Homotypic RANK signaling differentially regulatesproliferation, motility and cell survival in osteosarcomaand mammary epithelial cells

Alexander G. Beristain, Swami R. Narala, Marco A. Di Grappa and Rama Khokha*Ontario Cancer Institute, Princess Margaret Hospital, 610 University Avenue, Toronto Ontario, M5G 2M9, Canada

*Author for correspondence (rkhokha@uhnres.utoronto.ca)

Accepted 28 October 2011Journal of Cell Science 125, 1–13� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.094029

SummaryRANKL (receptor activator of NF-kB ligand) is a crucial cytokine for regulating diverse biological systems such as innate immunity, bonehomeostasis and mammary gland differentiation, operating through activation of its cognate receptor RANK. In these normal physiological

processes, RANKL signals through paracrine and/or heterotypic mechanisms where its expression and function is tightly controlled.Numerous pathologies involve RANKL deregulation, such as bone loss, inflammatory diseases and cancer, and aberrant RANK expressionhas been reported in bone cancer. Here, we investigated the significance of RANK in tumor cells with a particular emphasis on homotypicsignaling. We selected RANK-positive mouse osteosarcoma and RANK-negative preosteoblastic MC3T3-E1 cells and subjected them to

loss- and gain-of-RANK function analyses. By examining a spectrum of tumorigenic properties, we demonstrate that RANK homotypicsignaling has a negligible effect on cell proliferation, but promotes cell motility and anchorage-independent growth of osteosarcoma cellsand preosteoblasts. By contrast, establishment of RANK signaling in non-tumorigenic mammary epithelial NMuMG cells promotes their

proliferation and anchorage-independent growth, but not motility. Furthermore, RANK activation initiates multiple signaling pathwaysbeyond its canonical target, NF-kB. Among these, biochemical inhibition reveals that Erk1/2 is dominant and crucial for the promotion ofanchorage-independent survival and invasion of osteoblastic cells, as well as the proliferation of mammary epithelial cells. Thus, RANK

signaling functionally contributes to key tumorigenic properties through a cell-autonomous homotypic mechanism. These data also identifythe likely inherent differences between epithelial and mesenchymal cell responsiveness to RANK activation.

Key words: Autocrine, Cancer, RANKL

IntroductionThe RANK [receptor activator of nuclear factor kappa B (NF-

kB)] signaling pathway, which is activated in response to its

ligand, RANKL, has been shown to regulate many cellular

processes in a diverse set of tissues. Most notably, RANK

signaling is essential for bone homeostasis because it controls

the differentiation and activation of bone-resorbing osteoclasts

(Bucay et al., 1998; Dougall et al., 1999; Kong et al., 1999).

RANK also plays a crucial function in immunity through

promotion of dendritic cell survival (Anderson et al., 1997),

induction of thymic epithelial progenitor cell differentiation (Lee

et al., 2008) and lymph node development (Dougall et al., 1999;

Kong et al., 1999). In the mammary gland, RANK regulates

progesterone-mediated responses during pregnancy and the

estrous cycle (Fata et al., 2000; Joshi et al., 2010). In these

normal biological systems, RANK activation is facilitated through

paracrine and/or heterotypic RANK–RANKL interactions that

generate regulatory feedback mechanisms that help maintain

normal physiology. It is therefore not surprising that deregulation

of RANK signaling underlies the progression of multiple human

diseases (Dougall et al., 2007; Leibbrandt and Penninger, 2008).

The development of molecular therapies aimed at inhibiting the

RANK pathway has been significant in the past decade, and

therefore a better understanding of this pathway and the molecular

mechanisms directed by RANK is imperative for best utilization

of these evolving drugs.

In cancer, the importance of RANKL has been highlighted in

multiple reports that demonstrate its role in regulating tumor cell

metastasis to bone (Morony et al., 2001; Feeley et al., 2006;

Armstrong et al., 2008; Canon et al., 2008). However, recent

studies have provided new insights into the involvement of

RANK signaling in tumorigenesis beyond that of metastasis. It

has been shown that RANK signaling in mammary epithelial

cells dictates cell proliferation (Cao et al., 2001; Fernandez-

Valdivia et al., 2009), as well as mammary stem cell expansion

during the reproductive cycle (Joshi et al., 2010). Consistent with

these findings, mouse models of progestin-driven mammary

tumorigenesis demonstrate a crucial function of RANK in breast

cancer (Schramek et al., 2010; Gonzalez-Suarez et al., 2010). A

role for RANK signaling in primary bone cancer has also been

acknowledged, where aberrant production of RANKL by tumor

cells leads to unregulated osteoclast-mediated bone destruction

and increased tumor burden (Lamoureux et al., 2007). Through

the generation of a transgenic mouse model of osteosarcoma

(Molyneux et al., 2010), we have obtained evidence for RANKL

involvement in primary bone tumorigenesis. Specifically, we

found that dysregulated PKA signaling due to the loss of the

Prkar1a bone tumor suppressor gene resulted in copious amounts

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JCS online publication date 15 March 2012

of RANKL and greatly accelerated tumorigenesis. Furthermore,aberrant RANK expression and signaling has been noted in

human (Mori et al., 2007) and mouse (Wittrant et al., 2006)osteosarcoma cell lines. Together, these studies open thepossibility that RANK, signaling through an autocrine or

homotypic mechanism, regulates tumorigenic phenotypes intumor cells of both epithelial and mesenchymal origin.

The RANK receptor is a type I transmembrane protein that

exists as a homotrimer at the cell surface and binds to RANKL,which is a type II membrane protein sharing close homology tothe tumor necrosis superfamily (TNFSF) members FasL, TRAIL

and TNF (Anderson et al., 1997; Wong et al., 1997; Lacey et al.,1998). RANK signal transduction is mediated by the TNFreceptor-associated factor (TRAF) family of adaptor proteins,with TRAF6 being the best described in regulating the

downstream effectors NF-kB, Akt (PKB) and the mitogen-activated protein kinases (MAPKs) JNK1, p38 (MAPK14) andErk1/2 (MAPK2) in osteoclasts and mammary epithelial cells

(Wong et al., 1998; Darnay et al., 1999; Kobayashi et al., 2001;Jones et al., 2006; Lamothe et al., 2007). Of these effectors, NF-kB is activated upon RANK stimulation during bone remodeling

and in other tissue systems, and has become a hallmark of theRANK-signaling pathway. However, other signaling pathwaysmodulated by TRAF6 could serve as crucial effectors of RANKsignaling during tumorigenesis. Identifying the role of specific

pathways downstream of RANK activation in tumor cells will fillan important knowledge gap, and might provide opportunities tocombine existing RANK inhibiting therapies with drugs targeting

crucial effectors downstream of RANK.

In this study, we interrogate the importance of aberrantRANK expression in osteosarcoma cells and test the significance

of homotypic RANKL–RANK activation in directing specifictumorigenic parameters using RANK-expressing osteosarcoma cellsderived from primary mouse bone tumors, as well as systems

engineered to ectopically express RANK or RANKL. We show inosteosarcoma cells that homotypic RANK activity promotes cellmotility and anchorage-independent viability, but not proliferation.

