Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

Abstract

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.

Constitutive Activation of Smoothened Leads to Impaired Developments of Postnatal Bone in Mice

“…This indicated that Smo activation leads to altered proliferation and differentiation. It was also reported that the frequency of regenerative stem cells in MMTV-SmoM2 mutant epithelia is decreased compared to the wild-type (Visbal et al, 2011). Here we demonstrated that a SmoM2;OCN-Cre mutant with constitutive Smo expression represses skeletal bone development and is involved in the retarded growth phenotype.…”

“…Eventually, the Smo-mediated Gli activation leads to the expression of specific genes that promote cell proliferation and differentiation. Smo is also frequently and ectopically expressed in human breast cancers (Visbal et al, 2011), while altered hedgehog signaling is implicated in approximately 20-25% of all cancers (Briscoe and Therond, 2005). Considerable evidence has shown that sustained Smo-mediated activation of hedgehog signaling leads to increased proliferation and abnormal ductal morphology of the mammary gland, although Smo activation in luminal mammary epithelial cells was found only in a small percentage (-5%) of glands (Moraes et al, 2007).…”

“…Considerable evidence has shown that sustained Smo-mediated activation of hedgehog signaling leads to increased proliferation and abnormal ductal morphology of the mammary gland, although Smo activation in luminal mammary epithelial cells was found only in a small percentage (-5%) of glands (Moraes et al, 2007). It was also suggested that Smo stimulates proliferation via a non-cell autonomous paracrine or juxtacrine mechanism (Visbal et al, 2011). However, the precise mechanism by which altered Smo expression affects cell proliferation and postnatal development is not completely defined.…”

Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila

“…25 Recent work in a mouse model for breast cancer that induced ligand-independent Hh signaling by expression of a constitutively active smo allele demonstrated a non-cell autonomous effect on proliferation that may support tumor growth. 49 Furthermore, in samples from human breast cancer patients, cells with deregulated Hh activity did not label for the proliferation marker Ki67, implying a non-cell autonomous effect of deregulated Hh signaling. 50 Although these studies focused on proliferation, we demonstrate here that deregulated Hh signaling also increases resistance to apoptosis non-cell autonomously.…”

Identification of Novel HIF1A Target Genes That Regulate Tumor Progression and Metastasis

“…The mean SFE among biological replicate experiments varied from 0.06% to 1.55% for WT cells and from 0.015% to 0.85% for KO cells. Variability in the sphere assay among biological replicates by more than a log 10 factor is not unusual [196]. More importantly, SFE within each experiment was always greater for WT cells than for KO cells.…”