A JAK2-binding Long Noncoding RNA Promotes Breast Cancer Brain Metastasis

Shouyu Wang1,11,#, Ke Liang1,#, Qingsong Hu1,#, Jian Song5, Yuedong Yang13, Jun Yao1,

Selanere Mangala6, Chunlai Li1, Wenhao Yang1, Peter K. Park1, David H. Hawke4, Jianwei

Zhou11, Yan Zhou12, Weiya Xia1, Mien-Chie Hung1,2,10, Jeffrey R. Marks9, Gary E. Gallick5,

Gabriel Lopez-Berestein8, Elsa R. Flores14, Anil K. Sood2,6, Suyun Huang7, Dihua Yu1, Liuqing

Yang1,2,3* and Chunru Lin1,2,*

1
Department of Molecular and Cellular Oncology
2
The Graduate School of Biomedical Sciences
3
Center for RNA Interference and Non-Coding RNAs
4
Department of System Biology
5
Department of Genitourinary Medical Oncology
6
Department of Gynecologic Oncology and Reproductive Medicine
7
Department of Neurosurgery
8
Department of Experimental Therapeutics

The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
9
Department of Surgery, Duke University School of Medicine, Durham, NC, 27710
10
Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical

University, Taichung, 404, Taiwan

11
Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing

Medical University, 140 Hanzhong Road, Nanjing 210029, China

12
Department of Oncology, Yixing People’s Hospital, 75 Zhenguan Road, Yixing 214200, China

1
 13
Institute for Glycomics, Griffith University, Parklands Dr, Southport, Queensland 4222,

Australia
14
Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612
#
Denotes equal contributions
*
To whom correspondence should be addressed: lyang7@mdanderson.org and

clin2@mdanderson.org

Abstract

Conventional therapies for breast cancer brain metastases (BCBMs) have been limited due to

chemoresistance and blood-brain barrier impermeability. Circumventing this resistance will

require comprehensively understanding the underlying mechanism. We report that expression of

a long non-coding RNA, Lnc-BM, is prognostic of brain metastasis progression in breast cancer

patients. Elevated Lnc-BM expression drives BCBM while targeting Lnc-BM with nanoparticle-

encapsulated siRNAs appears to effectively treat BCBM in preclinical models. Mechanistically,

Lnc-BM modulates Janus kinase 2 (JAK2) kinase activity to mediate Oncostatin M- (OSM) and

Interleukin 6- (IL6) triggered STAT3 phosphorylation. In breast cancer cells, STAT3-dependent

expression of Intercellular Adhesion Molecule 1 (ICAM1) and Chemokine (C-C Motif) Ligand 2

(CCL2) mediate vascular co-option and macrophage recruitment respectively, leading to a vicious

cycle where brain-resident macrophage produced OSM/IL-6 further activates the Lnc-

BM/JAK2/STAT3 pathway to enhance BCBM. Collectively, our results define the roles of Lnc-

BM and JAK2 in promoting BCBMs with important clinical implications.

2
KEYWORDS: Breast Cancer, Brain Metastasis, JAK2, STAT3, Long Non-coding RNA, Lnc-

BM, ICAM1, CCL2, macrophage, Nanoparticle

Introduction

Breast cancer brain metastasis (BCBM) is a serious health condition that negatively affects a

patient’s quality of life, with median survival times between 4 to 6 months (1). Brain metastasis

occurs in about 15% of women with newly diagnosed metastatic breast cancer (2). Clinically,

patients with either the HER2-positive or triple-negative type of breast cancer have significantly

higher incidences of brain metastasis, ranging between 20-50% (3, 4). Although modern

multidisciplinary care have been applied to treat cases of brain metastasis (5), the benefit of these

treatments is limited due to their palliative and localized tendencies (6). Therefore, there is an

imperative need for the development of novel therapies based on biological and molecular

mechanisms of brain metastatic lesions.

The blood-brain barrier (BBB), which is composed of non-fenestrated capillaries and

astrocytes, effectively limits circulating tumor cells (CTCs) from entering the brain parenchyma

(7). Animal models of brain metastasis show that metastatic cells arrested within brain capillary

beds encounter brain microvascular endothelial cells, a necessary step for growth and invasion (8).

Recent studies have revealed that L1 Cell Adhesion Molecule (L1CAM) promotes breast cancer

cell adhesion to brain capillaries and subsequent brain metastasis (9). Brain metastatic breast

cancer cells secrete CCL2, to recruit myeloid cells, which promote the outgrowth of metastatic

tumors (10). These studies signify the complicated and reciprocal cross-talk between tumor cells

and the metastatic niche.

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 Long noncoding RNAs (lncRNAs) are a class of transcripts that are longer than 200

nucleotides with low protein-coding potential and that are involved in a wide range of

physiological and pathological processes (11). Many lncRNAs have been shown to modulate

breast cancer-derived metastasis to the lung, liver, and lymph nodes (12-14). However, the role of

lncRNAs in mediating BCBM is still unclear. Targeting lncRNAs using locked nucleic acids

(LNAs) or an siRNA-based strategy has been successfully applied in several preclinical models

(13, 15).

Here, we report that elevated expression of Long non-coding RNA associated with Breast

Cancer Brain Metastasis (Lnc-BM) in primary breast cancer exhibit higher rates of recurrence to

the brain. High Lnc-BM expression promotes breast cancer cells to metastasize to the brain in

preclinical mouse models. Furthermore, targeting Lnc-BM by nanoparticle-coated siRNAs

knocked down Lnc-BM in vivo and alleviated tumor burden in mouse brains. Mechanically, Lnc-

BM interacts with and activates the non-receptor tyrosine kinase Janus kinase 2 (JAK2). The

activated JAK2 phosphorylates signal transducer and activator of transcription 3 (STAT3),

triggering activation of the downstream signaling pathway that includes the proteins intercellular

adhesion molecular 1 (ICAM1) and chemokine (C-C motif) ligand 2 (CCL2). ICAM1 is

responsible for breast cancer cell adhesion to blood vessels of the brain and ultimately to

extravasation to form metastatic lesions within the brain. CCL2 is a chemokine that is released

into the microenvironment and attracts macrophages to the cancer cell. The attracted macrophages

release the cytokines oncostatin M (OSM) and interleukin 6 (IL-6) both of which activate JAK2,

triggering a positive feedback loop that perpetuates the Lnc-BM/JAK2/STAT3 signaling axis that

is crucial to the metastatic potential of breast cancer cells to the brain.

4
Results

Identification of Lnc-BM as a biomarker of BCBM

To identify BCBM-relevant lncRNAs, we assessed expression profiles of lncRNAs in parental

MDA-MB-231 (231-Par) cells and isogenic brain metastatic cells (231-BM), isolated from brain-

seeking 231-Par cells by LncRNA array (v.3.0) (ArrayStar) (Figure 1A). There were 1,019

upregulated and 987 downregulated lncRNAs with significant differential expression (≥2 fold)

(Figure 1B and GEO ID:GSE79540). Nine lncRNAs were upregulated in 231-BM compared to

231-Par according to the following criteria: 1) the ratio of 231-BM/231-Par ≥ 2.5; 2) raw signaling

intensity ≥ 2000; and 3) lncRNA length ≥ 300 (Supplemental Figure 1A). RP11-355I22.7

(CRCh37/hg19, also known as AK055647), the most highly upregulated lncRNA, was renamed to

Lnc-BM (long non-coding for Brain Metastasis). Compared to parental cells (231-Par), Lnc-BM

expression was greatly upregulated in brain metastatic cells, but not in lung metastatic LM2 or

bone metastatic BoM-1833 (Supplemental Figure 1B-D). In GRCh38, a splicing variant of

SYT16 overlap with AK055647 (Lnc-BM), which is non-detectable in brain metastatic or non-

brain metastatic cells we tested (Supplemental Figure 1B-D). Lnc-BM sequence exhibited low

protein coding potential (coding potential score, -1.18283, Coding Potential Calculator). In vitro

translation assays indicated that neither the sense nor the antisense of Lnc-BM transcripts encodes

protein (data not shown). RNA In situ hybridization (FISH) and nuclear/cytoplasmic fractionation

showed that Lnc-BM is mostly located in the cytoplasm (Supplemental Figure 1E-F).

Using RNAScope 2.0 HD technology, we found only 6.9% of adjacent normal breast

tissues while 54.9% of breast cancer tissues were Lnc-BM positive (Figure 1C and Supplemental

Table 1). TissueScan Cancer qPCR Arrays indicated that Lnc-BM was significantly overexpressed

in breast cancer tissues vs. normal tissues (p < 0.001); in stages III-IV vs. I-II of breast cancer (p

5
= 0.034); in metastatic (TnN > 0M ≥ 0) vs. non-metastatic breast cancer (TnN0M0) (p = 0.022)

(Figures 2D-F). Consistently, Lnc-BM expression was elevated in TNBC cell lines and HER2

positive cell lines (BT474 and MDA-MB-361), both with brain metastatic potential (16, 17), when

compared to normal mammary epithelial cells or estrogen receptor (ER) positive cells with low

metastatic potential (Supplemental Figure 1G).

High Lnc-BM expression was also negatively correlated with recurrence-free survival in

breast cancer patients (p < 0.0001) (Figure 1I). Importantly, primary breast cancer tissues with

high Lnc-BM expression exhibited higher recurrence rates to the central nervous system (CNS)

than local/regional or other distant organs (Figure 1J), suggesting that Lnc-BM expression in the

primary tumor may indicate an increased risk for brain metastasis. Furthermore, 92.9% of breast

cancer brain metastatic tissues showed positive Lnc-BM staining (Figure 1K). These data suggest

the importance of Lnc-BM in BCBM development and progression.

Lnc-BM promotes breast cancer brain metastasis

Experimental brain metastatic xenograft models via intracardiac or intra-arterial injections are the

well-established anima models to study brain metastasis due to the lack of allograft murine models

(9). To determine the functional role of Lnc-BM in brain metastasis, we first stably knocked down

Lnc-BM in 231-BM cells (Supplemental Figure 2A). The role of Lnc-BM in breast cancer cell

extravasation was examined using an in vitro model for the BBB, which is comprised of human

endothelial cells and astrocytes on each side of a Transwell membrane (Figure 2A). Lnc-BM

knockdown significantly inhibited transmigration of 231-BM cells through this barrier, but not cell

proliferation (Figure 2B and Supplemental 2B). The role of Lnc-BM in promoting brain

metastasis was examined using pre-clinical animal models by intracardiac injection of 231-BM

6
cells labeled with GFP and luciferase. Remarkably, four weeks post-injection, mice in the control

group developed overt and pervasive brain metastasis as detected by magnetic resonance imaging

(MRI) and in vivo/ex vivo bioluminescence imaging (BLI) (Figures 2C-D). Lnc-BM knockdown

decreased the number and size of brain metastatic lesions (Figures 2C-D) but did not affect bone

metastatic lesions (Supplemental Figure 2C). Interestingly, ex vivo GFP revealed that

extravasated 231-BM cells bound to and spread along the surface of brain capillaries in the control

group and that outgrowth occurred mainly on co-opted vessels, which were both abolished by Lnc-

BM depletion (Figure 2D, middle panel). H&E staining of mouse brain tissues indicated that Lnc-

BM knockdown diminished micrometastases, macrometastases and increased cleaved caspase-3

(Figures 2E and Supplemental 2D and 2G).