Overexpression of RANK in non-tumorigenic osteoblastic andmammary epithelial cells further demonstrates that RANKsignaling differentially regulates motility, proliferation, anchorage-

independent viability and growth in these two cell types. In-depthanalyses of the major signaling pathways and small-moleculeinhibitors provide evidence of the involvement of Erk1/2 and Akt inmediating specific tumorigenic properties downstream of RANK

activation. Thus, cell-autonomous RANK signaling leads to distinctadvantages for mammary epithelial and mesenchymal osteoblasticcells through activation of Erk1/2.

ResultsCharacterization and generation of experimental systemsto study homotypic RANK signaling in osteosarcoma cells

To elucidate the role of homotypic and or autocrine RANK

signaling in cancer, we took advantage of our recently reportedtransgenic mouse model of osteosarcoma designated as MOTO(Molyneux et al., 2010). Comparison of Rankl, Opg and Rank

mRNA levels in normal bone, early osteosarcoma lesions andadvanced osteosarcomas revealed elevated expression of Rankl andRank at both early and late stages of bone tumor development

(Fig. 1A). Specifically, Rank expression was significantly higher in3 of 4 bones from 10-week-old MOTO mice; Rankl was elevated inall samples; whereas 3 of the 4 samples expressed significantly less

Opg (Fig. 1A). In 28-week-old mice with advanced tumors, 8 or 12

of 13 tumors showed high Rankl and Rank levels, respectively(Fig. 1A), and 5 tumors within this cohort expressed Rank at .50-fold higher levels than normal bone. Immunohistochemical analysis

of RANK in MOTO tumors demonstrated specific expression of thisprotein in osteosarcoma cells, whereas RANK was absent in normalosteoblasts and osteocytes but was detectable in osteoclasts andmacrophages residing in the bone marrow (Fig. 1A). Thus, MOTO

osteosarcomas express high levels of Rank mRNA at initial stages oftumor development when no gross tumors are observed, as well as atmore advanced stages of tumorigenesis. Furthermore, RANK

protein is localized to osteosarcoma tumor cells and osteoclastswithin the osteosarcoma tumors.

We have previously reported the expression status of Rankl

and Opg in cell lines (designated Moto1.1, Moto1.2) derived

from MOTO osteosarcomas (Molyneaux et al., 2010). In additionto these two cell lines, we analyzed Rank and Rankl expressionin nine other MOTO-derived cell lines (designated Moto1.3–

Moto1.10. and Moto2.1). We also included the immortalized butnon-tumorigenic osteoblastic MC3T3-E1 and 7F2-OSB cells ascontrols in this analysis (Fig. 1B). RANKL was expressed at

significantly higher levels relative to 7F2OSB osteoblasts in 4 of11 MOTO cell lines (Moto1.2, Moto1.3, Moto1.4 and Moto2.1)whereas Rank mRNA was overexpressed in 5 of the 11 cell lines

(Moto1.1, Moto1.2, Moto1.4, Moto1.5 and Moto2.1), indicatingthat the overexpression of these molecules is a common featureof osteosarcoma cells. MC3T3-E1 and 7F2-OSB cells expresslow amounts of Rankl and undetectable levels of Rank. RANK

and RANKL protein expression was confirmed in Moto1.1 andMoto1.2 cell lines (Fig. 1B). Specifically, immunocytochemicalanalysis showed expression of RANK in both Moto1.1 and

Moto1.2. In these cells, RANK protein displayed a diffusecytoplasmic pattern that was more intense in Moto1.1 than inMoto1.2. MC3T3-E1 cells did not show any positive RANK

signal. Further, immunoblot analysis of RANKL demonstratedelevated protein levels in Moto1.2 cells and low levels ofRANKL in Moto1.1, 7F2OSB and MC3T3-E1 cells. These

findings are consistent with the mRNA expression data of Rank

and Rankl in these osteosarcoma cell lines.

We next established a system to study the effects of homotypicRANK signaling in normal osteoblasts by using gain- and loss-

of-function strategies. We stably transduced the MC3T3-E1 and7F2-OSB cell lines with a Rank overexpression vector generatingMC3T3-pRANK and 7F2-pRANK cells. Overexpression of

RANK in these cells was confirmed by immunoblotting using aRANK-specific antibody and by qRT-PCR analysis (Fig. 1C;supplementary material Fig. S1). To generate a loss-of-RANKscenario in MOTO osteosarcoma cells, Rank-specific shRNA

knockdown expression constructs were stably transduced intoMoto1.2 cells. The extent of knockdown was measured by qRT-PCR for four distinct Rank targeting constructs (pLKO.1-shRNA-

2, -7, -8 and -10); pLKO.1-shRNA-7 was found to be the mosteffective and selected for loss-of-function experiments, whereascells transduced with a non-specific sequence (pLKO.1) or GFP

(pLKO.1-GFP) served as controls (Fig. 1D).

RANKL does not affect proliferation of osteosarcoma orosteoblastic cell lines

Because we have observed expression of both Rank and Rankl atearly stages of tumor development in MOTO mice and in Motoosteosarcoma cells, we tested whether induction of RANK activity

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could regulate proliferation in Moto1.1 and Moto1.2. Increasing

concentrations of recombinant mouse RANKL (RL) did not affect

cell proliferation in either cell line assayed over 96 hours as shown

in (supplementary material Fig. S1). Moto1.2 cells express

endogenous RANKL; we therefore treated these cells with

increasing concentrations of the RANKL inhibitor, OPG-Fc,

which also failed to alter cell proliferation (supplementary

material Fig. S1). Finally, stimulation of RANK-overexpressing

MC3T3-E1 preosteoblasts and 7F2-OSB osteoblasts (MC3T3-

pRANK and 7F2-pRANK) with RL did not affect cell proliferation

(supplementary material Fig. S1). These studies show that

RANK signaling does not regulate cell proliferation in either

RANK-expressing osteosarcoma cells or in osteoblastic cells

overexpressing RANK.

Fig. 1. Characterization and

generation of experimental gain- and

loss-of-function RANK signaling

systems. (A) Scatter-plot histograms

depicting mRNA levels of Rankl, Opg

and Rank assayed by real-time PCR in

10- or 28-week-old wild-type or MOTO

osteosarcoma bone specimens (i). Each

scatter point represents expression from

a distinct mouse. Photomicrographs of

wild-type bone (Wt) and MOTO

osteosarcoma (MOTO) tissue sections

stained with an anti-RANK antibody

(ii). Arrowheads denote areas of

positive immunostaining for RANK.

Scale bars: 20 mm. (B) Bar graphs

represent mRNA expression levels of

Rankl and Rank in 7F2-OSB and

MC3T3-E1 osteoblastic cells and in

Moto1.1-1.10 and Moto1.2 primary

osteosarcoma cells propagated from

MOTO tumors (i). Photomicrographs

show immunocytochemical analysis of

RANK protein expression in MC3T3-

E1, Moto1.1 and Moto1.2 cells (ii). The

representative immunoblot depicts

RANKL and b-actin protein levels in

Moto1.1, Moto1.2, 7F2OSB and

MC3T3-E1 cells (iii). (C) Bar graph

depicting mRNA levels of Rank in

Moto1.1 and Moto1.2 osteosarcoma

cells and in MC3T3 cells stably

transduced with a control (pLVX) or

RANK-expression (MC3T3-pRANK)

lentiviral expression construct (i). The

representative immunoblot shows Rank

and b-actin protein levels from lysates

extracted from MC3T3-pLVX or

MC3T3-pRANK (ii). (D) Bar graph

representing expression levels of Rank

mRNA in Moto1.2 cells stably

transduced with lentiviral control

(pLK0.1 and pLK0.1-GFP) and RANK

shRNA (pLKO.1-shRNA-2, -7, -8 and

-10) expression constructs. All real-time

PCR reactions were normalized to

endogenous b-actin. Protein molecular

mass (kDa) is shown to the left of

immunoblots. The results are presented

as mean 6 s.e.m. in the histograms

(*P#0.05, compared with pLK0.1

control).

