c-MYC Drives Breast Cancer Metastasis to the Brain, but ...€¦ · 28/9/2018 · Brain metastasis in breast cancer is particularly deadly, but effective treatments remain out of

1

c-MYC Drives Breast Cancer Metastasis to the Brain, but Promotes

Synthetic Lethality with TRAIL

Running title:

Achilles' Heel of Brain-metastatic Breast Cancer

Ho Yeon Lee 1,6, Junghwa Cha* 2,6, Seon Kyu Kim*3, Jun Hyung Park1,6,

Ki Hoon Song4, Pilnam Kim 2,6, Mi-Young Kim 1,5,6

1 Department of Biological Sciences, 2 Department of Bio and Brain Engineering, 3

Personalized Genomic Medicine Research Center, Korea Research Institute of

Bioscience & Biotechnology (KRIBB), 4 Ajou University, 5KAIST Institute for the

BioCentury, Cancer Metastasis Control Center, 6Korea Advanced Institute of Science

and Technology (KAIST)

* Authors contributed equally.

Correspondence: Mi-Young Kim

Rm 5204, Department of Biological Sciences, KAIST

291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

Phone: 82-42-350-2615 Email: [email protected]

Conflict of interest

The authors declare no conflict of interest.

on October 15, 2020. © 2018 American Association for Cancer Research.mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 28, 2018; DOI: 10.1158/1541-7786.MCR-18-0630

mailto:[email protected]

http://mcr.aacrjournals.org/

2

Abstract

Brain metastasis in breast cancer is particularly deadly, but effective treatments

remain out of reach due to insufficient information about the mechanisms underlying

brain metastasis and the potential vulnerabilities of brain-metastatic breast cancer

cells. Here, human breast cancer cells and their brain-metastatic derivatives (BrMs)

were used to investigate synthetic lethal interactions in BrMs. First, it was

demonstrated that c-MYC activity is increased in BrMs and is required for their brain-

metastatic ability in a mouse xenograft model. Specifically, c-MYC enhanced brain

metastasis by facilitating the following processes within the brain microenvironment:

1) invasive growth of BrMs, 2) macrophage infiltration, and 3) GAP-junction formation

between BrMs and astrocytes by up-regulating connexin 43 (GJA1/Cx43).

Furthermore, RNA-sequencing (RNA-seq) analysis uncovered a set of c-MYC-

regulated genes whose expression is associated with higher risk for brain metastasis

in breast cancer patients. Paradoxically, however, increased c-MYC activity in BrMs

rendered them more susceptible to TRAIL (TNF-related apoptosis-inducing ligand)-

induced apoptosis. In summary, these data not only reveal the brain metastasis-

promoting role of c-MYC and a subsequent synthetic lethality with TRAIL, but also

delineate the underlying mechanism. This suggests TRAIL-based approaches as

potential therapeutic options for brain-metastatic breast cancer.

Implication

This study discovers a paradoxical role of c-MYC in promoting metastasis to the

brain and in rendering brain-metastatic cells more susceptible to TRAIL which

suggests the existence of an Achilles' heel; thus, providing a new therapeutic




3

opportunity for breast cancer patients.

Key Words; c-MYC, brain metastasis, breast cancer, TRAIL




4

Introduction

To successfully form distant metastases, migrating cancer cells must adapt to new

microenvironments within secondary organs. This process of adaptation typically

involves changes in gene expression and in the signal transduction profiles of cancer

cells that will eventually give rise to the distinct features of organ-tropic metastatic

cells (1). While several genes that contribute to organ-specific metastasis have been

identified, little is known about whether these genes can cause synthetic lethality in

organ-specific metastatic cancer cells when combined with anti-cancer agents.

Synthetic lethality in cancer drug discovery refers to the selective killing of cancer

cells harboring a specific mutation by compromising the function of a secondary gene

(2). This approach is widely used as an alternative to directly targeting the oncogene,

which is often challenging. For example, inhibition of PLK1, STK33, or TBK1 can

induce death in several types of cancer cells containing KRAS mutations (3-5).

Synthetic lethal approaches are also being used to discover therapeutic agents that

cause lethality in cancer cells containing molecular lesions in tumor suppressor

genes. The best-known example is the synthetic lethality of cancers lacking breast

cancer 1 or 2 (BRCA1 or 2) induced by treatment with an inhibitor of poly (ADP-

ribose) polymerase1 (PARP1) (6,7).

While the studies mentioned above provide insight into the synthetic lethality of

cancers with specific genetic lesions, synthetic lethal interactions in organ-tropic

metastatic cancer cells are largely unexplored.

Breast cancer metastasizes to several organs including lungs, brain, bones and liver

and a majority of breast cancer mortality is attributed to metastasis (8,9). Especially,

brain metastasis is generally associated with shorter survival (10) and there are no




5

effective treatments for this disease. Thus, in the present study, we investigated

synthetic lethal interactions in brain-metastatic breast cancer. We found that c-MYC

function is essential for breast cancer metastasis to the brain, but increased c-MYC

activity makes brain-metastatic breast cancer cells (BrM-BCCs) highly sensitive to

TRAIL-induced apoptosis. Thus, our study provides evidence of a synthetic lethal

interaction between c-MYC-induced brain-metastatic potential and TRAIL.




6

Materials and Methods

Expression constructs

c-MYC overexpression construct : pCDH-puro-c-MYC (Addgene No. 46970) (11). c-

MYC shRNA construct: pRetrosuper-puro-shMYC (Addgene No. 15662) (12).

MISSION shRNA (TRCN 0000039640, Sigma-Aldrich) GJA1 overexpression

construct : pLVX-IRES-hygro-GJA1 (13).

Cell viability assays

MDA-MB-231, MCF7, and their metastatic derivatives were treated with TRAIL of for

24 and 72 h, respectively. Cell viability was measured using CellTiter-Glo system

according to manufacturer’s protocol. (Promega).