231-Par cells exhibited minimal brain metastasis post intracardiac injection; however,

stable overexpression of Lnc-BM in these cells (Supplemental Figure 2H) showed increased

brain metastasis (Figures 2F-G), but metastatic burden in bone was not affected (Supplemental

Figure 2I). Further, ex vivo GFP indicated that the majority of 231-Par cells were congested in the

vascular lumen but cells with overexpression of Lnc-BM penetrated through the BBB and formed

metastatic lesions (Figure 2G, middle panel). Consistently, high Lnc-BM expression resulted in

reduced cleaved caspase-3 staining in mouse brain tissues (Supplemental Figures 2J and 2M).

Chitosan nanoparticle-coated Lnc-BM siRNA suppresses BCBM in vivo

The impermeability of the BBB complicates drug transport to the brain by limiting the delivery of

the majority of currently available therapeutics to the brain metastatic lesions (6). Previous

research works have indicated that chitosan nanoparticles (NPs) can efficiently transport BBB-

impermeable siRNAs to the brain (18), which might have potential value in targeting BCBM.

7
 We first confirmed the in vivo efficacy of siRNA delivery into the brain by NPs. NP-

conjugated control siRNAs with or without Cy3 labeling were intravenously injected. 48 hours

post-injection, we detected abundant Cy3 signals in the perinuclear regions of mouse brain tissues

after subtracting the background (Supplemental Figure 2N).

Then NP-conjugated control siRNAs or siRNAs targeting Lnc-BM were delivered to brain

metastasis-bearing mice (Figure 2H, left panel). NP-siRNA treatment (150 μg/kg) began on day

3 (early treatment) or day 9 (late treatment) (19) after tumor cell injection via the tail-vein and

continued two times per week until nine doses had been given (Figure 2H, right panel). By week

4, NP-Lnc-BM siRNAs had significantly reduced brain metastatic burden compared to the control

siRNA or PBS (Figures 2I-J), however, bone metastatic lesions were not affected (Supplemental

Figure 2O). The knockdown efficiency of NP-siRNAs was confirmed by RNAscope staining of

brain metastatic mice tissues (Figure 2K and Supplemental 2P). The median survival time (MST)

of brain metastasis-bearing mice treated with PBS or the control siRNA was about 35d. On the

contrary, treatment with NP-siRNAs targeting Lnc-BM significantly prolonged the survival time

of these mice (MST = 46d, 72d and 56d for si#1, si#2 and si#2 (Late), respectively, p < 0.001)

(Figure 2L). Moreover, application of NP-siRNAs did not induce detectable toxicity to organs we

examined (Supplemental Figure 2Q). These results demonstrate that NP-delivered Lnc-BM

siRNAs efficiently depleted Lnc-BM in vivo and suppressed BCBM in the preclinical model.

Lnc-BM-dependent brain metastatic cancer cell vascular co-option impedes fasL-induced

apoptosis

Lnc-BM knockdown dramatically inhibited cancer cell vascular co-option in the preclinical model

(see Figure 2D). To investigate the underlying mechanism, we examined the attachment of cancer

8
cells to endothelial cells of blood vessels using a cancer cell adhesion assay. The primary cultured

endothelial cells (HUVECs) formed tube structures when cultured on Matrigel, which mimics

blood vessel features. Over 80% of brain metastatic cancer cells spread along these tubes of

endothelial cells. Notably, Lnc-BM depletion significantly reduced this attachment (Figure 3A).

In brain metastatic mice tissues, GFP labeled 231-BM cells spread on the abluminal surface of

capillaries in mouse brains and formed a sheath around the vessels, whereas cells with Lnc-BM

knockdown were round and failed to co-opt with vessels (Figure 3B).

To examine the role of Lnc-BM in promoting cancer cell vascular co-option in the brain

parenchyma, mouse brain slices were co-cultured with 231-BM cells (Figure 3C). Cancer cell-

bearing mouse brain slices were subjected to confocal imaging. The surface of brain slices (X-Y

facet) were used to determine blood vessel co-option by cancer cells; Z-stacking (Y-Z facet) was

used to measure the depth of invasion into mouse brain slices (Figure 3C). 231-BM cells harboring

control shRNAs adhered to the capillaries and stretched over their surfaces (Figure 3D, left panel),

followed by infiltration into mouse brain tissues (Figure 3D, middle and right panel). In contrast,

Lnc-BM deficient cells could not adhere to vessels or invade into brain tissues. Furthermore, Lnc-

BM knockdown cells underwent apoptosis, as revealed by cleaved caspase-3 staining in brain

slices, while the control cells did not (Figure 3E), which were confirmed in the in vivo brain

metastasis model (Figure 3F).

Activated astrocytes secrete FasL, which causes apoptosis of brain metastatic cancer cells

via Fas-mediated apoptosis (9). Lnc-BM knockdown facilitated FasL-induced apoptosis and

increased cleavage of caspase-8, caspase-3, and PARP1 in 231-BM cells, but did not affect

caspase-9 activity (Figure 3G-H and Supplemental Figure 3A and). These data suggest that Lnc-

9
BM is important for promoting vascular co-option and antagonizing FasL-triggered apoptosis

pathways in brain metastatic cells.

Lnc-BM mediates JAK2-STAT3 signaling that is triggered by OSM

To understand the mechanisms that underlie the role of Lnc-BM in BCBM, we sought to identify

the protein binding partners of Lnc-BM using RNA pulldown followed by mass spectrometry (MS).

The sense transcript, but not the antisense transcript, of Lnc-BM associated with Interleukin-6

receptor subunit beta (IL6R-beta, also known as gp130), JAK2, and suppressor of cytokine

signaling 3 (SOCS3) (Figure 4A and Supplemental Figure 3B-C and Supplemental Table 2).

Homodimerization or heterodimerization between gp130 and other cytokine receptors

triggers phosphorylation of JAK2 and of signal transducer and activator of transcription (STATs)

upon ligand binding (20). We screened eight known gp130-associated cytokines (21) and

surprisingly found that OSM was the most potent ligand that triggered phosphorylation of JAK2

and of STATs in 231-BM cells (Figure 4B). OSM bound with gp130 and Oncostatin M Receptor

(OSMR) to form a heterodimer (21). Knockdown of either gp130 or OSMR abolished OSM-

induced JAK2 phosphorylation and the activation of JAK2 downstream targets (Supplemental

Figure 3D). Notably, 231-BM cells exhibited higher OSMR protein levels and stronger

phosphorylation of JAK2/STATs upon OSM treatment than parental cells (Figure 4C). The

OSMR protein is more stable in 231-BM than in 231-Par cells without a change in OSMR gene

transcription (Supplemental Figures 3E-F). Further, we found that OSMR expression is elevated

in breast cancer tissues in comparison to adjacent normal tissues (Figure 4D) and that higher

OSMR expression is associated with shorter recurrence times for breast cancer patients (p = 0.034)

(Figure 4E).

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 Lnc-BM knockdown substantially reduced OSM-induced phosphorylation of JAK2 and

STATs (Figure 4F). In contrast, overexpression of Lnc-BM in 231-Par cells enhanced OSM-

induced JAK2-STATs activation (Supplemental Figure 3G). These data were validated in MCF7

cells that have low Lnc-BM expression and in BT-474 cells that have high Lnc-BM expression

(Supplemental Figures 3H-K). In brain metastatic tissues from the in vivo model, p-JAK2 and p-

STAT3 staining were decreased in the Lnc-BM knockdown group but increased in the Lnc-BM

overexpression group in comparison to the respective control groups (see Supplemental Figures

2D-F and 2J-L). Further, Lnc-BM knockdown did not affect interaction between JAK2 and OSMR

or gp130 (Supplemental Figure 3L), suggesting that Lnc-BM regulates JAK2/STATs signaling

through modulation of JAK2 kinase activity.

JAK2 is required for Lnc-BM-mediated breast cancer brain metastasis

We used CRISPR/Cas9-mediated gene editing to knock out JAK2 (JAK2 KO) in 231-BM cells,

which were confirmed by protein level (Supplemental Figure 3M-N). JAK2 KO cells failed to

associate with blood capillaries and exhibited reduced mobility (Figure 4G and Supplemental

Figure 3O). The in vivo brain metastasis model showed that JAK2 depletion inhibited

development of metastatic lesions in mouse brains (Figure 4H-I), but bone metastatic burden was

not affected (Supplemental Figure 3P). Moreover, MST levels of brain metastasis-bearing mice

with JAK2 KO cells were approximately double those of mice with JAK2 WT cells (Figure 4J).

Collectively, we conclude that JAK2 is required for BCBM.

Next, we evaluated phospho-JAK2 levels in human breast cancer by IHC and found

elevated levels of p-JAK2 in breast cancer tissues (Figure 4K), which was significantly correlated

with shorter recurrence times for breast cancer patients (p = 0.0149) (Figure 4L). Most importantly,

11
breast cancer patients with high Lnc-BM expression exhibited increased JAK2 phosphorylation

status and vice versa (Figure 4M), further confirming that Lnc-BM promotes BCBM by activating

JAK2.

Lnc-BM modulates the kinase activity of JAK2

In vitro RNA-protein binding assay revealed that Lnc-BM directly interacted with JAK2, but not

with gp130 or SOCS3 (Supplemental Figure 4A). We further confirmed that, in 231-BM cells,

JAK2 was specifically interacted with Lnc-BM, but not with LINK-A (22) or other LncRNAs

upregulated in brain metastatic cells (Supplemental Figure 4B). Moreover, basal Lnc-BM-JAK2

interaction was robustly enhanced upon OSM stimulation (Figure 5A). Domain mapping studies

indicated that the JH2 domain (a.a. 545-809) mediated Lnc-BM-JAK2 interaction (Supplemental

Figure S4C-D). We also identified the sequence of Lnc-BM that was responsible for JAK2

binding by in vitro RNA pull-down followed by dot blot assay. The motif containing nt. 961-1020

of Lnc-BM was required for this interaction (Figure 5B). Deletion of this binding motif (referred

to as Lnc-BM ∆JAK2) failed to associate with JAK2 in vitro (Supplemental Figures 4E-F).