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Activation of RANK increases osteosarcoma cell motility

There has been increasing attention assigned to the role of

RANKL in regulating cell motility (Jones et al., 2006; Mori et al.,

2007; Armstrong et al., 2008; Chen et al., 2010; Hsu et al., 2010;

Sabbota et al., 2010) and metastasis (Jones et al., 2006; Miller

et al., 2008; Canon et al., 2008; Tan et al., 2011), with a

significant emphasis on prostate and breast cancer cell metastasis

to bone. Nonetheless, the role of RANK signaling through a

homotypic scenario in the context of normal cell motility has yet

to be investigated. Also, it is not clear whether co-expression of

RANKL and RANK in cancer cells will promote their invasive

capacity. We therefore measured the effect of RANK signaling

on cell migration and invasion using Moto1.1 and Moto1.2

osteosarcoma cells, MC3T3-E1 preosteoblasts and 7F2-OSB

osteoblasts. Comparison of baseline invasion of Moto1.1 and

Moto1.2 cells showed that motility of both cell lines significantly

differed (Fig. 2A). Specifically, Moto1.2 cells were more

invasive than Moto1.1 cells. Upon treatment with RL, the

invasive capacity of both tumor cell lines increased significantly;

exogenous RL treatment in Moto1.1 cells lead to a ,sixfold

increase in invasion, whereas this increase was twofold for

Moto1.2 cells. This difference might arise in part from the

presence of endogenous RANKL in Moto1.2 cells, resulting in

basal RANK activity. Consistent with this, perturbation of

endogenous RANKL by OPG-Fc in Moto1.2 cells significantly

ablated cell migration and invasion as assessed by scratch-wound

motility (data not shown) and Matrigel-coated Transwell invasion

assays (Fig. 2B). Similarly, shRNA-mediated knockdown of

endogenous RANK in Moto1.2 cells (pLKO.1-shRNA-7)

inhibited cell invasion through both Matrigel and rat-tail

collagen I (Fig. 2C). In non-tumorigenic MC3T3-E1 and 7F2-

OSB cells overexpressing RANK (MC3T3-pRANK or 7F2-

pRANK), treatment with RL promoted cell migration and

invasion through collagen I matrix (Fig. 2D; supplementary

material Fig. S2). Note that the baseline increase in migration and

invasion in MC3T3-E1 cells was independent of RL treatment,

suggesting that low levels of endogenous RANKL, leading to an

autocrine or homotypic RANK-RANKL interaction, is sufficient

to elicit a motile phenotype. These data clearly show that

RANKL signaling induces motility in osteoblastic cells.

RANK impacts anchorage-independent growth andsurvival in osteosarcoma cells

The role for RANK signaling in promoting dendritic cell and

osteoclast survival has been well established (Dougall et al., 1999;

Lum et al., 1999); however, its effects on mediating tumor cell

survival or resistance to apoptosis is less clear. To address whether

a RANKL stimulus could provide a survival mechanism in

osteosarcoma cells, we performed anchorage-independent cell

viability assays on Moto1.1, Moto1.2 and MC3T3-pRANK cells.

Addition of RL to Moto1.1 and Moto1.2 osteosarcoma cells led to

an increase in cell viability when cultured on ultra-low-attachment

microplates over a 72 hour time course (Fig. 3A). Although the

kinetics of cell viability varied among these two cell lines, RANK

signaling provided a significant survival advantage to both Moto1.1

and Moto1.2 cells, whereas other genetic inherent differences

between these cancer cell lines might account for these differences.

Furthermore, treatment of MC3T3-pRANK cells with RL resulted

Fig. 2. RANK signaling promotes a motile phenotype on mesenchymal osteoblastic cells. (A) Representative photomicrographs and histograms describe the

invasive capacities of Moto1.1 and Moto1.2 cells unstimulated (-) or stimulated with 30 ng/ml RL through Matrigel. (B) Representative photomicrographs and

histograms show the effects of loss-of-RANK signaling by OPG-Fc (200 ng/ml) on Moto1.2 invasion through Matrigel. (C) Bar graphs depicts the invasive

capacity of Moto1.2 cells transduced with by shRNA (pLKO.1-shRNA-7) through Matrigel and rat-tail collagen I matrices. Moto1.2 cells transduced with an

expression construct expressing scrambled shRNAs served as a control (pLKO.1). (D) Representative photomicrographs and histograms show cell invasion

through collagen I matrix of MC3T3-pLVX or MC3T3-pRANK cells in response to 30 ng/ml RL. Cell invasion was performed for 48 hours. Each cell line was

plated in triplicate with the experiment repeated on at least three independent occasions (n53). The results are presented as mean 6 s.e.m. in the bar graph

(*P#0.05, **P#0.01 compared with the control).

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in increased cell viability over the entire 72 hour time course; the

histogram representing the 48 hour time-point highlights

this observation. Even in RL-unstimulated MC3T3-pRANK cells,

a baseline increase in anchorage-independent cell survival

was evident, again suggesting the significance of endogenous

RANKL in activating RANK through an homotypic mechanism.

One means by which cells facilitate anchorage-independent

viability is through resistance to anoikis (Westhoff and Fulda,

2009). To investigate whether RANK signaling could provide a

resistance mechanism to anchorage-independent cell death, we

analyzed levels of active caspase-3 by immunoblot analysis of

protein lysates obtained from Moto1.1 and Moto1.2 cells cultured

for 48 hours on ultra-low-attachment plates. Moto1.1 and

Moto1.2 cells showed lower cleaved caspase-3 levels following

RL treatment, and this observation was more pronounced in

Moto1.1 than in Moto1.2 cells (Fig. 3B). Intriguingly, MC3T3-

pRANK cells cultured on ultra-low-attachment plates displayed a

remarkable reduction in cleaved caspase-3 levels in the presence

or absence of RL (Fig. 3B). These data strongly implicate

RANK in providing a mechanism for resistance to anoikis

in osteosarcoma and RANK-expressing osteoblastic cells.

Next, we performed a standard transformation assay by

subjecting Moto1.2 pLKO.1-shRNA-7 and MC3T3-pRANK

cells to soft-agar colony formation. Osteosarcoma Moto1.2

cells transduced with control vector (pLKO.1) grew numerous

large colonies over 14 days and knockdown of endogenous

RANK in pLKO.1-shRNA-7 cells significantly compromised

their colony-forming ability (Fig. 3C). MC3T3-pRANK cells

overexpressing RANK did not form colonies upon RL treatment,

as assessed over 21 days in culture (data not shown).