Animal studies

All animal works were done in accordance with a protocol approved by the KAIST

Institutional Animal Care and Usage Committee (IACUC). For subcutaneous

injections, 4 × 105 of 231-BrM cells were mixed with growth factor-reduced matrigel

(3:1), followed by injection into the lower flanks of 6-8 weeks-old BALB/c Nude mice

(Orient Bio). Tumor volumes were calculated using (width)2 × (length) / 2. For brain

metastasis assays, 1 × 105 of 231-BrM or 2 × 105 of 231-Par cells were intra-

cardiacally injected and extracted brains were imaged with IVIS system. 1 × 106

MCF7-HER2 cells were intra-cardiacally injected after pre-treating mice with 10 μg of

β-Estradiol (Sigma Aldrich) for 3 days. β-estradiol was administrated everyday of

imaging until the completion of the experiments. Brain metastases was assessed

based on the weight loss as well as the presence of behavioral abnormalities. For

TRAIL treatment assays, Mice were treated with 500 μg of APO2/TRAIL by intra-

peritoneal bolus injection everyday for 14 days, starting 8 days post intra-cardiac




7

injection of 231-BrM.

Cell culture and cell line generation

MDA-MB-231 and MCF7-HER2 cell lines were cultured in Dulbecco’s modified

Eagles medium (DMEM) and RPMI1640 with 10% FBS, respectively. Human

astrocytes were purchased from ScienCell and cultured in complete Astrocyte Media

(ScienCell). c-MYC knockdown and rescued 231-BrM cell lines were generated by

using retro- and lenti-viral system, respectively. c-MYC-overexpressing 231-Par and

MCF7-Par and Cx43 rescued cell lines were generated by a lenti-viral system. Cell

lines were recently authenticated by DNA fingerprinting analysis and Mycoplasma

was periodically tested by e-myco plus detection kit (Intron).

Glutamine consumption assay

Conditioned media (CM) were collected 24h post cell plating and glutamine

concentration were measured with Glutamine/Glutamate-GloTM Assay kit (Promega).

according to manufacturer’s protocol. Glutamine consumption rate was calculated by

[glutamine] control media- [glutamine] CM and normalized by cell numbers.

Bax oligomerization assays

Cells were homogenized by using a 23-gauge syringe and centrifuged at 2,500 RPM

for 10 minutes at 4 °C. Supernatant was collected, re-centrifuged at 8,000 RPM for

10 minutes at 4 °C. Pellet was re-suspended with DPBS and treated with BMH (final

1 mM) for 30 minutes in ice. Samples were quenched with 25 mM DTT for 10

minutes at room temperature and subjected to western blotting analysis.

Immunostaining and microscopic analysis

Brains were isolated and embedded in OCT (Leica). 10 μm-thick sections were

subjected to immune-fluorescent staining with CD45 and F4/80 antibodies and




8

microscopic analysis was performed using Zeiss Imager M1 microscope at x20

magnification.

Microwell-based three-dimensional tumor sphere formation assay

The agarose was dissolved by boiling in DMEM and stamped to fabricate microwell

to generate non-adherent surfaces. Single cells were seeded onto microwell and

cultured for 7 days. Microscopic analysis was performed with phase contrast

microscope (Leica). The area and perimeter of each sphere was measured by using

ImageJ software. The circularity of spheres was calculated by 4π × (area) /

(perimeter)2.

3D invasion assays

Tumor spheres were embedded in hyaluronic acid (HA)-collagen1 hydrogels. After 72

h, spheres were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized

with Triton X-100 (Sigma-Aldrich). Spheres were then stained with DAPI and

Phalloidin (Sigma-Aldrich). Microscopic analysis was performed with phase contrast

microscope (Leica) and a confocal microscope (Nikon). The invading area for initial

and final time-point was measured using ImageJ software. The invasion was

quantified by calculating the ratio of invading area changes from initial to final time-

point, comparing to the initial invading area (A = (Afinal - Ainitial) × 100 / Ainitial).

Macrophage (RAW 264.7) recruitment assay

Tumor spheres and RAW 264.7 cells were labeled with CellTrackerTM Green CMFDA

and Red CMTPX, respectively. Tumor spheres were embedded with hyaluronic acid

(HA)-collagen type I hydrogels. RAW 264.7 cells mixed with the same matrix were

added onto tumor sphere-containing HA-collagen matrix. RAW cell infiltration was

observed for 24 h using confocal microscope (Nikon). The infiltrated Raw 264.7 cells




9

were quantified by counting the infiltrated cells area inside the boundary of microwell.

Dye Transfer assay

Suspended cancer cells were labeled with 3 μM of Calcein Red-Orange, AM dye

(Invitrogen) for 30 minutes at 37 °C. Labeled cancer cells were mixed with unlabeled

human astrocytes at a ratio of 4 : 1 and incubated for 5 h at 37 °C. Dye transferred

astrocytes were analyzed FACS LSRFortessa .

qRT-PCR

4 ng of cDNA were subject to qRT-PCR by using an CFX-96 Real-Time PCR System

(BioRad). HPRT was used as an endogenous control. Primer sequences are

provided in Supplemental Material and Methods.

Genome-wide RNA-seq analysis and MYC-BrMGS derivation

RNAs isolated from 231 cell lines were subjected to strand-specific whole

transcriptome analysis by Illumina Next-seq 500 using 75 nt single-end methodology

(SE75). The RNA sequence reads were aligned to the human GRCh38 using the

STAR software (ver. 2.5). For quantification of mRNA, the counts per million

fragments mapped (CPM) of each sample were calculated. EdgeR package that

uses a negative binomial model was used to detect differentially expressed genes

from RNA-seq count data. Genes with the fold difference of >1.5.and P-value <0.001

between shcntr and shMYC as well as shMYC-Vec and rescue groups were

selected. A gene set composed of these genes (total 125) and c-MYC was defined as

MYC-BrMGS. The data is available in the NCBI Gene Expression Omnibus (GEO)

public database under accession number GSE106312.

Clinical sample analysis

EMC 192 (GSE12276) and EMC 286 (GSE2034) data sets were used to analyze




10

association between c-MYC-BrMGS and brain metastasis-free survival. To divide

breast cancer patients into subgroups based on expression levels of c-MYC-BrMGS,

a hierarchical clustering algorithm was applied that used the centered correlation

coefficient as the measure of similarity and centroid linkage clustering. For cluster

analysis, gene expression data were normalized by the quantile method, log2-

transformed, and median-centered across genes and samples. The Kaplan-Meier

method was used to calculate the time to metastasis, and differences between the

times were assessed using log-rank statistics.

