MMTV-Wnt1 and -ΔN89β-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin activate canonical Wnt signaling within distinct cell-types. ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER−PR−CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand

Gli activity is critical at multiple stages of embryonic mammary and nipple development.

Gli3 is a transcriptional regulator of Hedgehog (Hh) signaling that functions as a repressor (Gli3(R)) or activator (Gli3(A)) depending upon cellular context. Previously, we have shown that Gli3(R) is required for the formation of mammary placodes #3 and #5. Here, we report that this early loss of Gli3 results in abnormal patterning of two critical regulators: Bmp4 and Tbx3, within the presumptive mammary rudiment (MR) #3 zone. We also show that Gli3 loss leads to failure to maintain mammary mesenchyme specification and loss of epithelial Wnt signaling, which impairs the later development of remaining MRs: MR#2 showed profound evagination and ectopic hairs formed within the presumptive areola; MR#4 showed mild invagination defects and males showed inappropriate retention of mammary buds in Gli3(xt/xt) mice. Importantly, mice genetically manipulated to misactivate Hh signaling displayed the same phenotypic spectrum demonstrating that the repressor function of Gli3(R) is essential during multiple stages of mammary development. In contrast, positive Hh signaling occurs during nipple development in a mesenchymal cuff around the lactiferous duct and in muscle cells of the nipple sphincter. Collectively, these data show that repression of Hh signaling by Gli3(R) is critical for early placodal patterning and later mammary mesenchyme specification whereas positive Hh signaling occurs during nipple development

<i>Tbx3</i> mRNA expression in <i>Gli3^xt/xt</i> embryos.

<p>Whole-mount <i>in situ</i> hybridization for <i>Tbx3</i> in wt (A, B) and <i>Gli3^xt/xt</i> (C, D) embryos. At the 40-somite stage, wt embryos (A) express <i>Tbx3</i> mRNA in the mesenchyme between the fore- and hind-limb. This band of expression is narrower in <i>Gli3^xt/xt</i> embryos at this stage (yellow bracket). At the 45-somite stage <i>Tbx3</i> is reduced within the mesenchyme but is induced within the epithelium of developing mammary placodes #3 (white arrow) in wt embryos (B). <i>Gli3^xt/xt</i> embryos (D), show a narrower band of <i>Tbx3</i> expression between the fore- and hind-limb and fail to concentrate epithelial expression within placode #3 (white arrow). The control <i>in situ</i> hybridization for <i>Tbx3</i> sense probe is shown in (E). Abbreviations: F – fore-limb, H – hind-limb, s – somite.</p

Percentages of MRs showing phenotypic abnormalities in female <i>Gli3^xt/xt</i> embryos at E18.5.

<p>Numbers represent percentages of MRs showing loss, evagination or impairment in invagination from a total ‘n’. Abbreviations: Imp. Invag: Impaired Invagination; MR: mammary rudiment; xt: extra toe mutation; n: total number of MRs analyzed.</p

Expression and modulation of <i>Gli1-LacZ</i> in the adult nipple during the pregnancy cycle.

<p>Gli1-LacZ expression is visible at the neck of the mammary sprout (red arrow) in skin whole-mounts of E18.5 <i>Gli1^lzki/+</i> embryos at low (A) and higher power (A’). Histological section through the sprout shows expression of Gli1-LacZ within the stroma (red arrow) surrounding the sprout (black arrow) underneath the nipple sheath (black arrowheads) at low (B) and higher power (B’). <i>Gli1-lacZ</i> is expressed within the dermal component but not the epithelium in virgin (C), mid pregnant (D), lactating (E) and involuting (F) nipples. Immunohistochemistry for desmin (G, J), SMA (H, K) and vimentin (I, L) on serial sections of a 15.5 day pregnant nipple demonstrated that <i>Gli1-lacZ</i> was expressed in both smooth muscle cells and fibroblasts but not myofibroblasts. <i>Gli1-lacZ</i> was also found near and surrounding peripherin positive nerve tracts (M) and both Von Willebrand positive vessels (N).</p

Percentages of MRs showing phenotypic abnormalities in female <i>Gli2^1ki/1ki; Gli3^xt/+</i> embryos at E18.5.

<p>Numbers represent percentages of MRs showing loss, evagination or impairment in invagination from a total ‘n’. Abbreviations: Imp. Invag: Impaired Invagination; n: total number of MRs analyzed.</p

Evagination of MR#2 and encroachment of hair follicles in <i>Gli3</i>^<i>xt</i>/xt embryos.

<p>Analysis of outer surface of E18.5 skin whole-mounts (A–D) stained with carmine (C, D) and sections stained with nuclear fast red (E, F) and X-Gal (blue) to detect hair follicles expressing the Gli1-LacZ reporter (A–F). MR#1 (A) from <i>Gli3^xt/xt; Gli1^lzki/+</i> mutants and MR#2 from control <i>Gli1^lzki/+</i> (C) embryos show normal invagination and appropriate exclusion of hair follicles. In contrast MR#2 from <i>Gli3^xt/xt; Gli1^lzki/+</i> mutants (B, D) showed prominent evagination and encroachment of hair follicles.</p

Percentages of MRs showing retention in male <i>Gli3^xt/xt</i> embryos at E16.5 and E18.5.

<p>Numbers represent percentages of MRs retained on male skin whole-mounts from a total ‘n’. Abbreviations: E: embryonic day; n: total number of putative sites for MRs on male skin whole mounts.</p

Misactivation of Hh signaling detrimentally affects MR invagination and hair follicle suppression in females and eradication of MRs in males.

<p>X-Gal stained whole-mounts (A–E) and sections (F-H) of <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos were examined at E18.5. In whole-mounts of female skins, MR#2 protruded prominently and showed encroachment of hair follicles inappropriately close to the evaginated MR (A). Examination of male skin whole-mounts revealed retention of MR#1, #2 and #4 at low (B) and high power (C, D, E respectively), similar to that seen in <i>Gli3^xt/xt;Gli1^lzki/+</i> embryos. Serial sections through a male MR#2 from <i>Gli2^1ki/1ki;Gli3^xt/+;Gli1^lzki/+</i> embryos showed Gli1-LacZ-positive hair follicles close to the protruding bud (F; NFR counterstain) and weak expression of Tenascin C (G) and loss of AR (H) by immunohistochemistry.</p