Together, these results show that constitutive RANK signaling

in osteosarcoma cells provides a transformation advantage.

However, the establishment of a RANK-signaling system in

non-transformed osteoblastic cells is not sufficient to promote

anchorage-independent growth and/or cell transformation, but

does provide a survival advantage.

Fig. 3. Anchorage-independent survival is

facilitated by RANK in osteosarcoma. (A) Line

graphs depict the effect of RL treatment (30 or 100 ng/

ml) on cell viability of, Moto1.1 and Moto1.2

osteosarcoma (i,ii) and MC3T3-pLVX or -pRANK

osteoblastic cells cultured on ultra-low-adherence

plates for up to 72 hours (iii). A histogram depicting the

48-hour anchorage-independent survival of MC3T3-

pLVX/-pRANK is additionally shown as a bar graph

(iv). Anchorage-independent cell viability was

evaluated by a Cell-titer Glo ATP-based luminescence

assay performed in triplicate on three separate

occasions. (B) Representative immunoblots of protein

lysates showing cleaved caspase-3 and b-actin levels

from Moto1.1 and Moto1.2 (i) or in MC3T3-pLVX and

MC3T3-pRANK cells (ii) cultured for 48 hours on

ultra-low-adherence plates and treated with or without

RL (30 ng/ml). (C) Representative photomicrographs

of soft-agar colonies formed by Moto1.2 pLKO.1 or

pLKO.1-shRNA-7 cultured over 14 days. The

molecular mass markers (kDa) are shown to the left of

immunoblots. The results are presented as mean 6

s.e.m. in the bar graphs (*P#0.05, compared with

pLVX control).

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Autocrine RANK signaling promotes distinct

transformation properties in mammary epithelial cells

The above series of experiments identified that RANK regulates

properties of cell transformation, i.e. cell motility and anchorage-

independent viability of osteosarcoma and non-tumorigenic

osteoblastic cells. These findings in mesenchymal cells

prompted us to ask whether a RANKL–RANK homotypic

mechanism would similarly impact epithelial cells. We selected

non-transformed and non-tumorigenic mammary epithelial

NMuMG cells, as counterparts to MC3T3-E1 cells, because the

importance of RANK in mammary epithelial cell growth,

differentiation and tumorigenesis is now evident. The widely

used NMuMG cells have been derived from normal murine

mammary epithelial tissue and retain characteristics of primary

mammary epithelial cells (David et al., 1981; Soriano et al.,

1995). Our measurements of the relevant molecules by qRT-PCR

showed that NMuMG cells express Rank, the RANKL decoy

inhibitor Opg, but not Rankl mRNA, relative to adult murine

mammary gland (Fig. 4A). We then established a system to study

homotypic RANK signaling. The mammalian expression vector

(pcDNA3.1) containing a full-length mouse Rankl cDNA was

stably transfected into NMuMG cells to overexpress RANKL

Fig. 4. Autocrine RANK-signaling in

mammary epithelial cells promotes

proliferation and anchorage-independent

growth but not motility. (A) Bar graphs

representing Rank, Opg, or Rankl mRNA

levels in 15-week-old mouse mammary gland

tissue, wild-type NMuMG mammary

epithelial cells (i) or in NMuMG-pRL cells

stably expressing RANKL (ii).

(iii) Representative immunoblot containing

protein lysates extracted from NMuMG-

pLacZ or NMuMG-pRL cells probed with an

anti-FLAG antibody directed against a

FLAG-epitope located at the C-terminus of

exogenously expressed RANKL. The blot

was re-probed with a monoclonal antibody

against b-actin. (B) Line graphs depict cell

proliferation of NMuMG mammary epithelial

cells treated with 0 or 100 ng/ml RL (i) or

stably transfected with control (pLacZ) or

RANKL (pRANKL) expression construct

(ii) for up to 72 hours of culture; cells were

additionally treated with or without 200 ng/

ml OPG-Fc. (C) Line graph depicts the effect

of RL treatment (30 or 100 ng/ml) on cell

viability of NMuMG epithelial cells cultured

on ultra-low-adherence plates for up to

72 hours. A subset of RL-treated NMuMG

cells were additionally treated in combination

with OPG-Fc (200 ng/ml).

(D) Representative immunoblots of protein

lysates showing cleaved caspase-3 and b-

actin levels from NMuMG cultured for

48 hours on ultra-low-adherence plates and

treated with or without RL (30 ng/ml). The

molecular mass markers (kDa) are shown to

the left. (E) Representative

photomicrographs of soft-agar colonies

formed by NMuMG-pLacZ or NMuMG-

pRANKL cells cultured over 21 days.

NMuMG-pRANKL cells were separately

treated with recombinant OPG-Fc (200 ng/

ml). The results are presented as mean 6

s.e.m. in the bar graphs. Cell viability was

evaluated by a Cell-titer Glo ATP-based

luminescence assay performed in triplicate

on three separate occasions (n53; *P#0.05,

compared with controls).

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(NMuMG-pRL). We verified ectopic RANKL expression byqRT-PCR using Rankl-specific Taqman primers and by

immunoblot analysis using an anti-RANKL antibody (Fig. 4A).

The functional importance of RANK signaling in NMuMG cellswas next tested using methods identical to those applied to the

osteosarcoma cells. RANKL treatment has been reported topromote proliferation of mammary epithelial cells cultured ex vivo(Kim et al., 2006). Beyond mammary epithelium, recent evidence

shows proliferative effects directed by RANKL in other tissues,including the hair follicle pilosebaceous gland (Duheron et al.,2011) and thymus (Lee et al., 2008). Addition of RL stimulated

proliferation of NMuMG cells in a dose-dependent manner over72 hours of culture (Fig. 4B). Furthermore, NMuMG-pRL cellsoverexpressing RANKL displayed a significant increase inproliferation at 48 and 72 hours compared with control NMuMG

cells expressing b-galactosidase (NMuMG-pLacZ) (Fig. 4B).Treatment with OPG-Fc reversed the proliferative effect of RLin parental cells, and of transfected RANKL in NMuMG-pRL

cells, thus demonstrating specificity of RANKL-induced mammaryepithelial cell proliferation. This result was in contrast to thatseen in osteosarcoma and RANK-overexpressing osteoblastic

cells.

We next determined whether stimulation of RANK inmammary epithelial cells altered cell motility and invasion.

Treatment of NMuMG cells with increasing concentrations of RLproduced a negligible effect on cell migration or invasion(supplementary material Fig. S3). Consistent with this finding,

the invasion of NMuMG-pRL cells through Matrigel did notdiffer from control cells (supplementary material Fig. S3). Weconclude that homotypic activation of RANK in mammaryepithelial cells does not alter migration or invasion.

The role of RANK signaling was finally tested in anchorage-independent viability and growth. Recombinant mouse RANKL

treatment led to a significant increase in cell viability of NMuMGcells cultured on ultra-low-attachment plates over a 72 hour timecourse, whereas concomitant treatment with the inhibitor OPG-Fc reversed this effect (Fig. 4C). Intriguingly, RL treatment did

not result in a reduced level of cleaved caspase-3 in NMuMGcells (Fig. 4D), which we observed in both Moto1.1 andMoto1.2 osteosarcoma cells and RANK-overexpressing MC3T3

preosteoblasts (Fig. 3B). However, subjecting NMuMG-pRLcells to soft-agar colony assays clearly demonstrated thatoverexpression of RANKL generated multiple colonies over 14

days (Fig. 4E). This growth was suppressed by treatment withOPG-Fc, suggesting that activation of RANK is sufficient toelicit cell transformation in mammary epithelial cells.