FACS analysis for apoptosis

Cells were treated with 10 ng / ml of TRAIL for 2 h and stained with Annexin V–

Alexa488 (Invitrogen) and 7-AAD solution (BD), followed by flow cytometry analysis.

Statistical analysis

Statistical analysis was performed by Unpaired Student’s t-test, Two-way ANOVA,

Mann–Whiney tests, or Log-Rank test as indicated in each Figure legend. P-values

of < 0.05 were considered as statistically significant. Results are reported as mean ±

SEM.

Detailed information is available in Supplemental Materials and Methods.




11

Results

Elevated c-MYC activity in highly brain-metastatic breast cancer cells (BrM-

BCCs) is required for their brain-metastatic ability

The oncogenic function of c-MYC is well established and its role in cancer metastasis

has been documented in some cancer types (14-17). In the case of breast cancer

metastasis, both lung metastasis-promoting and suppressive functions of c-MYC

have been reported (18-20). However, whether or not c-MYC activity is altered in

BrM-BCCs and this is required for breast cancer metastasis to the brain remains

mostly unexplored.

To investigate this, we first examined whether highly brain-metastatic BCCs have

differential expression levels of c-MYC compared to poorly brain-metastatic BCCs.

We used MDA-MB-231 and MCF7-HER2 (Erb-B2 Receptor Tyrosine Kinase 2)

human breast cancer cell lines as model systems because of the availability of the

low metastatic parental population (231-Par and MCF7-Par) and highly brain-

metastatic sublines (231-BrM and MCF7-BrM3) (21,22). c-MYC protein levels were

increased in 231-BrM cells compared to 231-Par cells with no increase in its

transcript levels (Fig. 1A). Increased c-MYC in 231-BrM #2 cells, another

independent brain-metastatic variant of MDA-231, was also observed but from the

transcript level (Supplementary Fig. S1A). In MCF7-HER2 system, MCF7-Par and

BrM3 showed similar total c-MYC protein levels but MCF7-BrM3 exhibited an

increased pS62/pT58-MYC ratio compared to MCF7-Par (Supplementary Fig. S1B).

It has been shown that activity of c-MYC can be regulated by relative levels between

phospho-S62-MYC (pS62-MYC) and phospho-T58-MYC (pT58), in which a high

pS62/pT58-MYC ratio indicates the activated c-MYC pathway (23). Thus, our data




12

suggest that c-MYC pathway is more active in MCF7-BrM3 compared to the Par

cells. Further supporting our this, MCF7-BrM3 showed increased glutamine

consumption rates, which is consistent with previous data that overexpression of c-

MYC promotes glutamine consumption in MCF7 (24,25) (Supplementary Fig. S1C).

Based on our data indicating the activated c-MYC pathway in BrM-BCCs, we tested

whether c-MYC is required for breast cancer metastasis to the brain. c-MYC-

knockdown in 231-BrM had no effect in their general growth rate as subcutaneous

tumors (Fig. 1B). However, c-MYC knockdown led to a significant decrease in the

formation of brain metastases (Fig. 1C and Supplementary Fig. S1D). Consistent

with this, overexpression of c-MYC in 231-Par cells increased their ability to form

brain metastases (Fig. 1D). Similar results were observed with c-MYC-

overexpressing MCF7-Par (Supplementary Fig. S1E). Collectively, our data suggest

that c-MYC is a key player in breast cancer metastasis to the brain.

Increased c-MYC function in BrM-BCCs is essential for invasive outgrowth in

the brain microenvironment

Next, we investigated the mechanism by which increased c-MYC activity enhances

the ability of BrM-BCCs to form brain metastases. Brain-metastatic cancer cells

exhibit expansive growth when growing as spheres and this phenotype is well

correlated with their invasiveness in a 3-dimensional (3D) matrix (26,27). To

determine whether c-MYC contributes to this process, we used a non-adherent

microwell platform, each well of which is designed to contain a single tumor sphere

(Fig. 2A schematic). In this assay, while control 231-BrM cells formed spheres with

disorganized morphologies (i.e., expansive or grape-like structures (27)), c-MYC-




13

depleted 231-BrM cells produced much more compact and circular spheres, which

was reversed upon c-MYC rescue (Fig. 2A and B, Supplementary Fig. S2A).

Consistent with this, overexpression of c-MYC in MCF7-Par cells led to formation of

disorganized spheres as demonstrated by decreased circularity (Supplementary Fig.

S2B).

Based on the aforementioned association between expansive growth and

invasiveness, we next investigated the role of c-MYC in the invasive growth of BrM-

BCCs. We used a published spheroid-based invasion assay in which tumor spheres

are embedded in a 3D matrix composed of collagen (type 1) and hyaluronic acid

(HA), one of the major components in brain extracellular matrix (28,29). 231-BrM

cells exhibited more invasive behavior in this HA-collagen matrix compared to the

231-Par (Supplementary Fig. S2C), but invasive growth of 231-BrM was significantly

reduced upon knockdown of c-MYC and restored upon c-MYC rescue (Fig. 2C and

2D). Together, these data suggest c-MYC promotes the invasive growth of BrM-

BCCs in the brain microenvironment.

c-MYC creates a favorable brain microenvironment by promoting macrophage

infiltration

In addition to changes in their intrinsic properties such as invasive growth, BrM-BCCs

can also modify their own microenvironment by modulating the infiltration of

inflammatory cells (e.g., macrophages) to improve their survival and growth in the

brain (10,30). To determine whether c-MYC also plays a role in modulation of brain

microenvironment, we looked for changes in the stromal composition of brain lesions

arising from c-MYC-depleted 231-BrM cells. Similar to our observation in vitro (refer




14

to Fig. 2A), lesions formed by control 231-BrM cells exhibited more disorganized and

expanded shape whereas those formed by c-MYC-depleted BrM cells showed more

compact structure (Fig. 3A, green). More importantly, immuno-stained brain sections

revealed that lesions formed by control 231-BrM cells contained large numbers of

leukocytes (CD45+), including macrophages (F4/80+) (Fig. 3A, red). In contrast, brain

lesions formed by c-MYC-depleted 231-BrM cells only contained very few CD45+ and

F4/80+-cells (Fig. 3A, red).