To quantitatively assess the interaction between Lnc-BM and JAK2, we performed an

Alpha Assay using biotinylated Lnc-BM and GST-tagged JAK2 (JH2-JH1 domain) as the donor

and acceptor pair (Figure 5C). Saturation curve indicated that 269.7 nM of Lnc-BM reach 50%

maximal binding with JAK2, but not other lncRNAs that are highly expressed in breast cancer,

including H19 (23) and LINK-A (22) (Figure 5D).

Prediction of Lnc-BM secondary structure suggests that the 60mer RNA fragment (nt. 961-

1020) of Lnc-BM forms three stem-loop structures (Supplemental Figure 4G, dashed circles,

referred to as #1, #2 and #3). We attempted to determine the nucleotides of Lnc-BM that are

12
required for Lnc-BM-JH2 interaction. The three stem loops were deleted individually, and we

found that all three fragments contribute to the Lnc-BM-JH2 interaction (Supplemental Figure

4H).

To characterize the JH2 domain’s RNA binding propensity, computational calculations

have been performed using Bindup (24), which indicate that the JH2 domain harbors a large patch

of positive regions that could be involved in protein-RNA binding (Supplemental Figure 4I). The

predicted binding site was confirmed by RBRpred (25), another sequence-based method.

Positively-charged amino acids are essential for mediating RNA-protein interactions (26). Thus,

we individually mutated 11 positively-charged amino acids to alanine and found that R715 and

K752 were important for mediating Lnc-BM-JH2 interaction (Figure 5E and Supplemental

Figure 4J).

Using alpha assay, we measured the binding affinity of Lnc-BM and JH2 domain with

mutants. Competition assay indicated that 298.4 nM of unlabeled Lnc-BM was needed to achieve

50% inhibition of the interaction between biotin-Lnc-BM and His-tagged JH2 domain, which was

significantly impaired by Lnc-BM JAK2 or JH2 R715A/K752A mutant (Figure 5F). The RNA

oligonucleotide representing Lnc-BM nt. 961-1020 exhibited insubstantial association with JH2

domain (Figure 5F), suggesting that the importance of three-dimensional structure of RNA in

RNA-protein binding.

The kinase domain of JAK2 (JH1 domain) is self-inhibited by interaction between JH2 and

JH1 domains (27). To test whether Lnc-BM modulate the enzymatic activity of JAK2, JAK2

immunoprecipitated from cells with Lnc-BM knocked down were subjected to an IP-kinase assay,

finding Lnc-BM knockdown abolished JAK2 kinase activity both with and without OSM

stimulation (Figure 5G). In vitro kinase assays using recombinant JH1/JH2 domains or JH1
13
domain only indicated that JH1 domain exhibited potent kinase activity, which was not affected

by Lnc-BM; however, the kinase activity of the JH1/JH2 domains were dramatically enhanced in

the presence of Lnc-BM FL but not the ∆JAK2 mutant transcript (Figure 5H-I). We reasoned that

Lnc-BM binding with the JH2 domain may interfere with the JH2-JH1 domain interaction, leading

to activation of JH1 tyrosine kinase activity (Figure 5J). To confirm our hypothesis, we co-

expressed Myc-tagged JH1, SFB- (S-protein, a FLAG epitope, and a streptavidin-binding peptide)

tagged JH2, and Lnc-BM in 293T cells and found that the interaction between exogenous Myc-

JH1 and FLAG-JH2 was impaired in the presence of full-length Lnc-BM (Figure 5K, lane 8, top

panel).

It has also been suggested that JH2-dependent phosphorylation at Tyr570 mediates JH2-

JH1 interaction and subsequent inhibition of JAK2 kinase activity (28) (Figure 5J). Lnc-BM

overexpression significantly reduced Tyr570 phosphorylation (Figure 5K, lane 8, bottom panel).

Lnc-BM knockdown resulted in increased Tyr570 phosphorylation but suppressed JAK2

Tyr1007/1008 phosphorylation in 231-BM cells (Figure 5L). Moreover, in vitro transcribed Lnc-

BM-FL sense transcript (sen.), but neither the antisense (a.s.) transcript nor Lnc-BM-∆JAK2,

abolished phosphorylation of Tyr570 (Figure 5M). These data suggest that Lnc-BM may facilitate

a potential conformational change in JAK2 from a "closed" (autoinhibited) to an "open" (activated)

configuration. Lnc-BM binding to the JH2 domain inhibits JH2 kinase activity (indicated in blue),

which leads to hypophosphorylation of Tyr570. These events result in a more accessible structure

and enzymatic activation of the JH1 domain (indicated in red) (Figure 5J), which is essential for

activation of downstream signaling cascades.

Mutations of JAK2 negative regulatory sites (Ser523 and Tyr570), JH2 domain kinase-

inactivating mutant (K581A) and a natural mutant (V617F) led to constitutive activation of the

14
JAK2 kinase by disrupting the interaction between the JAK2 JH1 and JH2 domains (28).

Expression of these JAK2 mutants in JAK2 null -2A cells led to elevated JAK2 phosphorylation

at Tyr1007/1008 sites and enhanced binding between Lnc-BM and the JH2 domain as indicated

by the RNA pulldown assay (Supplemental Figure 5A), suggesting that the potential “open”

conformation of JAK2 is more accessible to Lnc-BM binding.

Next, we examined if the Lnc-BM-JAK2 interaction and activation of STATs affects Lnc-

BM expression. The half-life of Lnc-BM was significantly reduced upon treatment with a JAK2

inhibitor (TG101348) (Supplemental Figure 5B) as well as in JAK2 KO cells (Supplemental

Figure 5C). Although the promoter region (3000 nucleotides upstream of the Lnc-BM

transcription start site) of Lnc-BM harbors potential STAT binding elements, siRNAs against

STAT1, STAT3, or STAT5 did not affect Lnc-BM expression (Supplemental Figure 5D-E),

which suggests that high levels of Lnc-BM expression found in brain metastatic cells are due to

stabilization as opposed to transcriptional regulation.

To confirm that Lnc-BM-dependent JAK2 kinase activation is functionally important, we

performed functional rescue experiments. Lnc-BM knockdown impaired OSM-triggered, JAK2-

dependent STAT3 phosphorylation (Supplemental Figure 5F, lane 8). We reintroduced Lnc-BM-

FL or the ∆JAK2 deletion mutant in 231-BM cells with Lnc-BM knockdown (Supplemental

Figure 5G). Knockdown of Lnc-BM abolished OSM-induced phosphorylation of JAK2, STAT3

and cell migration through BBB, which could be rescued by wild-type Lnc-BM but not the Lnc-

BM JAK2 deletion mutant (Figure 5N and Supplemental Figure 5H). In JAK2 depletion cells,

expression of exogenous wild-type JAK2, but not the R715A/K752A mutant, restored the OSM-

induced STAT3 phosphorylation and cell mobility (Figures 5O and Supplemental Figure 5I).

15
These data suggest that Lnc-BM-JAK2 interaction is critical for sustained STAT3 signaling

activation and is important in brain metastasis.

ICAM1 supports vascular co-option and invasion of breast cancer cells

JAK2 triggers activation of three downstream AKT, MAPK, and STAT1/3/5 signaling

pathways (29). To determine the signaling pathways that are regulated by Lnc-BM, we profiled

gene expression on cells with Lnc-BM depletion or JAK2 KO and compared to their corresponding

controls following OSM stimulation. Gene Set Enrichment Analysis (GSEA) and Gene Ontology

(GO) analysis indicated that 231-BM cells showed enrichment of STAT3 signature genes (30)

upon OSM treatment, which was abolished in cells deficient of Lnc-BM or of JAK2 (Figure 6A-

B and Supplemental Figure 6A, GEO ID: GSE79534). These observations were validated by

RT-qPCR (Figure 6C). In human brain metastatic breast cancer tissues, Lnc-BM expression was

correlated with phospho-STAT3 status (Figure 6D), signifying that Lnc-BM may govern STAT3

signaling in BCBM. Using brain slice culture, Lnc-BM deficient 231-BM cells infected with a

constitutively active (CA) mutant of STAT3 achieved significant restoration of the potential to

attach and stretch over vessels and enhanced subsequent invasion of cancer cells into brain tissues

(Supplemental Figure 6B).

We screened the expression of 84 molecules related to human extracellular matrix (ECM)

and adhesion, by which five of them were downregulated (> 4 fold) in cells upon Lnc-BM

knockdown in comparison to the control group (Figure 6E). ICAM1 is co-regulated by Lnc-BM

and STAT3 (Figure 6F), and we confirmed that mRNA and protein levels of ICAM1 were

decreased upon Lnc-BM knockdown (Figures 6C and Supplemental Figure 6C). Then we

restored the protein level of ICAM1 by stably expressing exogenous ICAM1 (Supplemental

16
Figure 6C). MMP9, another ECM adhesion molecule that is regulated by Lnc-BM, also be

included as a control (Supplemental Figure 6D). Although both exogenous ICAM1 and MMP9

partially rescued Lnc-BM-dependent cell invasion (Figure 6G), only ICAM1 rescued cancer cell

adhesion to blood vessels (Figure 6H). Consistently, restoration of ICAM1 expression in Lnc-BM

depleted cells allowed cancer cells to recover their ability to stretch over blood vessels and

penetrate into the brain slices (Figure 6I). These data suggest that the STAT3 target gene ICAM1

plays a key role in mediating Lnc-BM-dependent breast cancer vascular co-option and invasion.

Macrophage-secreted OSM and IL6 induced JAK2-STAT3 signaling in brain metastatic cancer

cells

To examine if OSM were autocrined, we measured the expression of gp130-associated cytokines

in 231-Par and 231-BM cells and found that both cell lines produce undetectable OSM

(Supplemental Figure 7A). For cancer cells, extravasation through the BBB is the first step of

metastasis to brain (6). However, BBB-constructing cells produce undetectable OSM when co-

cultured with cancer cells (Supplemental Figure S7B-C). Recent evidence has demonstrated that

brain-residential macrophages promote the outgrowth of metastatic cells (10). We therefore

examined mouse brain-residential macrophages (BV2 cells), finding higher OSM expression in

these cells than other mouse cell lines (Supplemental Figure 7D-E). Interestingly, conditioned

media from 231-BM cells robustly induced expression of IL-6 and OSM in BV2 cells (Figure 7A),

which was confirmed in human macrophages differentiated from PMA-primed human monocytes

(U937 cells) (Supplemental Figure 7F).