Active RANK induces signal transduction byphosphorylation of Erk1/2 and Akt

A number of signaling pathways have been implicateddownstream of RANK activity. To identify the signaltransduction pathways responsive to RANK stimulation in

MOTO osteosarcoma, MC3T3-E1 and NMuMG cell lines, weperformed immunoblot analysis for NF-kB p65, Akt, and theMAPKs Erk1/2, JNK and p38. Specifically, cells were serum

starved and then stimulated with RL for 0, 5, 10, 15, 30 and 60, orup to 120 minutes. First, a time-course analysis following RLtreatment of Moto1.2 cells showed a sharp peak of induction in

phosphorylated Erk1/2, phosphorylated p38 and phosphorylatedJNK within 5 minutes, whereas the phosphorylated p65 signalalso increased but showed a gradual induction over 60 minutes.

Treatment with RL had no effect on phosphorylated Akt.NMuMG cells displayed a few distinctions in the kinetics of

RANKL response when compared to Moto1.2. Specifically,Erk1/2, JNK and p38 and Akt were all induced within 5 minutesfollowing RL treatment. Interestingly, NF-kB, which is the most

studied pathway downstream of RANK, showed a negligibleinduction as measured by phosphorylated p65 levels (Fig. 5A).

We next determined the consequence of de novo RANK

activation in preosteoblasts and mammary epithelial cells.Comparison of MC3T3-pRANK with control MC3T3-pLVXcells showed that RL stimulation essentially captured the same

repertoire of signal transduction responses described in abovesection, with the main difference being a gradual inductionof phosphorylated Akt over 60 minutes post treatment(Fig. 5B). With respect to epithelial cells, NMuMG-pRL cells

endogenously expressing RANK coupled with ectopic RANKLestablished an autocrine RANK signaling system. When culturedin low serum (1% FBS), we observed an elevated baseline

of phosphorylated Akt, whereas levels of phosphorylatedErk1/2 and phosphorylated p65 remained unchanged, althoughphosphorylated Erk1/2 levels increased in response to OPG-Fc in

NMuMG-pLacZ control cells (Fig. 5C). The specificity of RL ininducing these signaling proteins was determined throughinhibition of RANK signaling by OPG-Fc. Addition of OPG-Fc dampened Akt phosphorylation in NMuMG cells whereas

incorporating OPG-Fc treatment to MC3T3-pRANK cells negatedactivation of phosphorylated Erk1/2 (Fig. 5C; supplementarymaterial Fig. S4). The signaling pathways found to be activated

by RANKL are summarized in Fig. 5D.

RANK promotes cell proliferation through Akt and Erk1/2

Given the importance of Erk1/2 and Akt in regulating cellproliferation (Meloche and Pouyssegur, 2007; Cheng et al., 2005)

and our findings that these signaling pathways are activatedby RANK in mammary epithelial cells, we examined theirsignificance as downstream effectors of RANK-inducedproliferation. The PI3K inhibitor wortmannin and MEK1

inhibitor U0126 were selected to block activation of Aktand Erk1/2 in NMuMG cells. The effectiveness of inhibitionwas verified by western blotting following RL treatment

(supplementary material Fig. S5). We observed that RLstimulated NMuMG proliferation as expected, and the co-treatment of RANKL with either wortmannin or U0126

abrogated this induction, demonstrating that both pathwaysunderlie RANK-mediated cell proliferation (Fig. 6A).

Erk1/2 plays a dominant role in RANK-induced cellinvasion and cell survival

Next, we tested the importance of both Akt and Erk1/2 signaling

pathways in RANK-regulated invasion using Moto1.2 cells.Matrigel-coated Transwell chamber assays were performedfollowing RL treatment in combination with wortmannin,

U0126 and OPG-Fc (Fig. 6B). The PI3K inhibitor wortmanninshowed no effect on RANK-induced invasion through Matrigel,whereas the Erk1/2 inhibitor U0126 clearly reduced invasion;

these two inhibitors exerted little effect in the absence ofRL. Strikingly, OPG-Fc completely prevented invasion.Representative images of invaded cells at the 24 hour time

point are depicted (Fig. 6B). These data show that Erk1/2, but notAkt, facilitates RANK-dependent cancer cell invasion, whereasthe powerful inhibition of invasion by OPG-Fc indicates that

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additional mechanisms are recruited by RANK during the process

of cell invasion.

Lastly, we sought to determine whether inhibition of Akt or

Erk1/2 in NMuMG and Moto1.1 cells affected the pro-survival

mechanism elicited by RANK. In both cell types, U0126

treatment in combination with RL significantly inhibited cell

viability over the entire time course of anchorage-independent

culture (Fig. 6C). Wortmannin, however, transiently inhibited

RANKL-induced NMuMG survival and had no effect on

Moto1.1 cells. Taken together, we conclude that the Erk1/2

MAPK pathway is the predominant signaling effector underlying

RANKL-induced anchorage-independent cell survival.

DiscussionIn this work we have investigated the functional importance of

RANK signaling in osteosarcoma cells, and also in non-

tumorigenic mesenchymal and epithelial cell lines engineered

to ectopically express RANK or RANKL. Our approaches were

generally designed to study homotypic RANK signaling through

ligand and receptor production by the same cell type, with some

experiments extending to autocrine RANK signaling with cells

expressing both ligand and receptor. This is in contrast to

the well-described heterotypic interaction among RANKL and

RANK expressed by distinct cell types. The gain- and loss-of-

function studies demonstrate that RANK activation differentially

Fig. 5. RANK activates multiple signaling pathways in mammary epithelial and osteoblastic mesenchymal cells. (A) Representative immunoblots depicting

activation of Erk1/2, JNK1, p38, Akt and p65 NF-kB after RL treatment in Moto1.2 (i) and NMuMG (ii) cells. Cells were starved for 24 hours, treated with 30 ng/

ml RL and subjected to immunoblot analysis. Phospho-specific antibodies were used to detect phosphorylated Erk1/2, phosphorylated JNK1, phosphorylated p38,

phosphorylated Akt and phosphorylated p65 in the whole-cell extracts. The membranes were stripped and probed with antibodies directed against non-

phosphorylated forms of the above proteins. (B) Representative immunoblots depicting activation of the aforementioned phosphorylated and total proteins in

MC3T3 cells transfected with control (pLVX) or RANK-expressing (pRANK) contructs starved for 24 hours and treated with 30 ng/ml RL. (C) Representative

immunoblots depicting activation of the aforementioned phosphorylated and total proteins in NMuMG pLacZ or pRL cells cultured for 24 hours in 0% or 1%

DMEM with or without 200 ng/ml OPG-Fc. One representative of three independent experiments is shown. (D) A schematic representation of the key signaling

molecules downstream of RANK depicting putative homotypic, autocrine and juxtacrine signaling mechanisms.