To further support c-MYC’s role in macrophage recruitment in the brain, we used a

3D macrophage recruitment assay (Fig. 3B, experimental scheme). Briefly,

macrophages (RAW 264.7) were added to a HA-collagen matrix in which tumor

spheres were embedded and their migration toward tumor spheres was monitored.

Consistent with our in vivo staining data, 231-BrM cells caused rapid infiltration of

RAW 264.7 toward tumor spheres in the HA-collagen matrix. Importantly, c-MYC

knockdown dramatically reduced this macrophage infiltration, but it was restored

upon c-MYC rescue (Fig. 3B). Similarly, overexpression of c-MYC in MCF7-Par

increased RAW cell recruitment (Supplementary Fig. S3). Our data suggest that

elevated c-MYC in BrM-BCCs induce the infiltration of macrophages within brain

metastases.

c-MYC promotes GAP junction formation between BrM-BCCs and astrocytes

via up-regulation of Cx43

To better understand how c-MYC promotes brain metastasis in breast cancer, we

compared the transcriptomes of 231-BrM cells expressing shcntr, shMYC, shMYC-

Vec, and c-MYC rescue constructs by using genome-wide RNA sequencing. Through




15

this analysis, we identified 20 and 105 genes whose expression were up- and down-

regulated by c-MYC, respectively (p<0.001 and fold change > 1.5) (Table S1).

To identify the genes that mediate c-MYC’s role in promoting brain metastasis, we

compared our gene set with a previously reported list of genes that are differentially

expressed in 231-BrM and CN34-BrM (BrM subline derived from CN34 breast cancer

cell line) compared to their corresponding Par lines (21). This analysis identified 4

genes (1 up and 3 down) that were differentially expressed in 231-BrM cells

compared to 231-Par and regulated by c-MYC (Fig. 4A). c-MYC-dependent

expression of these four genes was verified by qRT-PCR (Fig. 4B, Supplementary

Fig. S4A, S4B and Table S1). From this analysis, we found that major gap junction

protein GJA1 (Cx43, connexin43) was up-regulated by c-MYC in BrM-BCCs. Cx43-

mediated gap junction formation between BCCs and astrocytes has recently been

suggested to support BCC growth in the brain (13). However, the upstream regulator

of Cx43 in BrM-BCCs has not been identified. Thus, we hypothesized that c-MYC

may promote brain metastasis by stimulating gap junction formation between BrM-

BCCs and astrocytes and that Cx43 functions as a downstream mediator of c-MYC in

this process.

To test this, we performed dye (calcein) transfer assays, which allowed us to monitor

gap junction formation (13). While c-MYC knockdown significantly reduced dye

transfer from 231-BrM to astrocytes, c-MYC rescue restored the dye transfer activity

(Fig. 4C and D). This indicates that c-MYC function is required for the gap junction

formation between BrM-BCCs and astrocytes. Furthermore, the lost dye transfer

ability of c-MYC-depleted 231-BrM cells was fully recovered upon the ectopic

expression of Cx43 (Fig. 4E and F, and Supplementary Fig. S4C). Collectively, these

data suggest that c-MYC promotes gap junction formation through up-regulating




16

CX43 expression.

Activated c-MYC pathway predicts brain metastasis in breast cancer patients

Based on our discovery of a role for c-MYC in promoting brain metastasis, we next

asked whether c-MYC is clinically associated with the risk of brain metastasis in

breast cancer patients. To this, we used publically available breast cancer microarray

datasets (EMC192 and EMC286) that included information about brain metastasis-

free sutvival (21,31). In both EMC192 and EMC286, c-MYC transcript levels showed

no association with brain metastasis-free survival (BrMFS) (Fig. 5A).

We suspected that c-MYC transcript levels may not represent a sufficient reflection of

active c-MYC signaling because our experimental data suggest that c-MYC pathway

in BrM-BCCs can also be activated by increased c-MYC protein level and by

changes in phosphorylation status of the protein (refer to Fig 1A and Supplementary

Fig. S1B).

To address this, we performed a similar analysis as above but using a gene set that

is composed of 125 c-MYC-regulated genes, identified in our RNA-seq analysis

(Table S1), and c-MYC. We named this gene set the “c-MYC brain metastasis gene

set (MYC-BrMGS)”. Compared to c-MYC expression alone, MYC-BrMGS showed a

significant association with the risk of brain metastasis in EMC192 and EMC286 data

sets (Fig. 5B). In contrast to increased risks for brain metastasis in MYC-BrMGS-

positive patients, association between MYC-BrMGS and lung metastasis-free (LMFS)

was only observed in EMC 192 (Supplementary Fig. S5A). In case of bone

metastasis-free survival (BoMFS), MYC-BrMGS-positive group showed lower risks




17

for the bone metastasis in EMC 286, which is opposite of what we found with brain

metastasis (Supplementary Fig. S5B).

Thus, clinical data analyses in combination with our experimental data strongly

suggest that increased c-MYC activity is required for the brain metastasis in breast

cancer.

Increased c-MYC activity in BrM-BCCs sensitizes them to TRAIL-induced

apoptosis

Changes in molecular features of cancer cells can alter their drug responsiveness.

Since we found that the c-MYC pathway is highly activated in BrM-BCCs, we

wondered whether this causes changes in their sensitivity to anti-cancer drugs. To

test this, we first compared the responsiveness of 231-Par and BrM to the most

commonly used chemotherapeutic agents for breast cancer (docetaxel, doxorubicin,

and methotrexate) and observed no differences between 231-Par and BrM

(Supplementary Fig. S6A).

However, 231-BrM cells, but not 231-LM cells (a highly lung-metastatic MDA-MB-231

derivative), exhibited dramatically increased susceptibility to TRAIL-induced cell

death compared to 231-Par cells (Fig. 6A). FACS analysis confirmed a greater

increase in apoptotic cells in 231-BrM upon TRAIL treatment (Supplementary Fig.

S6B), which is in accordance with previous reports that c-MYC can activate TRAIL-

induced apoptosis in various cell types (2,25,32,33). Increased TRAIL sensitivity was

also observed with 231-BrM #2 as well as MCF7-BrM3 compared to the

corresponding Par cells (Supplementary Fig. S6C and D). These data together




18

suggest the synthetic lethal interaction between BrM-BCCs and TRAIL.