We examined macrophages that infiltrated metastatic lesions in vivo. IHC staining of

CD11b, a myeloid cell marker, showed robust recruitment of CD11b+ cells to brain metastatic

17
lesions. This recruitment was markedly reduced upon Lnc-BM depletion (Figure 7B, left panel).

Less neutrophil (Ly6G+ cells) recruitment was detected in brain metastatic lesions (Figure 7B,

right panel). Further, recruitment of Lnc-BM-dependent macrophages was confirmed using the

macrophage marker IBA1 (Figure 7C). Subsequently, the macrophage recruitment assay indicated

that 231-BM cells recruited more macrophages compared to the parental cells (Figure 7D-E). Lnc-

BM or JAK2 deficiency significantly reduced macrophage recruitment in comparison to control

cells (Supplemental Figures 7G-H).

We examined the expression of 84 human cytokines and chemokines and found that seven

of them were downregulated (> 4 fold) in Lnc-BM depleted cells (Figure 7F), by which CCL2

was co-regulated by Lnc-BM and STAT3 (Figure 7G). CCL2 expression was induced by OSM

or by IL6, which was abolished by Lnc-BM stable knockdown (Figures 6C and 7H). According

to Figure 7H (blue bar), we performed a CCL2 rescue assay by adding 400 pg/ml of recombinant

CCL2 to conditioned media of Lnc-BM depleted cells. Impaired macrophage recruitment upon

Lnc-BM knockdown was rescued by the addition of recombinant CCL2 (Figure 7I and

Supplemental Figure 7I). Collectively, these data demonstrate that CCL2, which is produced by

231-BM cells, facilitate Lnc-BM-dependent macrophage recruitment in BCBM lesions.

Next, 231-BM cells were treated with conditioned media from PMA-primed macrophage-

like U937 cells, which were pre-blocked by human OSM and/or IL6 antibodies. Conditioned

media from either primed U937 cells or BV2 cells potently induced phosphorylation of JAK2,

STAT3 and Lnc-BM-JAK2 interaction in 231-BM cells or mouse breast cancer 4T1 cells, which

were abolished by combined treatment with OSM and IL6 antibodies (Figures 7J-K and

Supplemental Figure 7J). Collectively, our data suggest that brain metastatic cancer cells

18
produced CCL2 to recruit macrophages, which in turn secreted OSM and IL6 synergistically to

activate Lnc-BM/JAK2/STAT3 signaling in cancer cells to promote BCBM.

Discussion

Development of effective preventative and therapeutic strategies for BCBM rely on a

comprehensive understanding of the mechanisms of the disease at the molecular level. Our

findings demonstrate that Lnc-BM-JAK2-STAT3-ICAM1 axis facilitates adhesion of breast

cancer cells to brain capillaries and extravasation into the brain parenchyma. Interestingly, Lnc-

BM promoted brain metastatic cell secretion of CCL2, which recruits macrophages to the brain

lesion. The recruited macrophages in turn increased local concentrations of OSM and IL6, which

reciprocally enhanced activation of this signaling axis (Figure 7L). These phenomena suggest that

Lnc-BM is important in mediating communication between breast cancer cells and brain

microenvironment.

JAK2 functions as a prototypical kinase to phosphorylate STAT3, which promotes

tumorigenesis and progression in a wide range of tumor types (31). Our data indicate that

JAK2/STAT3 signaling was hyperactivated in brain metastatic breast cancers and that depletion

of JAK2 diminished brain metastasis in vivo, which suggest that JAK2 may be a promising

therapeutic target for brain metastatic breast cancers. An unexpected finding was that Lnc-BM

bound to and inhibited the kinase activity of the JAK2 JH2 domain. These events result in potential

conformational change from a “closed” to an “open” structure for the JH2-JH1 domains, leading

to hyperactivation of JAK2. This mechanism may partially explain the paradox that human brain

metastatic breast cancer rarely harbors natural mutations in the JH2 domain (32), whereas JAK2

exhibits hyperphosphorylation in these tissues (33). Thus, lncRNA-dependent modulation of JAK2

19
kinase activity represents a novel mutation-independent mechanism that attenuates self-inhibition

of JAK2 in solid tumors.

Our data indicate that STAT3 mediates Lnc-BM-dependent BCBM. This notion is

consistent with previous reports suggesting the oncogenic role of STAT3 in promoting BCBM

(34). Furthermore, we demonstrated that a STAT3 target gene, ICAM1, mediates Lnc-BM-

dependent cancer cell vascular co-option and invasion, which is consistent with the report that

ICAM-1 promotes invasion in human breast cancer cells (35). Further, interaction between ICAM1,

VCAM-1, and activated moesin and ezrin is required leukocyte adhesion to the endothelium during

inflammation (36), which may partly explain why brain metastatic breast cancer cells adhere to

brain blood vessels with high ICAM1 expression.

A preclinical study has shown that the STAT3 inhibitor WP1066 can pass through the BBB and

into brain metastatic lesions (19). However, the side effects of this inhibitor need to be further

investigated because STAT3 is required for proliferation of normal cells (37). Emerging evidence

suggest lncRNA potential as therapeutic targets given that some of them are overexpressed in

cancer and facilitate cancer progression (38). Chitosan nanoparticles have been used for systemic

in vivo siRNA delivery due to their low immunogenicity, low toxicity (39, 40), and brain

permeability (18). Indeed, our data demonstrate that chitosan NP-coated Lnc-BM siRNAs

dramatically inhibited brain metastases in vivo and prolonged the survival of cancer-bearing mice.

Our studies show the importance of Lnc-BM in promoting BCBM and the therapeutic value of

targeting Lnc-BM in fighting this difficult disease.

20
METHODS
Clinical samples

Fresh frozen primary tumor and some paired normal breast tissues were obtained from individuals

with breast cancer diagnosed at Yixing People’s Hospital in China (Yixin cohort) or Duke

University (Duke cohort), and Normal Tissue cDNA Arrays I-IV were purchased from Origene.

The clinical and pathological features of all tissue specimens are listed in Supplemental Table 1.

BCBM tissue samples were previously described (34). The protocol was approved by the

Institutional Review Board of Nanjing Medical University, Duke University Health System and

MD Anderson Cancer Center (MDACC). All tissue samples were collected in compliance with

informed consent policy. Detailed clinical information is summarized in Supplemental Table 1.

Cell culture, transfection, treatments, and lentiviral or retroviral transduction

MDA-MB-231, MCF-7, HEK293T, U937, NIH-3T3, primary Human Umbilical Vein Endothelial

Cells (HUVEC) obtained from the American Type Culture Collection (ATCC), were cultured

under standard conditions. Parental BT474 and its brain metastatic derivative (BT474-BM),

parental MDA-MB-231 (231-Par) and its brain metastatic derivative (231-BM) were previously

described (16). The lung and bone metastatic cell lines MDA-MB-231 LM2 and BoM-1833 (41)

were kindly provided by Dr. Jianming Xu and Dr. Xiang Zhang (Baylor College of Medicine)

respectively. The γ-2A (JAK2-null) cell line was kindly provided by Dr. George Stark (Cleveland

Clinic). The BV2 cell line and the mouse astrocyte cell line were kindly provided by Dr. Zhimin

Lu and Dr. Isaiah J. Fidler (M.D. Anderson Cancer Center) respectively.

siRNA transfections were performed using DharmaFECT4 (GE Healthcare). Plasmid

transfections were performed using Lipofectamine3000 (Invitrogen). For treatment, cells were

21
serum starved overnight followed by treatment with IL6, IL11, IL27, CNTF, LIF, Oncostatin-M

(OSM), Cardiotrophin-1 (CT1), NNT-1 (Peprotech) at 50 ng/ml for 30 minutes or as indicated.

Virus-containing supernatant was collected 48 h and 72 h after co-transfection of pCMV-

VSV-G, pCMV ∆8.2 (for lentivirus production; pUMVC was used for retrovirus production) and

the shRNA- or ORF-containing vector into HEK293T cells, and then added to the target cells.

Twenty-four hours later, the infected cells were selected with 10 μg/ml blasticidin S HCl (Gibco,

A11139-03, for pLOC vector) or 2 μg/ml puromycin (Gibco, A11138-03, for pLKO.1 vector and

the pBabe retroviral vector).

siRNA, shRNA and Plasmid Constructs

Lincode siRNAs targeting Lnc-BM were designed and synthesized from GE Healthcare

Dharmacon, detailed siRNA sequences were listed in the Oligonucleotide Sequences, Probes

and Primers section. Lincode non-targeting control siRNAs (D-001320), and ON-TARGET plus

SMART pool siRNA targeting GP130 (L-005166), OSMR (L-008050) from GE Healthcare

Dharmacon were used in this study. The knockdown efficiency and specificity of all siRNAs were

validated by either RT-qPCR or immunoblotting.

The oligonucleotides for shRNA targeting Lnc-BM were designed based on the siRNA

sequence and cloned into pLKO.1-puro vector; the two shRNAs that produced the best knockdown

efficiencies were used in the following functional studies.

The full-length pDONR223-JAK2 was purchased from Addgene (plasmid #23915) and

then subcloned into the pBabe-SFB or Myc-vector using the Gateway system (Invitrogen).

Mammalian expression vector for wild type Lnc-BM and mutants were constructed by subcloning

the gene sequences into pBabe or pCDNA3.1 backbone (Addgene). pLOC-ICAM1, pLOC-MMP9,

22
and pLOC-RFP cDNA clones were obtained from Open Biosystems through the shRNA and

ORFeome Core facility of The University of Texas MD Anderson Cancer Center. Human JAK2

CRISPR/Cas9 KO plasmid (sc-400246) (detailed sgRNA sequences were listed in the

Oligonucleotide Sequences, Probes and Primers section), JAK2 HDR plasmid (sc-400246-

HDR) and control CRISPR/Cas9 plasmid (sc-418922) were obtained from Santa Cruz Biotech. To

generate Lnc-BM shRNA#2-resistant mammalian expression vectors used in the rescue

experiments, shRNA#2 targeting sequences CCA AGA TTT CAT AGC AAT A were mutated to

CCA AGA CTC CGT GGC AAT A. The point or domain deletion mutants were generated from

the wild type sequence using QuikChange™ Lightning Site-Directed Mutagenesis Kit (Agilent

Technologies).