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impacts individual tumor cell characteristics of epithelial and

osteoblastic origin, and identify the involvement of Erk1/2 as a

major downstream effector of RANK. We also uncover the

ability of RANK overexpression to impact cell transformation

and anchorage-independent viability of MC3T3 preosteoblasts

and NMuMG epithelial cells. This indicates that de novo

RANK expression and activation might be sufficient for

cell transformation. The implications of RANK signaling on

tumorigenic characteristics through homotypic, autocrine

and heterotypic (paracrine) mechanisms in osteosarcoma and

mammary epithelial cells are modeled in Fig. 7.

The TNFR superfamily member RANK is widely expressed in

a number of tissues, whereas its ligand has a more restricted

expression pattern. RANKL is abundantly expressed in normal

thymus, lymph node and lung, and at lower levels in spleen, bone

marrow and mammary gland (Boyce and Xing, 2007). In

Fig. 6. Erk1/2 and Akt/PKB are crucial downstream effectors of RANK signaling. (A) Line graph depicts the cell proliferation of NMuMG cells treated in

combination with RL, U0126 or wortmannin over 72 hours of culture (i), whereas bar graph shows proliferation of NMuMG cells at 72 hours (ii). (B) The bar

graph and representative photomicrographs show Moto1.2 cell invasion through Matrigel in response to treatment in combination with RL (100 ng/ml),

OPG-Fc (200 ng/ml) U0126 (1 mg/ml) or Wortmannin (1 mg/ml). Invasion experiments were performed in triplicate with the experiment repeated on three

independent occasions (n53). (C) Line graphs depict anchorage-independent viability of NMuMG (i) or Moto1.1 (ii) cells treated in combination with RL

(100 ng/ml), U0126 (10 mM) or wortmannin (0.2 mM) over 72 hours. The horizontal bar graph to the right shows the effect of these compounds on

anchorage-independent viability at 48 hours of culture. The results are presented as mean 6 s.e.m. (*P#0.05, compared with cells treated with RL).

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autoimmune disorders such as osteoarthritis, osteoporosis and

bone loss induced by cancer metastasis, RANKL is aberrantly

produced by synovial cells (Nakano et al., 2004), activated T

cells (Kotake et al., 2001) and bone stromal cells (Kitazawa and

Kitazawa, 2002). In these degenerative diseases, paracrine

RANKL–RANK signaling is recognized to have a pivotal role

in osteoclast-mediated bone destruction. Consistent with this

concept, we observed paracrine RANK-mediated effects when

the Moto1.1 cell line was treated with exogenous RANKL, which

provides a strong stimulus for cell motility and survival. Beyond

this, we demonstrated homotypic RANK function. First, we

found that primary osteosarcoma bone lesions expressed

significant levels of both RANKL and RANK at early stages of

tumorigenesis, and that this expression pattern becomes

exaggerated in late-stage tumors. Second, we show that

RANK localizes to osteoblastic tumor cells residing within

osteosarcomas. Last, through the use of Moto1.2 cells that co-

express RANK and RANKL and through strategies that inhibit

endogenous RANKL (OPG-Fc and RANK shRNA), we confirm

the importance of homotypic and autocrine RANK function.

Similar effects of RANK activation on SaOS-2 human

osteosarcoma cell line have been reported (Akiyama, et al.,

2010). We propose that autocrine or homotypic RANK signaling

is crucial for early stages of osteosarcoma development, and

that this in conjunction with heterotypic signaling between

osteoclasts, osteoblasts and tumor cells, operates during tumor

progression. These complex interactions create a vicious cycle to

accentuate bone tumor burden.

We further show that establishment of a RANK overexpression

system in normal osteoblasts is able to enhance tumorigenic

properties including migration and anchorage-independent

survival but not soft-agar colony growth. These data suggest

that RANK activation provides substantial advantages to cancer

cells, but might not be sufficient for overt transformation of

normal osteoblastic cells. However, clinical correlations have

been made showing a positive relationship between RANK

and RANKL expression, and outcome of human osteosarcoma

patients, such that RANK-positive tumors exhibit a poor chemo-

response and increased tumor burden (Mori et al., 2007; Lee et al.,

2011), supporting a role for RANK signaling in primary bone

tumorigenesis. Of notable interest is our observation that

activation of RANK led to a greater cell survival advantage in

NMuMG cells, as demonstrated by its dual ability to enhance

anchorage-independent viability and soft-agar growth. 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. In this study, RANK was shown to be preferentially

expressed in basal type breast cancers with a predisposition to

metastasis. As mentioned previously, RANK has also been

assigned a crucial role in promoting progestin-mediated breast

cancer (Schramek et al., 2010; Gonzalez-Suarez et al., 2010).

Taken together, the RANK signaling pathway is emerging as

important in primary mammary tumorigenesis, although whether

RANK or RANKL can be considered as proto-oncogenes is still

premature. Further studies are required to elucidate whether

aberrant expression of RANKL in primary, non-immortalized

mammary epithelial cells is sufficient to initiate tumorigenesis.

At least seven signaling pathways are activated by RANK in

osteoclasts; four directly mediate osteoclastogensis (IKK–NF-

kB, JNK1, Myc, calcineurin–NFAT), two regulate osteoclast

activity (Src–Akt, p38) and two promote osteoclast survival (Src–

Akt, Erk1/2) (Boyce and Xing, 2007; Walsh and Choi, 2003).

How these signaling molecules relate to osteoclasts is now well

understood. The function of RANK in conditions other than bone

homeostasis and immunity is exemplified in the mammary gland,

where IKKa–NF-kB signaling has been shown to be a crucial

downstream pathway in regulating epithelial cell proliferation

(Cao et al., 2001; Schramek et al., 2010; Tan et al., 2011). Our

thorough examination of the signaling pathways activated

through RANK showed specific kinetics of phosphorylation for

Fig. 7. RANK signaling through homo- and heterotypic

cellular mechanisms regulates survival, proliferation and

motility. A schematic model representing the putative RANK–

RANKL signaling interactions pertaining to osteosarcoma cells

and mammary epithelial cells. In osteosarcoma, RANK signaling

is facilitated through paracrine, autocrine and homotypic

mechanisms where Erk1/2 plays a dominant role in promoting

cell survival and motility. The source of RANK in the tumor

microenvironment is osteoclasts and osteosarcoma cells,

whereas RANKL is produced in osteoblasts and tumor cells.

RANK-induced mammary epithelial cell survival and

proliferation are mediated through Akt and Erk1/2 signals.

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Erk1/2, Akt, JNK, p38 and p65–NF-kB. Because Akt and Erk1/2

family members have been widely implicated in tumorigenicparameters such as cell migration (Kim et al., 2001; Schafer et al.,2004) and survival (Bao and Stromblad, 2004), even extending tosome osteosarcoma cell lines (Wittrant et al., 2006; Mori et al.,

2007; Akiyama et al., 2010), their significance was furtherprobed with specific biochemical inhibitors. RANK activation inNMuMG cells resulted in increased cell proliferation, and

inhibition of either Akt or Erk1/2 countered this effect.Furthermore, we found that the effects of RANK activation onosteosarcoma cell migration and survival were mediated through

Erk1/2. Our data provide new evidence for the functionaldependence of RANK on Erk1/2 signaling for regulatingseveral tumor cell phenotypes.