TRAIL-induced apoptosis is initiated by the binding of TRAIL to death receptor 4 and

5 (DR 4 and 5). This triggers activation of caspase-8 and the downstream effector

caspases (caspase-3, 6 and 7) through cell type-specific mechanisms (34). In type I

cells, direct activation of caspase-3/6/7 by caspase-8, a.k.a. extrinsic apoptotic

pathway, is sufficient to execute apoptosis (35). On the other hand, apoptosis in type

II cells requires activation of the mitochondrial (intrinsic) pathway. In this pathway,

caspase-8-mediated cleavage of BID (BH3 interacting Domain Death Agonist)

induces BAK/BAX oligomerization and subsequent release of cytochrome C from

mitochondria. This is followed by activation of caspase-9 and caspases 3/6/7 (36)

Upon TRAIL treatment, 231-BrM exhibited faster cleavage of caspase-8, indicating

activation of extrinsic pathway (Fig. 6B). 231-BrM also showed increased BID

cleavage (Fig. 6B), faster oligomerization of BAX and BAK (Fig. 6C and

Supplementary Fig. S6E) as well as accelerated cleavage of caspase-9 and

caspase-3 (Fig. 6B). This suggests that increased TRAIL sensitivity in 231-BrM also

involves activation of the intrinsic pathway.

Based on this, we next examined 1) whether c-MYC is responsible for the higher

TRAIL sensitivity in BrM-BCCs and 2) if so, which apoptotic pathway(s) is regulated

by c-MYC. c-MYC-depleted 231-BrM cells exhibited reduced TRAIL sensitivity and

restoring c-MYC expression re-sensitized them to TRAIL-induced apoptosis (Fig.

6D). Furthermore, overexpression of c-MYC in 231-Par cells increased their TRAIL

sensitivity (Supplementary Fig. S6F). In 231-BrM cells, however, c-MYC

overexpression had no effect on TRAIL sensitivity (Fig. 6D). This is probably because

endogenous levels of c-MYC are sufficient to induce maximum TRAIL sensitivity.




19

Consistent with results in 231-BrM cells, c-MYC knockdown in MCF7-BrM3 cells

reduced their TRAIL sensitivity (Supplementary Fig. S6G). Taken together, this

suggests the essential role of c-MYC in increased susceptibility of BrM-BCCs to

TRAIL.

Next, we investigated molecular mechanisms underlying c-MYC-mediated increase

in TRAIL-sensitivity of BrM-BCCs. By monitoring activation of apoptotic pathways, we

found that c-MYC knockdown in 231-BrM attenuated cleavage of caspase-8, BID,

caspase-9 and caspase-3, which was restored upon c-MYC rescue (Fig. 6E). These

indicate that c-MYC activates both extrinsic and intrinsic pathways. Further

supporting this, DR5 and caspase-8 inhibitor FLIP was increased and decreased in

231-BrM compared to the 231-Par, respectively (Supplementary Fig. S6H) and this

was reversed by c-MYC knockdown in 231-BrM (Fig. 6F). Finally, accelerated BAX

and BAK oligomerization observed in 231-BrM was impaired upon c-MYC

knockdown and restored by c-MYC rescue (Fig. 6G and Supplementary Fig. S6I).

This explains attenuated activation of caspase-9 in 231-BrM upon c-MYC knockdown

which we showed in Fig. 6E. Therefore, these data together suggest that increased

c-MYC in BrM-BCCs sensitizes them to TRAIL-induced apoptosis via activating both

extrinsic and intrinsic pathways.

Finally, as our data indicate that TRAIL may be an effective death-inducer of BrM-

BCCs, we tested this possibility in vivo. To this, 231-BrM cells were introduced into

the arterial circulation of immune-deficient mice and mice were treated with either

vehicle (water) or TRAIL. Brain metastases were monitored by using bioluminescent

imaging (BLI). TRAIL treatment reduced the growth of brain metastases (Fig. 6H),

suggesting TRAIL is effective in the treatment of brain-metastatic breast cancer.




20

Discussion

Evidence is accumulating that BCC subpopulations acquire brain-metastatic potential

via the activation of distinct molecular pathways (21,37). Despite significant advances

in our understanding of the molecular mechanisms that govern breast cancer

metastasis to the brain, there are no effective therapeutic agents currently available.

This necessitates new approaches to target this disease.

In this study, we demonstrate that BrM-BCCs exhibit increased c-MYC activity, which

promotes brain metastasis, and this subsequently leads to increased susceptibility to

TRAIL-induced apoptosis. To the best of our knowledge, our study is the first to

report a link between c-MYC, brain-metastatic potential, and TRAIL sensitivity, thus

providing evidence of a novel synthetic interaction in BrM-BCCs.

We found that c-MYC confers brain-metastatic potential on BCCs without affecting

their general tumor-forming capability. This suggests c-MYC may function as a brain

virulence gene—a gene that promotes BCC colonization within the brain— rather

than providing BCCs with an advantage to expand within the primary tumor.

How, then, does c-MYC enables BCCs to form metastatic lesion within the brain?

The brain contains a specialized extracellular matrix (ECM) and cell types including

astrocytes (38). To colonize the brain, incoming BCCs must be able to cope with this

unique brain microenvironment. Our data indicate c-MYC contributes to the ability of

BCCs to colonize the brain not only by promoting their invasive growth within the

brain ECM, but also by modulating and exploiting stromal components such as

macrophages and astrocytes. In this regard, c-MYC in BrM-BCCs enhances their

ability to induce macrophage recruitment. This is consistent with previous reports that

c-MYC promotes macrophage infiltration into pancreatic cancers (39,40). Our results




21

along with these published studies strongly suggest c-MYC modulates the tumor

microenvironment, eventually leading to cancer cell survival and growth. We suspect

this is accomplished via c-MYC-mediated changes in cytokines and chemokines.

Future studies will provide more insight into the molecular mechanisms by which c-

MYC-expressing BCCs induce macrophage influx.

We also discovered a novel role for c-MYC in the formation of gap junctions between

BCCs and astrocytes, which is essential for the growth and chemo-resistance of

brain metastases (13). We further showed that this c-MYC-mediated increase in gap

junction formation is accomplished via an upregulation of Cx43. A previous study

reported that Cx43 is upregulated in RAS-expressing NIH3T3 cells due to increased

occupancy of the Hsp90 and c-MYC complex at the Cx43 promoter (41), which is

consistent with our results.