Antibodies

Anti-phospho-JAK2 (Y1007/1008) (3776), anti-JAK2 (3220), anti- phosphor-STAT3 (Y705)

(9145), anti-STAT3 (9139), anti-phospho-STAT1 (Y701) (7649), anti-STAT1 (9172), anti-

phospho-STAT5 (Y694) (9351), anti-STAT5 (9363), anti-phospho-AKT (S473) (4060), anti-AKT

(2920), anti-GP130 (3732), anti-SOCS3 (2932), anti-cleaved caspase-3 (9661), anti-caspase-3

(9665), anti-cleaved caspase-8 (9496), anti-caspase-8 (9746), anti-caspase-9 (9508), anti-cleaved

caspase-3 (9661), anti-PARP1 (9542), anti-GST tag (2624), anti-His tag (2365) and anti-Myc tag

(2276) were from Cell Signaling Technology; anti-MMP9 (ab76003), anti-ICAM1 (ab53013),

anti-GFP (ab13970) and anti-phospho-JAK2 (Y1007/1008) (ab32101) were from Abcam; anti-

IBA1 (019-19741) from Wako; anti-collagen IV (AB756P) from Millipore; anti-OSMR (sc-30010)

and anti-GAPDH (sc-32233) from Santa Cruz Biotech; anti-FLAG tag (F3165) from Sigma-

Aldrich; anti-phospho-JAK2 (Y570) (PA5-35830) from Thermo Scientific; anti-mIL6 (MAB406),

23
anti-hIL6 (MAB206), anti-mOSM (AF495) and anti-hOSM (AF295) from R&D systems; anti-

JAK2 (S523) was kindly provided by Dr. Olli Silvennoinen (University of Tampere, Finland).

Biotinylated RNA preparation

The Lnc-BM lncRNA sequence was cloned into pGEM-3Z vector (Promega) for in vitro

transcription using Biotin RNA Labeling Mix (Roche) and MEGAscript® Transcription Kit (Life

Technologies). Biotinylated RNAs were purified by RNA Clean & Concentrator™-5 (Zymo

Research).

RNA isolation, quantitative real-time PCR

Total RNA was isolated from cultured cells using the RNeasy Plus Mini Kit with QIAshredder

columns (Qiagen). Cytoplasmic and nuclear RNAs were purified from 1x106 231-BM cells using

SurePrep Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific). cDNA was prepared

using the iScript Reverse Transcription Supermix (Bio-Rad). RT-qPCR was performed with iTaq™

universal SYBR® Green supermix (Bio-Rad) and detected on a CFX Connect Real-Time PCR

Detection System (Bio-Rad). For TissueScan™ Cancer and Normal Tissue cDNA Arrays, the

median value of Lnc-BM expression in NBT was normalized as 1.

Image analysis and quantification for Hematoxylin and eosin staining (H&E)

immunohistochemistry staining

The immunohistochemistry were performed as previous described (13). The images were

visualized with Zeiss Axioskop2 plus Microscope, and the slides were scanned on the Automated

Cellular Image System III (ACIS III, Dako, Denmark) for quantification by digital image analysis.

24
 To define the positive staining, the IHC staining were categorized into five grades 0, 1+,

2+, 3+ and 4+, according to the following criteria: 0) no staining or less than 5% tumor cells in

each field (3 fields) examined; 1+) 5%-10% tumor cells have staining in each field (3 fields)

examined; 2+) 10%-25% tumor cells have staining in each field (3 fields) examined; 3+) 25%-50%

tumor cells have staining in each field (3 fields) examined; 4+) 50%-100% tumor cells have

staining in each field (3 fields) examined. The positive rate were calculated based on the case

numbers of positive staining (1+ or above) over total cases.

In vivo model, macrometastases lesion (macromets.) were calculated as long axis > 300

μm and micrometastasis (micromets.) were calculated as long axis < 300 μm.

For correlation analysis, the staining intensity of RNAScope® and IHC staining were

measured by Image-Pro plus 6.0 (Media Cybernetics) for each tissue sample.

Immunofluorescence

Tissue for immunofluorescence was obtained after overnight fixation with PFA 4% at 4 oC.

Organotypic slice cultures or slicing of the mouse brain using microtome (Fisher) were blocked in

5% normal goat serum (NGS), 1% BSA, PBS with 0.25% Triton X-100 in for 2 h at room

temperature (RT). Primary antibodies were incubated overnight at 4 oC in the blocking solution

and the following day for 30 min at RT. After extensive washing in PBS-Triton 0.25%, the

secondary antibody was added in the blocking solution and incubated for 2 h. After extensive

washing in PBS-Triton 0.25%, nuclei were stained with Hoechst 33258 (2 μg/ml; Life

Technologies) for 10 min at RT. Slices were mounted with ProLong Gold anti-fade reagent

(Invitrogen) and imaged with confocal microscopy (Zeiss). For brain slice immunofluorescence,

confocal 3D set up: X (length, 450 μm), Y (width, 450 μm) and Z (depth, 30 slices × 2 μm interval

25
= 60 μm) axis was employed to investigate stretched cancer cells over blood vessel (X and Y facet)

and invasive depth of cancer cells (Y and Z facet).

RNA Fluorescence In Situ Hybridization

RNA FISH was performed using LNA FISH technology according to the manufacturer’s

instructions (Exiqon) with minor modifications, which have been described previously (13). The

probe was listed in the Oligonucleotide Sequences, Probes and Primers section.

RNA pull-down and mass spectrometry analysis and in vitro RNA pulldown assay

RNA pull-down followed by mass spectrometry analysis and in vitro RNA-protein binding assay

were performed as described before (13). For in vitro RNA pulldown assay, magnetic Dynabeads

M-280 streptavidin beads were washed twice with a 2× volume of nuclease-free 0.1 M NaOH, 50

mM NaCl followed by wash once in 100 mM NaCl and then immediately subjected to capture in

vitro transcribed RNA (2 µg) as described in the RNA pull-down section. To pull-down

recombinant proteins, the RNA-capture beads were incubated with recombinant proteins (1 µg) in

binding buffer [50 mM Tris-HCl pH 7.9, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-

ME 0.1% NP- 40] for 1 hour at 30ºC.

For mass spectrometry analysis, the cell lysates were freshly prepared using ProteaPrep

Zwitterionic Cell Lysis Kit, Mass Spec Grade (Protea®) with Anti-RNase, Protease/ Phosphatase

Inhibitor Cocktail, Panobinostat and Methylstat supplemented in the lysis buffer. The BcMag™

Monomer avidin Magnetic Beads (Bioclone) were first prepared according to manufacturer’s

instructions and then immediately subjected to RNA (20 µg) capture in RNA capture buffer [20

mM Tris-HCl pH 7.5, 1M NaCl, 1mM EDTA] for 30 minutes at room temperature with agitation.

26
The RNA-captured beads were washed once with NT2 buffer [50 mM Tris-HCl pH 7.4, 150 mM

NaCl, 1 mM MgCl2, 0.05% NP-40] and incubated with 30 mg of the cell lysates diluted in NT2

buffer supplemented with 50 U/mL Anti-RNase, 2 mM dithiothreitol, 30 mM EDTA and Heparin

0.02 mg/ml for 4 hours at 4°C with rotation. The RNA-binding protein complexes were washed

sequentially with NT2 buffer (twice), NT2-high salt buffer containing 500 mM NaCl (twice), NT2-

high salt buffer containing 1 M NaCl (once), NT2-KSCN buffer containing 750 mM KSCN (twice),

and PBS (once) for 5 minutes at 4°C and eluted by 2 mM D-biotin in PBS. The eluted protein

complexes were denatured, reduced, alkylated, and digested with immobilized trypsin (Promega)

for MS analysis at MD Anderson Cancer Center Proteomics Facility.

In vitro RNA pulldown coupled with dot-blot assay

The in vitro binding of Lnc-BM with recombinant proteins and subsequent purification of protein-

bound Lnc-BM sequence was performed as previously described (13). Briefly, in vitro transcribed

biotinylated Lnc-BM was incubated with various recombinant proteins in binding buffer [50 mM

Tris-HCl pH 7.9, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-ME 0.1% NP- 40]. The

reactions were ultraviolet irradiated and subsequently the RNA was partially digested by RNase I

at 1:50 and 1: 500 dilutions for 5 minutes. RNA-protein complexes of interest were then partially

purified by His-tag or GST-tag magnetic beads and the purified RNA-protein complexes were

treated with proteinase K for 30 min. The recovered RNAs were hybridized to BrightStar® -Plus

positively charged nylon membrane (Ambion) spotted with 60-mer antisense DNA

oligonucleotides tiling along Lnc-BM (sequences are available upon request) at 37ºC overnight.

The hybridized membrane was washed three times and the protein-bound RNA sequence was

visualized by detection of streptavidin-HRP signals.

27
Microarray analysis and Gene set enrichment analysis (GSEA)

RNA was extracted from 231-Par and 231-BM6 cells as mentioned above. RNA samples were

subjected to human genome‐wide lncRNA microarray 3.0 analyses at ArrayStar Inc.

Differentially expressed lncRNAs with statistical significance were identified. The

thresholds we used to screen upregulated or downregulated lncRNAs were fold change (≥3) and P

value<0.05. Gene expression profiles of the Lnc-BM and JAK2 deficient 231-BM6 cells treated

with or without OSM (50 ng/ml) compared with their corresponding control were determined with

the “Human Whole Genome Oligo Microarray Kit V2” from Agilent (Agilent Technologies, Alto,

CA) following the manufacturer’s instructions.

Gene set enrichment analysis (GSEA) was performed using GSEA v2.1.0 software

downloaded from the Broad Institute (http://software.broadinstitute.org/gsea/index.jsp) using the

MSigDB v3.0 gene set signature database following the author’s instructions (42).

RT2 profiler PCR array analysis

The human Extracellular Matrix & Adhesion Molecules or human Cytokines & Chemokines RT2

Profiler PCR Array, consisting of 84 genes known to be involved in cell-cell and cell-matrix

interactions or 84 key secreted proteins, was used to profile Lnc-BM deficient cells according to

the manufacturer’s instructions. In brief, total RNA was extracted and reverse transcribed into

cDNA using an RT2 First Strand Kit (Qiagen). The cDNA was combined with an RT2 SYBR Green

qPCR Master Mix (Qiagen), and then equal aliquots of this mixture (25 μl) were added to each

well of the same PCR Array plate that contained the pre-dispensed gene-specific primer sets. Real-

time PCR and data collection were performed on a CFX96 instrument (Bio-Rad).
28
Cell lysis, immunoprecipitation (IP), immunoblotting, and RNA immunoprecipitation (RIP)

assay

Cells were homogenized in 1× RIPA buffer (EMD Millipore) supplemented with Protease/

Phosphatase Inhibitor Cocktail (Pierce, Thermo Scientific), Panobinostat (Selleck Chemicals), and

Methylstat (Sigma). Lysates were cleared by centrifugation at 13,000 rpm for 15 minutes at 4ºC.