Src is known to activate both Erk1/2 and Akt (Scheid andWoodgett, 2003; Walsh and Choi, 2003) and RANK stimulation

leading to Src-mediated PI3K–Akt–NF-kB or Erk1/2 activationis facilitated by the adaptor protein TRAF6 (Wong et al., 1999;Xing et al., 2001). Activation of MAPKs and NF-kB through

TRAF6 requires distinct protein-interacting regions (Kobayashiet al., 2001). For example, site-directed mutation of the first zinc-finger domain of TRAF6 abrogates NF-kB activation whereastransduction of MAPK remains unaffected. Thus, it seems that

although RANK can stimulate both Erk1/2 and NF-kB, the exactmechanism diverges at the level of TRAF6, as modeled inFig. 5D. Hence, RANK-directed effects might be channeled

through distinct downstream signaling effectors depending uponthe cell type.

This study highlights the advantages conferred by homotypicand autocrine RANK signaling on individual tumor cell types and

delineates the downstream effectors of RANK signaling. RANKhas been the focus of several recent studies, which defineits fundamental importance in mammary tissue biology andmammary tumors. Moreover, RANKL has been shown to be

regulated by the loss of a tumor suppressor in a genetic bonetumorigenesis model, beyond its deregulation in several humandiseases. Accordingly, the broad potential of RANK therapy

has garnered considerable pharmaceutical interest with thesubsequent development of RANK inhibitors. These agentswill achieve their optimal effects if we better understand

cell-autonomous RANK signaling in addition to heterotypicinteractions mediating its paracrine effects. This study providesnew cellular and molecular insights into the causal effects ofRANK on tumorigenic properties and identifies the MAPK

pathways that can be targeted to curb pathologies linked toRANK signaling.

Materials and MethodsTissues and cell cultureMouse osteosarcoma tumor samples from 10- and 28-week-old MOTO (murineosteocalcin promoter-driven T-antigen osteosarcoma) transgenic mice wereharvested and snap-frozen in liquid nitrogen before RNA or protein extraction.The MOTO transgenic mouse model of osteosarcoma has been describedpreviously (Molyneux et al., 2010). Moto osteosarcoma cell lines Moto1.1,Moto1.2 and Moto2.1, propagated from MOTO tumors, have been described indetail elsewhere (Molyneux et al., 2010). The MOTO cell lines Moto1.3, Moto1.4,Moto1.5, Moto1.6, Moto1.7, Moto1.8, Moto1.9 and Moto1.10 were propagatedfrom MOTO tumors as described (Molyneux et al., 2010). Mouse osteoblastic cells(7F2OSB, MC3T3-E1) and the mammary epithelial cell line (NMuMG) wereobtained from ATCC (CRL-12557, CRL-2593, CRL-1636). Ongoing cultureswere maintained in DMEM (Moto1.1, Moto1.2, NMuMG) or aMEM (7F2OSB,MC3T3-E1) containing 25 mM glucose, L-glutamine, antibiotics (100 U/mlpenicillin, 100 mg/ml streptomycin) and supplemented with 10% FBS.Recombinant mouse RANKL (RL) and OPG-Fc were purchased from R&DSystems Proteins (Minneapolis, MN). The MEK1/2 inhibitor U0126 and PI3K

inhibitor wortmannin were generously provided by Vuk Stambolic, Ontario CancerInstitute, Toronto, Canada. For cell-signaling experiments, cells were washed inPBS and starved in DMEM medium for 24 hours; cells were treated with 0, 10, 30or 100 ng/ml recombinant mouse RANKL or 200 ng/ml OPG-Fc for the indicatedtimes. In experiments using the pharmacological inhibitors U0126 (10 mM) orwortmannin (0.2 mM), cells were pre-treated in medium containing these inhibitorsfor 1 hour before RANKL stimulation; control cells were pre-treated in mediumcontaining 1% DMSO.

RNA extraction and qPCR analysis

Total RNA was prepared from MOTO bones (flushed to remove bone marrow,flash frozen and pulverized) or cell lines using TRIzol (Invitrogen, Carlsbad, CA).RNA purity was confirmed using a NanoDrop Spectrophotometer (ThermoScientific, West Palm Beach, FL) and by running RNA on formaldehyde agarosegels to observe the integrity of 18S and 28S ribosomal RNA species. Onemicrogram of RNA was reverse-transcribed using a first-strand cDNA synthesis kit(GE Healthcare, Pittsburgh, PA) and subjected to qPCR (DDCT) analysis, using anABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Carlsbad,CA) and TaqMan gene expression assay mix containing unlabeled PCR primersand FAM-labeled TaqMan MGB probes (RANKL Mm01313944_g1, OPGMm01205928_m1, RANK Mm01286484_m1, b-actin 4352933E). All raw datawere analyzed using Sequence Detection System software version 2.1 (AppliedBiosystems). The threshold cycle (CT) values were used to calculate relative RNAexpression levels. Values were normalized to endogenous b-actin transcripts.

Immunohistochemical analysis

Bone and osteosarcoma tissues were fixed in 10% formalin, decalcified withImmunocal (Decal Chemical, Tallman, NY), and sectioned at 5 mm. For cellimmunocytochemical analysis, cells cultured on eight-well chamber slides werefixed in 4% paraformaldehyde. RANK immunostaining was performed followingHRP-AEC System protocol (R&D Systems) with a few modifications.Specifically, antigen retrieval in tissue sections was achieved by Proteinase K(Dako, Carpinteria, CA) digestion for 10 minutes at 37 C followed by 7 minutesincubation at room temperature in a humidified chamber. Anti-RANK antibody(R&D Systems, clone AF692) diluted as 1:100 in 5% horse serum was incubatedovernight at 4 C. Biotinylated horse anti-goat secondary antibody (BA9500 VectorLaboratories, Southfield, MI) diluted 1:1000 in PBS containing 2% mouse serumwas incubated on sections at room temperature for 60 minutes.

Cell lysis and immunoblot analysis

Cells were washed in PBS and incubated in RIPA cell extraction buffer (20 mMTris-HCl, pH 7.6, 1% Triton X-100, 0.1% SDS, 1% NP-40, 1% sodiumdeoxycholate, 5 mM EDTA, 50 mM NaCl) supplemented with 200 mMNa3VO4, 2 mM PMSF, and an appropriate dilution of Complete Mini, EDTA-free protease inhibition cocktail tablets (Roche, Indianapolis, IN), for 30 minutes.Protein concentrations were determined using a BCA kit (Pierce Chemicals,Rockford, IL). For immunoblotting, 30 mg of cell protein lysate was resolved bySDS-PAGE and transferred to nitrocellulose membranes. The membranes wereprobed using the following mouse antibodies against: FLAG M2 (Sigma), RANK(AF692; R&D Systems), RANKL (L300), phosphorylated JNK (p-JNK, Thr183/Tyr185), JNK, phosphorylated p44/42 MAPK (p-Erk1/2, Thr202/Tyr204), p44/42MAPK (Erk1/2), phosphorylated p38 (p-p38, Thr180/Tyr182), p38 MAPK,phosphorylated Akt/PKB (p-Akt, Ser473), Akt, phosphorylated p65-NF-kB(Ser536), p65-NF-kB and cleaved caspase-3 (all from Cell SignalingTechnology, Boston, MA). The blots were stripped and reprobed with an HRP-conjugated monoclonal antibody directed against mouse b-actin (Santa CruzBiotechnology, Santa Cruz, CA).