Our clinical analyses indicate that MYC-BrMGS can be a prognostic marker for brain

metastasis in breast cancer patients. We expect that this is due to the regulation of c-

MYC at the post-transcriptional level (42-44) as we observed in our model system.

Consistent with this, one study found elevated levels of c-MYC protein in brain

metastases compared to the matched primary breast tumors (45).

Elevated c-MYC function in BrM-BCCs increases susceptibility of these cells to

TRAIL-induced apoptosis. Our data demonstrate that the underlying mechanisms of

elevated TRAIL sensitivity include c-MYC-regulated expression of DR5 and FLIP as

well as activation of the BAX and BAK oligomerization. Consistent with this, previous

studies reported that c-MYC exerts its pro-apoptotic function by upregulating DR5 in

mammary epithelial cells (2), by activating BAX in rat fibroblasts and mammary

epithelial cells (25,46) and by promoting BAK oligomerization in MCF7 cells (25).




22

Therefore, c-MYC induces TRAIL sensitivity in BrM-BCCs via a mechanism that is

similar to the one employed by mammary epithelial cells.

If c-MYC promotes brain metastasis but enhances TRAIL sensitivity as our data

suggest, how do c-MYC-expressing BCCs survive within the brain in the first place?

TRAIL can be secreted by the BCCs themselves and by various organs. Interestingly,

TRAIL is downregulated in 231-BrM compared to Par cells (21), suggesting BrM-

BCCs that overexpress c-MYC may protect themselves from apoptosis by

suppressing the expression of TRAIL. In addition, the brain microenvironment

produces less TRAIL than bone and lungs (31). This may help BCCs overexpressing

c-MYC survive in the brain microenvironment.

The important clinical implication of our study is that TRAIL-mediated apoptosis may

be used to target brain metastases. Despite the efficacy of TRAIL-based therapies in

preclinical models, the clinical benefits of these therapies are not yet proven. This is

typically blamed on TRAIL’s short half-life and rapid clearance from the body (47).

Although recent studies have suggested solutions for increasing the efficacy of

TRAIL-based therapies (48 ,49), insufficient delivery of TRAIL to the brain due to the

blood-brain barrier (BBB) has remained as a major impediment for treatment of brain

metastases (50). Several innovative approaches for resolving this problem have

recently proven effective in preclinical models of glioblastoma and brain metastases

of breast cancer. These include stem cell-mediated delivery of TRAIL and

administration of a TRAIL-inducing compound (50). Thus, our findings together with

these studies suggest breast cancer patients with brain metastases may benefit from

TRAIL treatment.

Collectively, our results indicate that c-MYC plays an essential role in the successful




23

formation of brain metastases and that, as a bystander effect, these cells become

more susceptible to TRAIL-induced apoptosis.




24

Acknowledgement

This research was supported by KIB Cancer Metastasis Control Center (N1018001),

KIAST grand challenge grant (N11180008), the National Research Foundation of

Korea (NRF) (NRF-2016M3A9B4915818), Korea Health Technology R&D Project

through the Korea Health Industry Development Institute (KHIDI, HI14C1324) and by

KRIBB Research Initiative Program.

We thank Dr. Joan Massagué for MDA-MB-231 and its metastatic derivative cell lines

and Cx43 constructs; Drs. Park, Wang, and Eilers for c-MYC and shMYC construct,

respectively. MCF7-HER2-Par and BrM3 cells were kindly gifted from Dr. Steeg.

Genentech/Amgen provide APO2/TRAIL used in in vivo study.

Author contributions

H.-Y.L, K-H.S, P.K,.J C and J-H.P designed and performed research; S.K.K.

contributed to bioinformatic analysis; H.-Y.L. and M.-Y.K. wrote the manuscript.




25

References

1. Jacob LS, Vanharanta S, Obenauf AC, Pirun M, Viale A, Socci ND, et al. Metastatic Competence Can Emerge with Selection of Preexisting Oncogenic Alleles without a Need of New Mutations. Cancer Res 2015;75(18):3713-9 doi 10.1158/0008-5472.CAN-15-0562.

2. Wang Y, Engels IH, Knee DA, Nasoff M, Deveraux QL, Quon KC. Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway. Cancer Cell 2004;5(5):501-12.

3. Scholl C, Frohling S, Dunn IF, Schinzel AC, Barbie DA, Kim SY, et al. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 2009;137(5):821-34 doi 10.1016/j.cell.2009.03.017.

4. Smolen GA, Zhang J, Zubrowski MJ, Edelman EJ, Luo B, Yu M, et al. A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes & development 2010;24(23):2654-65 doi 10.1101/gad.1989110.

5. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 2009;462(7269):108-12 doi 10.1038/nature08460.

6. Chan SL, Mok T. PARP inhibition in BRCA-mutated breast and ovarian cancers. Lancet 2010;376(9737):211-3 doi 10.1016/S0140-6736(10)61119-1.

7. Rios J, Puhalla S. PARP inhibitors in breast cancer: BRCA and beyond. Oncology (Williston Park) 2011;25(11):1014-25.

8. Chiang AC, Massague J. Molecular basis of metastasis. N Engl J Med 2008;359(26):2814-23 doi 10.1056/NEJMra0805239.

9. Vanharanta S, Massague J. Origins of metastatic traits. Cancer Cell 2013;24(4):410-21 doi 10.1016/j.ccr.2013.09.007.

10. Zhang C, Yu D. Microenvironment determinants of brain metastasis. Cell Biosci 2011;1(1):8 doi 10.1186/2045-3701-1-8.

11. Cheng Z, Gong Y, Ma Y, Lu K, Lu X, Pierce LA, et al. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2013;19(7):1748-59 doi 10.1158/1078-0432.CCR-12-3066.

12. Popov N, Wanzel M, Madiredjo M, Zhang D, Beijersbergen R, Bernards R, et al. The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 2007;9(7):765-74 doi 10.1038/ncb1601.

13. Chen Q, Boire A, Jin X, Valiente M, Er EE, Lopez-Soto A, et al. Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 2016;533(7604):493-8 doi 10.1038/nature18268.

