaling pathway is active in over 50% of carcinomas. However, specific mutations that might account for this activation of signaling have not yet been identified.