Overexpression and shRNA knockdown systems

A full-length mouse RANKL cDNA (GenBank Accession No. NM011613) waspurchased from Origene (Rockville, MD) and cloned into the mammalianexpression vector pcDNA3.1 at the NotI and BamHI restriction sites (Invitrogen,Carlsbad, CA). A clone (NMuMG-pRL) containing the RANKL cDNA in theforward orientation was subsequently identified by DNA sequencing. A pcDNA3.1expression vector containing the b-galactosidase gene (pcDNA3-LacZ; Invitrogen)was used to determine transfection efficiency and served as a control for thesestudies. Stable transfections were performed to establish multiclonal NMuMGcells constitutively expressing RANKL. One mg/ml of plasmid DNA wastransfected using Lipofectamine 2000 transfection reagent (Invitrogen) accordingto the manufacturer’s protocol. Selection began 48 hours post transfection using400 mg/ml G418 sulphate in DMEM; after selection, cells were maintained inmedium containing 100 mg/ml G418 sulphate.

For the generation of a RANK overexpression system, a full-length mouseRANK cDNA construct (No. NM009399) was purchased from Origene and clonedinto the lentiviral expression vector pLVX-puro (Clontech, Mountain View, CA).Generation of virus particles was achieved using the Lenti-X HT packaging system(Clontech) following the manufacturer’s protocol. MC3T3 cells were infected with

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virus for 24 hours, after which cells were provided fresh medium and cultured foran additional 48 hours. At this point, transduced cells were selected in completeaMEM containing 2 mg/ml puromycin for 72 hours. After selection, cells weremaintained in aMEM containing 10% FBS.

A set of four lentiviral pLKO.1 short hairpin (sh)-RNA expression vectorsexpressing shRNA sequences directed against mouse RANK under the control ofthe U6-promoter were generously provided by Jason Moffat, RNAi Consortium,The University of Toronto, Canada. These constructs were designated pLKO.1-shRNA-2, -7, -8 and -10. Vectors containing a scrambled shRNA (pLKO.1) orGFP (pLKO.1-GFP) insert served as controls. Lentiviral particles were generatedusing standard molecular biology procedures. Briefly, 293T packaging cells wereseeded at 1.36105 cells/ml in 6 cm tissue culture plates containing DMEMsupplemented with 10% FBS and antibiotics. After 24 hours, cells were transfectedwith the packaging plasmid pCMV-dR8.91 (Addgene; Cambridge, MA, USA), theenvelope plasmid VSV-G/pMD2.G (Addgene) and one of the four RANK-directedpLKO.1-shRNA vectors. After 18 hours, cell medium was removed and replacedwith fresh DMEM medium containing 10% FBS and cells were cultured for afurther 24 hours, after which medium containing lentivirus was collected and usedto infect target cells. Selection of transduced cells was performed as describedabove using Puromycin.

Proliferation and soft-agar colony-formation assays

Cells were seeded at 16103 cells/well into opaque 96-well microplates containing100 ml of DMEM with 5% FBS. Cells were cultured for 0, 24, 48, 72 or 96 hoursin the presence of recombinant mouse RANKL, OPG-Fc, U0126 or wortmannin,after which cell proliferation was measured using the CellTiter-Glo LuminescentCell Viability Assay (Promega, Sunnyvale, CA) following the manufacturer’sinstructions on a FLUOstar Optima plate reader (BMG; Offenburg, Germany).Soft-agar colony formation assays were performed by seeding cells in six-wellculture plates within 0.35% agar containing DMEM medium supplemented with10% FBS over a base layer of 0.5% agar dissolved in DMEM. Complete DMEMmedium (2 ml) containing 10% FBS and supplemented with or withoutrecombinant mouse RANKL or OPG-Fc was placed on top of agar cultures andreplaced every 3 days. On days 14 or 21 after seeding, cells were fixed in 100%methanol and stained with 0.05% crystal violet. Colony formations were imagedusing a digital camera mounted to a light microscope (256 objective).

Migration and invasion assays

Cell motility assays were performed using uncoated or coated (growth factorreduced Matrigel, rat-tail collagen I; BD Biosciences, San Diego, CA) Transwellsfitted with Millipore membranes (BD Biosciences; 6.5 mm filters, 8 mm pore size)as described (Beristain et al., 2011). Briefly, 26104 cells/200 ml of DMEMsupplemented with 1% BSA were plated in the upper chambers and cultured forthe indicated times. Lower chambers contained 500 ml of DMEM supplementedwith 10% FBS and both upper and lower chamber medium contained the indicatedconcentrations of recombinant RANKL or inhibitors. Cells from the uppersurface of the Millipore membrane were removed by gentle swabbing,whereas transmigrated cells attached to the membrane were fixed in 4%paraformaldehyde and stained with eosin. The filters were rinsed with water,excised from the Transwells, and mounted upside down onto glass slides. Cellinvasion was determined by counting the number of stained cells in 10 randomlyselected, non-overlapping fields at 1006 magnification using a light microscope.Cell invasion was tested in triplicate wells, on three independent occasions. Forscratch-wound migration assays, Moto1.2 cells were cultured in 24-well plates andallowed to grow to confluency, when a wound was made across the well with ayellow pipette tip. The medium was then changed to DMEM containing 10% FBSwith or without 200 ng/ml OPG-Fc. The wound was photographed immediatelyand again 20 hours after scraping to document cellular migration across thewound.

Anchorage-independent cell-viability assays

Anchorage-independent growth was achieved by culturing 56103 cells/well inultra-low-adherence 96-well microplates (Costar, New York, NY) containingDMEM supplemented with 10% FBS for 0, 2, 8, 24, 48 or 72 hours. Cells weretreated in combination with recombinant mouse RANKL, OPG-Fc or the inhibitorsU0126 and wortmannin. Cell viability was measured using the CellTiter-GloLuminescent Cell Viability Assay (Promega) according to the manufacturer’sinstructions. For analysis of cleaved caspase-3, cells were cultured for 48 hours on10 cm ultra-low-attachment culture plates (Costar) in DMEM containing 10% FBSsupplemented with or without recombinant mouse RANKL (30 ng/ml). Afterwhich, suspended cells were pelleted by centrifugation, washed in ice-cold PBSand subjected to RIPA buffer protein extraction.

Statistical analysis

Data are reported as mean 6 s.d. or s.e.m. All calculations were carried out usingGraphPad Prism software (San Diego, CA). Comparisons were made using two-tailed Student’s t-test and ANOVA. Cellular invasion indices were analyzed

by one-way ANOVA followed by the Tukey multiple comparison test. Thedifferences were accepted as significant at P,0.05.

AcknowledgementsThe authors thank Jason Moffat, for providing reagents and advice.The authors also thank Paul Waterhouse and Sam Molyneux forcritical reading of the manuscript.

FundingThis work was supported by funding from the Canadian CancerSociety Research Institute, Ontario Cancer Research Network andGlobal Leadership Round in Genomics & Life Sciences (GL2) toR.K. A.G.B. holds a Canadian Breast Cancer Foundation Fellowship.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.094029/-/DC1

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