14. Wolfer A, Ramaswamy S. MYC and metastasis. Cancer Res 2011;71(6):2034-7 doi 10.1158/0008-5472.CAN-10-3776.

15. Cho H, Herzka T, Zheng W, Qi J, Wilkinson JE, Bradner JE, et al. RapidCaP, a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis. Cancer Discov 2014;4(3):318-33 doi 10.1158/2159-8290.CD-13-0346.

16. Rapp UR, Korn C, Ceteci F, Karreman C, Luetkenhaus K, Serafin V, et al. MYC is a metastasis gene for non-small-cell lung cancer. PLoS One 2009;4(6):e6029 doi 10.1371/journal.pone.0006029.




26

17. Lawson DA, Bhakta NR, Kessenbrock K, Prummel KD, Yu Y, Takai K, et al. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 2015;526(7571):131-5 doi 10.1038/nature15260.

18. Liu H, Radisky DC, Yang D, Xu R, Radisky ES, Bissell MJ, et al. MYC suppresses cancer metastasis by direct transcriptional silencing of alphav and beta3 integrin subunits. Nat Cell Biol 2012;14(6):567-74 doi 10.1038/ncb2491.

19. Wolfer A, Wittner BS, Irimia D, Flavin RJ, Lupien M, Gunawardane RN, et al. MYC regulation of a "poor-prognosis" metastatic cancer cell state. Proceedings of the National Academy of Sciences of the United States of America 2010;107(8):3698-703 doi 10.1073/pnas.0914203107.

20. Cappellen D, Schlange T, Bauer M, Maurer F, Hynes NE. Novel c-MYC target genes mediate differential effects on cell proliferation and migration. EMBO Rep 2007;8(1):70-6 doi 10.1038/sj.embor.7400849.

21. Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, et al. Genes that mediate breast cancer metastasis to the brain. Nature 2009;459(7249):1005-9 doi 10.1038/nature08021.

22. Gril B, Palmieri D, Qian Y, Smart D, Ileva L, Liewehr DJ, et al. Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis. Clinical cancer research : an official journal of the American Association for Cancer Research 2011;17(1):142-53 doi 10.1158/1078-0432.CCR-10-1603.

23. Myant K, Qiao X, Halonen T, Come C, Laine A, Janghorban M, et al. Serine 62-Phosphorylated MYC Associates with Nuclear Lamins and Its Regulation by CIP2A Is Essential for Regenerative Proliferation. Cell reports 2015;12(6):1019-31 doi 10.1016/j.celrep.2015.07.003.

24. Wise DR, DeBerardinis RJ, Mancuso A, Sayed N, Zhang XY, Pfeiffer HK, et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proceedings of the National Academy of Sciences of the United States of America 2008;105(48):18782-7 doi 10.1073/pnas.0810199105.

25. Nieminen AI, Eskelinen VM, Haikala HM, Tervonen TA, Yan Y, Partanen JI, et al. Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis. Proceedings of the National Academy of Sciences of the United States of America 2013;110(20):E1839-48 doi 10.1073/pnas.1208530110.

26. Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT, et al. The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol 2007;1(1):84-96 doi 10.1016/j.molonc.2007.02.004.

27. Cheung WK, Zhao M, Liu Z, Stevens LE, Cao PD, Fang JE, et al. Control of alveolar differentiation by the lineage transcription factors GATA6 and HOPX inhibits lung adenocarcinoma metastasis. Cancer Cell 2013;23(6):725-38 doi 10.1016/j.ccr.2013.04.009.

28. Park JB, Kwak HJ, Lee SH. Role of hyaluronan in glioma invasion. Cell adhesion & migration 2008;2(3):202-7.

29. Jin SG, Jeong YI, Jung S, Ryu HH, Jin YH, Kim IY. The effect of hyaluronic Acid on the invasiveness of malignant glioma cells : comparison of invasion potential at hyaluronic Acid hydrogel and matrigel. Journal of Korean Neurosurgical Society 2009;46(5):472-8 doi 10.3340/jkns.2009.46.5.472.

30. Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain




27

tumor microenvironment. Glia 2011;59(8):1169-80 doi 10.1002/glia.21136. 31. Zhang XH, Wang Q, Gerald W, Hudis CA, Norton L, Smid M, et al. Latent

bone metastasis in breast cancer tied to Src-dependent survival signals. Cancer Cell 2009;16(1):67-78 doi 10.1016/j.ccr.2009.05.017.

32. Ricci MS, Kim SH, Ogi K, Plastaras JP, Ling J, Wang W, et al. Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death. Cancer Cell 2007;12(1):66-80 doi 10.1016/j.ccr.2007.05.006.

33. Ricci MS, Jin Z, Dews M, Yu D, Thomas-Tikhonenko A, Dicker DT, et al. Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity. Mol Cell Biol 2004;24(19):8541-55 doi 10.1128/MCB.24.19.8541-8555.2004.

34. Gonzalvez F, Ashkenazi A. New insights into apoptosis signaling by Apo2L/TRAIL. Oncogene 2010;29(34):4752-65 doi 10.1038/onc.2010.221.

35. Fulda S, Meyer E, Debatin KM. Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression. Oncogene 2002;21(15):2283-94 doi 10.1038/sj.onc.1205258.

36. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17(6):1675-87 doi 10.1093/emboj/17.6.1675.

37. Nguyen DX, Chiang AC, Zhang XH, Kim JY, Kris MG, Ladanyi M, et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 2009;138(1):51-62 doi 10.1016/j.cell.2009.04.030.

38. Lau LW, Cua R, Keough MB, Haylock-Jacobs S, Yong VW. Pathophysiology of the brain extracellular matrix: a new target for remyelination. Nature reviews Neuroscience 2013;14(10):722-9 doi 10.1038/nrn3550.

39. Sodir NM, Swigart LB, Karnezis AN, Hanahan D, Evan GI, Soucek L. Endogenous Myc maintains the tumor microenvironment. Genes & development 2011;25(9):907-16 doi 10.1101/gad.2038411.

40. Soucek L, Lawlor ER, Soto D, Shchors K, Swigart LB, Evan GI. Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nature medicine 2007;13(10):1211-8 doi 10.1038/nm1649.

41. Carystinos GD, Kandouz M, Alaoui-Jamali MA, Batist G. Unexpected induction of the human connexin 43 promoter by the ras signaling pathway is mediated by a novel putative promoter sequence. Molecular pharmacology 2003;63(4):821-31.

