Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

Abstract

The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorageindependent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in ~50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.

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

“…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.…”

Detection and analysis of mammary gland stem cells

“…Studies from the neural field have demonstrated that neurosphere formation overestimates stem cell frequency (by about 30-50-fold) and that all but the largest neurospheres are derived from non-stem progenitor cells [34,35]. A similar phenomenon appears to be occurring when mammary cells are placed into culture, since transplantation of individual mouse-derived mammospheres into cleared mammary fat pads results in only a 15% engraftment success rate [36]. Unfortunately, it is not clear at this point which cells in the mammary epithelial cell hierarchy can function as mammosphere-initiating cells.…”

“…These frequencies have ranged widely from 1 MRU in 100 total cells to <1 in 4900 cells [9,16,18,36]. Presumably this large variation is due to the different methods used to dissociate the mammary tissue and to transplant the cells; however, it is not known whether these different dissociation and transplantation protocols are liberating different functional classes of MRUs.…”

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

“…Gli1 itself may induce early osteoblast differentiation in a Runx2-independent manner and play key roles with Gli2 and Gli3 in osteogenesis (Hojo et al, 2012). Shh signaling is also involved in tumorigenesis in several cancers, particularly in breast cancers (Moraes et al, 2007). Because of this, considerable interest in elucidating the cellular and molecular mechanisms involved in Smo-mediated signaling has recently emerged.…”

“…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). It was also suggested that Smo stimulates proliferation via a non-cell autonomous paracrine or juxtacrine mechanism (Visbal et al, 2011).…”