Supernatants were analyzed for immunoblotting or immunoprecipitation with the indicated

antibodies. The blotting signals were detected using Clarity Western ECL Substrate (Bio-Rad).

RIP assay was performed as previously described (13).

In vitro kinase assay and IP kinase assay

For in vitro kinase assay, recombinant FLAG-tagged JAK2 full length, JH1, or JH1-JH2 proteins

and GST-tagged STAT3 (abnova) were incubated with 50 µl in vitro kinase assay buffer I

(SignalChem) containing 100 µM ATP, in the presence of in vitro transcribed Lnc-BM full length

or deletion mutant for 1 hour at 30C. For the IP kinase assay, γ-2A cells transfected with 10 μg

SFB-JAK2-JH2 expression construct were subjected to FLAG tag pulldown using FLAG M2

Magnetic beads (Sigma, M8823). The immunoprecipitates were further subjected to IP kinase

assay using GST-tagged STAT3 and in vitro kinase assay buffer I (SignalChem) with addition of

20 mM MnCl2 and 100 µM ATP. The reactants were subjected to SDS-PAGE and immunoblotting

using indicated antibodies.

Cell proliferation and cell apoptosis assay

29
The CellTiter 96® AQueous One Solution (Promega) was applied for the cell proliferation assay.

Simply, equal numbers of cells (2 × 103) were plated in 96-well plates with 6 replicates containing

a final medium volume of 100 µl per well. For the following 1-week, 20 µl/well of One Solution

Reagent was added, and after 4 hours incubation at 37°C the absorbance at 490 nm was recorded

daily using an ELISA plate reader.

To measure apoptosis, the cells were stained by APC Annexin V apoptosis detection kit

with PI according to the manufacturer’s instructions (BioLegend) and then subjected to FACS

analysis.

Cell Adhesion Assays

TM
24-well plates were coated with Matrigel (BD Biosciences) and kept in 37°C for 2 h. Then,

4×104 HUVECs were suspended in 1 ml medium and applied to the pre-coated 24-well plate. After

incubation at 37°C for another 24 h to form tubes, 2×104 cancer cells with GFP were seeded on

the layer of HUVEC for 24 h. Adherent and spread cancer cells (green) and the HUVEC tubes

(bright field) were scored by fluorescence microscopy. The number of GFP + cancer cells that

adhered to and stretched over the HUVEC tubes was calculated.

Tans-BBB invasion assay

This assay was performed as previously described with some modifications (9). Primary human

umbilical vein endothelial cells (HUVEC) were co-cultured with mouse astrocytes, on opposite

sides of a polylysine-treated (10 μg/ml for overnight) and gelatin-coated (0.2% for 2 h) Transwell

inserts with 8 μm pores. Inserts were placed upside-down and 104 mouse astrocytes were plated

on the membrane surface. Astrocytes were fed every 15 min for 5 h, and the inserts were then

30
flipped and placed in 24-well plates. 5x105 endothelial cells were plated on the upper chamber of

the inserts and cultures were placed in the incubator for 3 days. Then 5x105 cancer cells were

seeded in FBS-free medium on the upper chamber and immersed into medium containing 10%

FBS for 24 h. Pictures of multiple fields from 3 inserts per group were taken and cancer cells with

GFP transmitting to bottom chambers were counted.

CCL2 and OSM ELISA

The levels of human CCL2 and mouse OSM in the conditioned media of cultured cells were

quantitatively determined in triplicate by ELISA kit according to the manufacturer’s protocol

(R&D Systems).

Macrophage Transwell Migration assays

U937 cells were cultured in the suggested medium 24 h before priming. U937 monocytes were

primed with 5 nM PMA (sigma) for 48 h to become monocyte-derived macrophages as described

previously (43). Transwell assays assessing U937 monocyte-derived macrophages or BV2 cell

(resident macrophage in the brain) migration potential were performed on 24-well plates with

inserts (BD Biosciences) according to the manufacturer’s instruction. Briefly, 5×104 BV2 or 1×105

primed U937 cells were cultured in the upper chamber with FBS-free medium, which were inserted

in the 24-well cultured with cancer cells or in the conditioned media obtained by cultured 231-

BM6 cells with or without the addition of CCL2 (Peprotech), the migrated cells were stained by

crystal purple 24 h later and then counted.

Image analysis and quantification for RNAScope® assay

31
RNAScope® 2.0 High Definition Assay were performed according to the manufacturer’s

instructions (Advanced Cell Diagnostics) as previously described (13). The images were

visualized with Zeiss Axioskop2 plus Microscope, and the slides were scanned on the Automated

Cellular Image System III (ACIS III, Dako, Denmark) for quantification by digital image analysis.

Brain Slice Assays

Organotypic slice cultures from mouse brains were prepared as previously described with some

modifications (9). Brains from 4- to 6-week-old female athymic mice (NCr nu/nu) were dissected

in the pre-cold minimum essential medium (MEM) supplemented with 0.2 mM glutamine, 100

U/ml penicillin, 100 mg/ml streptomycin, and 4.5 mg/ml glucose, and removed the frontal pole

and the cerebellum from the whole brain section. Then the brain was embedded in low-melting

agarose (sigma) preheated at 42 oC and sliced the brain sections horizontally to a 350 μm thickness

using a vibratome (Leica). Brain slices were placed with flat spatulas on top of 0.4 μm

polycarbonate transwell membrane (Millipore) insert in a six-well plate with 1 ml of culture media

(50% MEM, 25% Hank’s balanced salt solution [HBSS], 25% normal horse serum, 0.2 mM

glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 4.5 mg/ml glucose) in the lower well.

The brain slices were incubated at 37 oC and 5% CO2 for 1 h, and then 5×104 cancer cells with

GFP suspended in 2 μl of culture media were placed on the surface of the slice and incubated for

72 h. Brain slices were fixed in paraformaldehyde (4%) overnight at 4 oC and then

immunofluorescence staining of free floating brain sections was performed for indicated

antibodies.

Determination of Kd value using Alpha assay

32
Alpha binding assay was used to determine Kd for a Lnc-BM and JAK2 interaction. The Kd was

determined by a competition experiment in which unlabeled Lnc-BM full length was titrated (2-

fold dilution) from 10 µM to 0.1 nM. Streptavidin donor beads and anti-GST or His6 AlphaLISA®

acceptor beads were used in these assays (PerkinElmer). Triplicate samples containing RNA and

GST- or His-tagged JH2 at indicated concentrations diluted in RNA-protein binding buffer [50

mM HEPES (pH 7.0), 50 mM NaCl, 5 mM MgCl2, and 2 mM CaCl2] were transferred, 10 µL to

each well, to a ½ area 96-well assay plate then incubated at room temperature for 1 hr. A 10 µl of

5x anti-His6 AlphaLISA® acceptor beads (100 µg/ml) was added to each well. The plate was placed

on an orbital shaker for 10 min then incubated at room temperature for 1 hr. Following incubation,

10 µl of 5x Streptavidin donor beads (100 µg/ml) was added to each well and incubated 30 min at

room temperature. Following the final incubation period the plate was read on the EnSpire

Multimode Plate Reader (PerkinElmer). The competitive inhibition curves were calculated based

on Alpha signal readings by fitting to a “log (inhibitor) vs. response-Variable slope (four

parameters)” model (GraphPad Prism 6 software).

Animal studies

All animal experiments were performed in accordance with protocol approved by the Institutional

Animal Care and Use Committee of MD Anderson. The luciferase-labelled 231-BM (0.5×106)

cells in 50 µl 1× PBS were intracardially injected into the left ventricle of six-week-old female

nude mice using a 100-μl Hamilton Microliter™ syringe. To assess the effect of siRNA-

incorporated chitosan nanoparticles (NP-siRNA), the efficiency of NP-siRNA infiltration into

brain was firstly detected. The brains were taken out 48 h after tail vein injection of control NP-

siRNA labelled with Cy3 (5’-3’: Cy3-UUC UCC GAA CGU GUC ACG U[dT][dT]), then brain
33
slices were subjected to fluorescence microscopy. For NP-siRNAs therapeutic model in vivo, mice

were intravenously injected with NP-siRNAs (150 μg/kg body weight, twice per week) (control

siRNA sequence is described above, Lnc-BM siRNAs are described in the Oligonucleotide

Sequences and Primers section) 3 days or 9 days post 231-BM cells injection. Brain metastases

were examined by Bioluminescent Imaging every week using an IVIS Spectrum Xenogen Imaging

System (Caliper Life Sciences) or magnetic resonance imaging at the Small Animal Imaging

Facility of MDACC.

Oligonucleotide sequences and primers

qPCR primers for gene expression and RIP: Lnc-BM (forward, GAC TGT GCT GAG TTT

TGG TGG; reverse, AAC ACT GGG AGA GGA TTG GG), OSMR (forward, TCC ATG GTG

AAG GAA AAA TGA TGC; reverse, GAG GGA GCA GCT TCA AGT GT), ICAM1 (forward,

TGA CCG TGA ATG TGC TCT CC; reverse, TCC CTT TTT GGG CCT GTT GT), CCL2

(forward, GAT CTC AGT GCA GAG GCT CG; reverse, TTT GCT TGT CCA GGT GGT CC),

LBP (forward, CTG GCA TTG CTG CTT ACG TC; reverse, CTC AAG TCC CCG GTG AAG

TC), SerpinB3 (forward, CCT GGG TGG AAA GTC AAA CG; reverse, CCA ATG TGG TAT

TGC TGC CA), BCL3 (forward, CAG ATG AGG ACG GAG ACA CG; reverse, ATG TGG TGA

TCA CAG CCA GG), hOSM (forward, GGT ACT GCT CAC ACA GAG GAC; reverse, ATA

GGG GTC CAG GAG TCT GC), hIL6 (forward, ATA CCT AGA GTA CCT CCA GAA CAG;

