RANK Overexpression in Transgenic Mice with Mouse Mammary Tumor Virus Promoter-Controlled RANK Increases Proliferation and Impairs Alveolar Differentiation in the Mammary Epithelia and Disrupts Lumen Formation in Cultured Epithelial Acini

Abstract

RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins ␤-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.The tumor necrosis factor (TNF) family member RANKL and its receptor RANK are key regulators of osteoclast differentiation and activation (16,30). The balance between RANKL, RANK, and osteoprotegerin (OPG), a soluble decoy receptor that competes with RANK for binding to RANKL (32), regulates osteoclast differentiation and activation and therefore bone remodeling and mobilization of calcium from the skeleton (27). In mice, RANK and RANKL are also essential for the development of the mammary gland during pregnancy (18), in particular, for the formation of lobuloalveolar structures after pregnancy day 14 (P14) which are required for a functional lactating mammary gland.Deregulation in the RANKL/OPG system has been reported in malignant bone disease including bone metastasis and humoral hypercalcemia of malignancy (13,21,23). Numerous lines of evidence in animal models indicate that blocking RANK/RANKL interaction effectively prevents tumor-induced hypercalcemia or reduces tumor-induced bone lesions, due to its critical role in osteoclast differentiation and activation (reviewed in references 38 and 41). While RANK expression has been detected in breast cancer lines and in primary tumors including breast tumors (45) and may play a functional role in tumor cell migration and metastasis to the bone (24), it is not clear whether activation of the RANK/RANKL pathway may also contribute to primary tumor growth.The mammary gland defects observed in RANK-and RANKL-deficient mice support the notion that the RANK/ RANKL pathway plays an active role in mammary cell proliferation and survival; however, the direct effect of increased RANK signaling in mammary cells is unknown. To determine if activation of the RANK/RANKL pathway can directly increase proliferation or surv...

Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo

“…Asterisks represent significant differences (Po0.01) compared with pcDNA3-transfected cells. NFjB blockade promotes senescence of differentiating GICs L Nogueira et al signaling through the NFkB activator receptor activator of NFkB (RANK) not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells (Gonzalez-Suarez et al, 2007). Our data show that maintained NFkB blockade in GICs undergoing differentiation, promotes proliferation arrest, morphological changes (enlarged and flattened shape), polyploidy, increase in phospho-H2AX-containing nuclear foci and induction of b-gal, which are features of cellular senescence.…”

Homotypic RANK signaling differentially regulates proliferation, motility and cell survival in osteosarcoma and mammary epithelial cells

“…This apparent distinction between the ability of RANK activity to promote a transformative phenotype in normal NMuMG, but not in MC3T3-E1 cells, was unexpected, but highly relevant. Gonzalez-Suarez and colleagues (Gonzalez-Suarez et al, 2007) showed that overexpression of RANK in the epithelial cell compartment of the mammary gland led to profound hyperplasia and impaired alveolar differentiation, although overt tumor formation did not occur. Recently, Santini and co-workers (Santini et al, 2011) provided a correlation between breast cancers expressing high levels of RANK and poor patient outcome.…”

RANK Induces Epithelial–Mesenchymal Transition and Stemness in Human Mammary Epithelial Cells and Promotes Tumorigenesis and Metastasis

“…Impaired mammary gland development of the RANK and RANKL null mice is due to defective proliferation and increased apoptosis of mammary epithelium (2). Conversely, overexpression of RANK or RANKL in the mammary gland leads to increased proliferation of the mammary epithelia (3,4). It has been postulated that paracrine signaling through RANK/RANKL is responsible for the expansion of mammary stem cells observed during pregnancy and luteal cycles (5,6).…”