reverse, TGG CAT TTG TGG TTG GGT CA), hLIF (forward, TTC TGT CTT ACA ACA CAG

GCT CC; reverse, TCC AGT GCA GAA CCA ACA GC), hIL11 (forward, ACA TGA ACT GTG

TTT GCC GC; reverse, AGC TGG GAA TTT GTC CCT CAG), hIL27 (forward, CAG GCG ACC

TTG GCT GG; reverse, GCT GAC TGT GAA CTC CCT CC), hCNTF (forward, TTC ACA GAG

34
CAT TCA CCG CT; reverse, CAG GCC CTG ATG CTT CAC AT), hCT1 (forward, TGA AGG

GAG CCG GGA TCA; reverse, ATC AGT CTG GGG GTC TTC CA), hNNT1 (forward, GCC

TGG CGG ATG GGA TTA TTA; reverse, CTA ACA TCC CCC ACG AGT CC), mOSM

(forward, GTG GCT GGG TTC CAA AGA GA; reverse, CGC CCA GAT CAG TGT TCC TT),

mIL6 (forward, CGG CCT TCC CTA CTT CAC AA; reverse, TCT GCA AGT GCA TCA TCG

TT), mLIF (forward, AAC GAT GGT GTC ACC CTG AAG; reverse, AGG AGA CCT TAG

ATG CAG GC), mIL11 (forward, TGG GGA CAT GAA CTG TGT TTG; reverse, CAG GAG

GGA TCG GGT TAG GA), mIL27 (forward, TGT CCA CAG CTT TGC TGA AT; reverse, CCG

AAG TGT GGT AGC GAG G), mCNTF (forward, TTT CAC CCC GAC TGA AGG TG; reverse,

TTC TGT TCC AGA AGC GCC AT), mCT1 (forward, GGG AGG GAA GTC TGG AAG ACC;

reverse, TCT CCC TGT TGC TGC ACG TA), mNNT1 (forward, CTG GCG GAT GGG ATT

ATT AAA GC; reverse, CCC ACG AGT CCC CTG CTC), B2M (forward, AGA TGA GTA TGC

CTG CCG TG; reverse, TCA TCC AAT CCA AAT GCG GC), GAPDH (forward, GCC GGT

GCT GAG TAT GTC; reverse, CTT CTG GGT GGC AGT GAT), mβ-Actin (forward, TGT CCA

CCT TCC AGC AGA TGT; reverse, AGC TGA GTA ACA GTC CGC CTA GA).

siRNA: Lnc-BM: #1(UGU CAA AGG CAG UCA GUA A); #2 (CCA AGA UUU CAU AGC

AAU A); #3 (GGU UCA AGA GAG AGA GAG A); #4 (GCA AAU AAU ACU UAA GGC A).

JAK2 sgRNA sequences: #1 (TGT ACT TCG ATG CAG TCC TA); #2 (ACC TGT GAC TCA

TGA AAC AC); #3 (TTA CCT GAT AGA GTT ATA GA).

Lnc-BM sgRNA sequence: # 1 pair (CACCGTGAAGGACCTTTTTTGGCAG;

CACCGGCAGACATTTTATACAAACT); # 2 pair

(CACCGAGTGAGGAAAGTCACTTGGG; CACCGAGCAGACATTTTATACAAAC); # 3

35
pair (CACCGGGAGATTGAAGGACCTTTTT; CACCGGCATCAACATGCAAATTTTA); # 4

pair (CACCGTGAAGGACCTTTTTTGGCAG; CACCGGCATCAACATGCAAATTTTA);

LNA FISH probes:β-actin (/56-FAM/CTC ATT GTA GAA GGT GTG GTG CCA) and Lnc-BM

(/5TEX615/TAT GTG CTA GAC TGT GCT GAG T).

Lnc-BM probes used in in vitro RNA pulldown coupled with dot-blot assay: 1) GGG AGG

GGA GAG ATG GAC AAG TTT GAT TCT TCA AAA GAA AAA TAA TAA AGG AGA TTG

AAG; 2) TGT GGG ATA GTA AAA GGG CAT TTA TGA AGA AAA TGA TTA CAT GAG

TGA GGA AAG TCA CTT; 3) AAT ATT GCT ATG AAA TCT TGG ATG TAC CTT AAA

AAT GAT TTT TTA AAT CTT TAC TTT TTC; 4) CAT ATG AAG ACC AAA AAT ATT GAG

AAA GTC AGA TTT ATC TTG CAC TGT GAA CAC AAA TAA; 5) GTA CAC AGT ATG

CAT ATG GTG AAA CAT AAA TGT GTG AAT GTG AAC AAC TGT TTC AGA TGC; 6)

CAC CCA TCA AAA GTT GAG ATA TAA AGG AAA ACC AAT AAA ATA TTT TAG AAC

AAC AGA GCT; 7) TAT GTT CAA AAT GAA TGA ATC ATT CAC ATT TAC ATT TTT TTC

ATG AAA GTT CTT CAA GGA; 8) CTA AGA ACA GTG TAC TCC ATG TAG GGT AAA

ATC TCC TGA AAA TCT CCT AAA ATA GTT ACA; 9) TGT TAA TAC ATC TGC AGC ATC

CAG CTG TGA GTA GAA TAG TAA TAT AAA GCA CAT TCC CTG; 10) CTA GCA CAT

ATA AAT AAT GTA TTA AAG TAA GCA TAG AAT AGA AAA AGT ACA TGT TTA CAA;

11) CAA AAG TGA AAT TTA TAA AAT TAC TGA CTG CCT TTG ACA ACC ACC AAA

ACT CAG CAC AGT; 12) ACT TAG AAA TCT AGA GGA AGA CCA GAT TTT ATA CTA

AAC CAT GTT ATA CTT AAC TAT CCA; 13) CAC CTT ACA ACT AAC AAA ACT GGC

CTT GAA CAA TCA ATT TGC CAA GAA ATT CTT CTA GTG; 14) ATA ATA GCA TCC

AGG TCT GAA AAT GCC TAT GAA ACA CTG GGA GAG GAT TGG GGT GGC TTC; 15)

CTC TCT CTT GAA CCT CTG GAC CAC ATA CGA ATT CTC TAC TGG AAA ACT AGA

36
GGA GGA ACT; 16) AAG ATT GTC AGA AGT TTC TAA AGT AAG GGT TCA ATT CTT

TTG AGG AGA AAT TTT TTC TCT; 17) CAC CAT GAA TCA ATA AGT CAT ACT ATG

AGA AAC ACA AGG ACA CCC ACA TGG AAT GTT CTG; 18) TTC TAC AGC TGT TTG

TTC TGG TAA CAG CTA GTG ATA CAT TCA TGC TTC TTT AAT AAG AGA; 19) TTC

CTA TAC ACA AAT TAT CCA CTG ATA GAA GTA AAT TTA GAA ATA GGA TGG GTC

ATT ACA; 20) AAG ACT ACA AAA GGT AGA AAG GTA TTC CAG TTT TTA ACA ATT

AAA AAA TTT CCA CAC CTT; 21) TTA AAC ACA ATA CAT AAT AGT AAG ATA GTA

ATA CAT TAA AAC CAT CAT TAT GAA ATC TTA; 22) TTT CAG AGG GCA TTT CTA

ACA AAA AGT ATT GAC ATT CAA TAT TTC ACT GAT TGG TCT TGT; 23) TAA TTC

TTC TAA TCA TGA ATT TAT TCC TTG TCA CAG GTA AAG TTT TAC ATT TTC TAA

CAT; 24) CCA ACA TCA CTG TGA GAC TGA ACA TCT GCC TTA AGT ATT ATT TGC

ACA AGA AAA ACA GTA; 25) TCC TCA TGC ATT TAA ATA TCT AAT ACA GAA ATA

GTG TTT AGA CTG CAT AAA ATA TGC TTT; 26) CAA ACT GGG GCT AAT GCT TCT

TCT ATG TTT CTG TTT TAA ACA AAA AGC AAT TAG AAT TTA; 27) TTG GAA TGA

TTT TCA GTT TTC ATG AAC TAT CAT GAA TTC AGT TGC AGC AGA CAT TTT ATA;

28) TAT AAG CCA TCT GCT AAA ACT TTA TTG ATA AAA CCA GTA GCA GCA GTT

TTA CAA AA.

Data analysis and statistics

Analyses of relative gene expression were determined using the 2-ΔΔCt method with GAPDH or

B2M as the internal reference genes. Results are reported as mean ± standard error of the mean

(S.E.M.) of at least three independent experiments. Each exact n values are indicated in the

corresponding figure legend. Comparisons were performed using two tailed paired Student’s t-test

37
or two-way ANOVA (n.s. p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001) as indicated in

individual figures. Pearson chi-square test or Fisher’s exact test was implemented for statistical

analyses of the associations between markers and clinical parameters, as indicated in the individual

figures. For survival analysis, the expression of Lnc-BM was treated as a binary variant and

divided into ‘high’ and ‘low’ level. The median expression level (50th percentile) of Lnc-BM was

used as the cutoff. Kaplan-Meier survival curves were compared using the log rank test with

GraphPad Prism (GraphPad Software). A p-value < 0.05 was considered statistically significant.

The investigators were not blinded to allocation during experiments and outcome assessment.

Author Contributions

S.W., C.Lin and L.Y. designed the research, and S.W. performed most of the experiments, with

participation of K.L., C. Li., Q. H. and W.Y.; D.H. executed mass spectrometry analysis. Clinical

specimens were ascertained and processed by S.W., J.Z., Y.Z., J.M. and S.H.; J.S. and G.G

performed in vivo BCBM experiments. S.M. and A.S. prepared with NP-siRNAs. The histological

staining and corresponding analysis were performed by K.L. and W.X. J.Y. helped with

bioinformatics analysis. Y.Y. performed computational structure modeling. P.P. helped with

manuscript preparation. M.H., E.R.F., and G.L.B. provided reagents and conceptual advices. S.W.,

L.Y., and C.Lin. wrote the manuscript.

Acknowledgments

We thank Dr. Oluf Dimitri Røe (Norwegian University of Science and Technology, Norway) for

revising the paper. We thank D. Aten for assistance with figure presentation. We thank the Small

Animal Imaging Facility at the MD Anderson Cancer Center for assistance in mice imaging and

38
tail vein injection. This work was supported by NIH R00 award (R00DK094981), UT Startup, UT

STARS grants, and CPRIT (150094) to C.Lin., and the NIH R00 award (R00CA166527), CPRIT

award (R1218), UT Startup, UT STARS and DOD breakthrough (BC151465) grants to L.Y.

39
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FIGURE AND FIGURE LEGENDS

42
Figure 1. Lnc-BM correlates with breast cancer and BCBM

(A-B) LncRNA profiling in 231-Par and 231-BM cells. (C) RNAScope detection of Lnc-BM

expression in human breast cancer and adjacent normal tissues. Left panel: representative images;

right panel: statistical analysis (29 normal breast tissues (NBT), 71 breast cancer tissues). Scale

Bar: 100 µM. (D-F) TissueScan Cancer Panels were analyzed by RT-qPCR for Lnc-BM

expression in human breast cancer and adjacent normal tissues. (G) Kaplan-Meier recurrence-free

survival (RFS) analysis of Lnc-BM expression in breast cancer patients. (H) Determination of

43
Lnc-BM expression in primary breast cancers with recurrence to local or distant sites by RT-qPCR.

∆Ct: Ct value of GAPDH was subtracted from the Ct value of Lnc-BM; ∆Ct: the median of ∆Ct

of Lnc-BM from all samples was subtracted from ∆Ct value of each sample. (I) RNAScope

detection of Lnc-BM expression in BCBM tissues (n = 14). Scale Bar: 100 µM.

44
Figure 2. Lnc-BM is required and sufficient to promote BCBM

(A-B) In vitro BBB transmigration activity of the 231-BM cells harboring control or Lnc-BM

shRNAs. The number of transmigrated cells relative to the control cells is plotted. Scale bars =

45
100 μm. (C-D) MRI imaging (n = 3) (C) or BLI imaging (n = 5) (D) of mouse, 4 weeks post

intracardiac injection of 231-BM cells harboring indicated shRNAs. Red arrows: metastatic lesions;

white arrows: brain blood vessels. (E) HE staining of mouse brain tumor burden at 35 days post

intracardiac injection of 231-BM cells harboring indicated shRNAs. Scale bars = 200 μm. n = 5

per group, 3 sections per brain. (F-G) Representative images of MRI (n = 3) (F) and BLI (n = 5),

brain ex vivo BLI and brain ex vivo GFP (G, left panel) 5 weeks after intracardiac injection of 231-

Par cells stably expressing indicated expression vector. Red arrows (F): metastatic lesions. White

arrows (G): brain blood vessels. (H) Left panel: schematic diagram of nanoparticle-siRNAs

treatment for brain metastasis-bearing mice; right panel: flow chart of the experiments. (I-J)

Representative BLI images (I) and quantification of BLI in the head region (J) 4 weeks after

intracardiac injection of 231-BM cells followed by intravenous administration of indicated

nanoparticle-siRNAs (n = 10). (K) RNAScope detection of Lnc-BM expression in the brain

metastatic lesion. Randomly selected mice were examined after 4th injection of NP-siRNAs (n =

2). Scale bars = 100 μm. (L) Kaplan-Meier plot of brain-metastasis-free survival in the experiment

of (J).

46
47
Figure 3. Lnc-BM is required for brain metastatic cancer cell vascular co-option and

impedes cancer cells from FasL-induced apoptosis

(A) 231-BM cells harboring indicated shRNAs were subjected to cancer cell adhesion assay. The

number of cancer cells attached and spread along HUVEC cells relative to total cancer cells were

plotted. Scale bars: 200 μm. (B) Immunofluorescence (IF) with indicated antibodies in brain

metastasis-bearing mice 35 days post-intracardiac injection of 231-BM cells harboring indicated

shRNAs. Scale bars: 50 μm (C) Schema of brain slice organotypic culture. (D) Representative

confocal images of cancer cell vascular co-option and quantification of infiltrated depth (right

panel) of brain slices co-culture with 231-BM cells harboring indicated shRNAs. Scale bars: 50

μm, white arrow: infiltrated cells. (E-F) IF staining of cleaved caspase-3 in brain slices co-cultured

with 231-BM cells harboring indicated shRNAs (E) or in brain metastatic lesions (related to Figure

2D), Scale bars: 50 μm. (G-H) FACS analyses (G) or Immunoblotting (IB) detection (H) of 231-

BM cells transfected with indicated siRNAs following by sFASL (200 ng/ml) treatment for 24 h.

48
49
Figure 4. Lnc-BM/JAK2-mediated signaling pathway triggered by OSM is required for

BCBM

(A) A list of top Lnc-BM-associated proteins identified by RNA pull-down and MS analysis in

231-BM cells. (B-C) IB detection using indicated antibodies in 231-BM cells treated with the

indicated cytokines for 30 min (B) or in 231-BM and 231-Par cells treated with OSM (50 ng/ml)

for indicated time. * unspecific band. (D) Immunohistochemistry (IHC) staining of OSMR in

human breast cancer and adjacent normal tissues (NBT). Scale Bars: 100 µM. (E) Kaplan-Meier

RFS analysis of OSMR expression in breast cancer patients detected by IHC. (F) IB detection

using indicated antibodies in 231-BM cells harboring indicated shRNAs treated with OSM (50

ng/ml) for indicated time. (G) Representative images and quantification of infiltrated depth of

brain slices co-cultured with indicated 231-BM cells. Scale bars = 50 μm, white arrows: infiltrated

cells. (H-I) MRI detection (H) or BLI image (I) of mice harboring BCBM 4 weeks post-

intracardiac injection of indicated cells. Red arrows: metastatic lesions; white arrows: mice brain

blood vessel. (J) Kaplan-Meier plot of brain-metastasis-free survival in the experiment of (I). (K)

IHC staining of p-JAK2 in human breast cancer and adjacent normal tissues (NBT). Scale bars:

100 μm. (L) Kaplan-Meier RFS analysis of p-JAK2 in breast cancer patients detected by IHC. (M)

High or low expression of Lnc-BM (related to Figure 1I) and p-JAK2 (related to Figure 4K) were

plotted for correlation in breast cancer tissues.

50
Figure 5. Lnc-BM modulate the kinase activity of JAK2

51
(A) RIP RT-qPCR detection of the indicated RNAs retrieved using indicated antibodies in 231-

BM cells treated with OSM (50 ng/ml) for indicated time. BCAR4 as a negative lncRNA control.

(B) In vitro RNA-protein binding, followed by dot blot assays using in vitro transcribed

biotinylated Lnc-BM sense (sen.) or antisense (a.s.) in the presence of indicated recombinant

proteins. Right panel: annotation for each dot. (C) Graphic illustration of Alpha Assay using

biotinylated Lnc-BM and recombinant JAK2 proteins. (D) Saturation curve to determine Kd for

interaction between indicated lncRNAs and recombinant GST-tagged JAK2 protein as indicated.

(E) Up panel: graphic illustration of the interaction between Lnc-BM and JAK2. Low panel: IB

detection of streptavidin pulldown using His-tagged JH2 wild-type or mutants and biotinylated

Lnc-BM. (F) Competition binding assay to determine Kd for interaction between Lnc-BM FL or

deletion mutants and recombinant JH2 wild-type or mutant, with unlabeled Lnc-BM RNA titrated

from 10 µM to 0.1 nM. (G) Immunoprecipitation (IP)-kinase assay using immunoprecipitates of

anti-JAK2 antibody retrieved from 231-BM cells transfected with indicated siRNAs followed by

OSM treatment. (H-I) In vitro kinase assay using recombinant JAK2 JH1/JH2 domain (H) or JAK2

JH1 domain (I), GST-STAT3 and in vitro transcribed RNA transcripts as indicated in the presence

and absence of ATP. (J) Graphic illustration of Lnc-BM mediated JAK2 JH1/JH2 domain

conformational alteration from auto-inhibition to activation. (K) FLAG-tag pulldown followed by

IB detection using antibodies in HEK-293T cells transfected with indicated vectors. (L) IP using

anti-JAK2 antibody followed by IB detection of indicated antibodies in 231-BM cells transfected

with indicated siRNA. (M) IP-kinase assay using immunoprecipitates of anti-FLAG affinity beads

in -2A cells transfected with SFB-JAK2 (aa. 482-809), in the presence of indicated RNA

transcripts and ATP. (N-O) IB detection using indicated antibodies in Lnc-BM deficient 231-BM

52
cells (N) or JAK2 deficient cells (O) transfected with indicated expression vectors followed by

OSM treatment.

53
Figure 6. ICAM1 facilitates breast cancer cell vascular co-option and invasion

(A) Heat map representation of the expression of STAT3 target genes in indicated cells treated

with 50 ng/ml OSM or vehicle for 4 h. The colors represent the fold changes of gene expression

induced by OSM over vehicle. (B) GSEA of STAT3 target gene signature in (A). NES: normalized

enrichment score. (C) RT-qPCR detection of STAT3-target genes expression in 231-BM cells

harboring indicated shRNAs treated with OSM. (D) Left panel, representative images of p-STAT3

and Lnc-BM staining in brain metastatic tissues of breast cancers. Right panel, Pearson’s

correlation analysis (n = 14), scale bar: 100 µm. (E) RT-qPCR analysis of ECM & adhesion

molecules expression (n = 84) in 231-BM cells transfected with indicated siRNAs. Blue dashed

lines, fold changes = 4. (F) Overlap between Lnc-BM regulated ECM/adhesion molecules (Figure

6E, n = 5, fold change > 4) and STAT3 target genes (Figure 6A, n = 28). (G-H) Trans-BBB assay

(G) or cancer cell adhesion assay (H) was performed in Lnc-BM deficient 231-BM cells stably

overexpressing ICAM1 and MMP9. Scale bars: 100 μm (G), 200 μm (H). (I) Representative

confocal images and quantification of infiltrated depth of brain slices co-cultured with Lnc-BM

deficient 231-BM cells overexpressing ICAM1. Scale bars: 50 μm, white arrow: infiltrated cells.

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Figure 7. Lnc-BM promotes recruitment of macrophages into the metastatic niche to form

positive feedback loop

(A) RT-qPCR analysis of gp130-associated cytokines expression in BV2 cells incubated with

conditioned media from indicated cells. (B-C) IHC detection of CD11b and Ly6G (B) or IF

staining of IBA1 (C) in brain metastatic lesions of 231-BM cells harboring indicated shRNAs

(related to Figure 2E). B, brain tissue; Met, metastatic cancer cells. Scale bars: 200 μm (B), 100

µM (C). (D-E) Macrophage recruitment assay examining the directional migration of BV2 cells

regulated by indicated cells. Scale bars: 200 μm. (F) RT-qPCR analysis of human cytokines &

chemokines expression (n = 84) in 231-BM cells transfected with indicated siRNAs. Blue dashed

lines, fold changes = 4. (G) Overlap between Lnc-BM regulated cytokines & chemokines (Figure

7F, n = 7, fold change > 4) and STAT3 target genes (Figure 6A, n = 28). (H) ELISA assay detection

of CCL2 concentration in conditioned media of 231-BM cells harboring indicated shRNAs treated

with OSM (50 ng/ml) or IL6 (50 ng/ml) for 12 h. (I) Macrophage recruitment assay examining the

migration of BV2 cells regulated by conditioned media from 231-BM cells harboring indicated

shRNAs with or without recombinant CCL2. Scale bars: 200 μm. (J-K) IB detection using

indicated antibodies (J) or RIP quantitative real-time PCR detection of the Lnc-BM retrieved by

indicated antibodies (K) in 231-BM cells treated with conditioned media from primed U937 cells

in addition of indicated human antibodies. (L) Model of the action of Lnc-BM in mediating

BCBM.

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