42. Sears RC. The life cycle of C-myc: from synthesis to degradation. Cell Cycle 2004;3(9):1133-7.

43. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, et al. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 2004;6(4):308-18 doi 10.1038/ncb1110.

44. Junttila MR, Westermarck J. Mechanisms of MYC stabilization in human malignancies. Cell Cycle 2008;7(5):592-6.

45. Singhi AD, Cimino-Mathews A, Jenkins RB, Lan F, Fink SR, Nassar H, et al. MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors. Mod Pathol 2012;25(3):378-87 doi 10.1038/modpathol.2011.171.

46. Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado JA, Penn LZ, Leber B, et al.




28

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 2005;24(12):2096-103 doi 10.1038/sj.emboj.7600675.

47. Stuckey DW, Shah K. TRAIL on trial: preclinical advances in cancer therapy. Trends Mol Med 2013;19(11):685-94 doi 10.1016/j.molmed.2013.08.007.

48. Graves JD, Kordich JJ, Huang TH, Piasecki J, Bush TL, Sullivan T, et al. Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity. Cancer Cell 2014;26(2):177-89 doi 10.1016/j.ccr.2014.04.028.

49. Holland PM, Miller R, Jones J, Douangpanya H, Piasecki J, Roudier M, et al. Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis. Cancer Biol Ther 2010;9(7):539-50.

50. Bagci-Onder T, Du W, Figueiredo JL, Martinez-Quintanilla J, Shah K. Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells. Brain 2015;138(Pt 6):1710-21 doi 10.1093/brain/awv094.




29

Figure legends

Figure 1. Elevated c-MYC activity in BrM-BCCs is required for their brain-

metastatic ability

A, Relative c-MYC protein (left) and mRNA levels (right) in 231-Par and -BrM cells by

immuoblotting analysis and qRT-PCR, respectively. P-values: Two-tailed unpaired

Student t-test.

B, Subcutaneous tumor growth rates of control (shcntr) and c-MYC knockdown

(shMYC) 231-BrM cells. Tumor volumes were measured at the indicated days.

C and D, Relative brain-metastatic abilities of c-MYC knockdown 231-BrM (C) and c-

MYC-overexpressing 231-Par (D), compared to their corresponding control cell lines.

(Top) c-MYC protein levels in the indicated cells prior to the injection. (Bottom) Cells

were injected into the left ventricle of immunodeficient mice. At the indicated days,

luminescent signals from extracted brains were measured by using bio luminescence

imaging (BLI) system. Normalized BLI signals in extracted brains and representative

BLI images of brains are shown. P-values: One-tailed Mann Whitney test. All data

represent mean ± SEM. *, P < 0.05.

Figure 2. Increased c-MYC function in BrM-BCCs is essential for invasive

outgrowth in the brain microenvironment

A and B, (A, top) A simplified schematic of the microwell system used in this study.

Single cells were seeded onto non-adherent microwell and bright field images were

taken at day 7. (A, Bottom) Representative images of spheres formed by the

indicated 231-BrM cells. (B) The quantification of sphere circularity from A. Lower




30

circularity indicates less compact and disorganized spheres. The circularity of

spheres was calculated by 4π × (area) / (perimeter)2. n = 3.

C and D, Comparison of invasive growth of the indicated 231-BrM cells. Tumor

spheres were embedded within the matrix composed of collagen (type1) and

hyaluronic acid (HA) and stained with Phalloidin . Images were taken by using a

confocal microscope (C). The invasion was quantified by calculating the changes of

invaded areas from 0 h (A0) to 72 h (A72) (D). Invaded area (A) = (A72 – A0) × 100 /

A0. n = 3.

P-values: Two-tailed unpaired Student t-test. All data represent mean ± SEM.. *, P <

0.05; **, P < 0.01; ****, P < 0.0001.

Figure 3. c-MYC creates a favorable brain microenvironment by promoting

macrophage infiltration

A, Mouse brain sections from metastasis assays shown in Fig. 1C were subject to

immunofluorescence staining with the indicated antibodies. GFP-positive cells

represent tumor cells. Representative microscopic images are shown.

B, Relative recruitment of macrophages (RAW 264.7 cells) by the indicated 231-BrM

cells in the HA-collagen matrix. Tumor spheres (green) were embedded in the matrix,

followed by addition of RAW 264.7 cells (red) mixed with the same matrix

(experimental scheme, top right). Images were taken by using a confocal

microscope. Representative top-view images (left) and the normalized RAW cell

recruitment (bottom right) are shown. P-values: Two-tailed unpaired Student t-test.

n = 3.




31

All data represent mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 4. c-MYC promotes GAP junction formation by upregulating Cx43

A, A Venn diagram showing the 125 c-MYC-regulated genes in 231-BrM from RNA-

seq analysis in the present study (left circle) and genes that were previously shown

to be differentially expressed in 231 and CN34-BrM compared to their corresponding

Par cell lines (right circle). The names of overlapping genes are indicated. The 125 c-

MYC-regulated genes were selected based on the fold change > 1.5 and p < 0.001.

B, Relative transcript levels of Cx43 in the indicated 231-BrM cells. n = 3.

C and D, (C, Top) A schematic diagram of dye transfer assay. 231-BrM cells were

labeled with calcein dye and incubated with unlabeled astrocytes. Calcein-positive

astrocytes were analyzed by flow cytometry. (C, Bottom) Quantification of dye

transfer from the indicated 231-BrM cells to astrocytes. Values are normalized to

shcntr-expressing 231-BrM cells. n = 3. (D) Representative histograms from

experiments in (C). The histograms represent red fluorescent signals from astrocytes

after incubation with the indicated 231-BrM cell lines. FL2 denotes emitted

fluorescence from calcein. The light grey box represents background signal from

astrocytes and 231-BrM mixture at 0 h.

E and F, Quantification of dye transfer from the indicated 231-BrM cells to astrocytes

(E) and representative histograms (F). n = 3.

All data represent mean ± SEM.. P-values: Two-tailed unpaired Student t-test. *, P <

0.05; **, P < 0.01; ***, P < 0.001.




32

Figure 5. The c-MYC-regulated gene set predicts brain metastasis in breast

cancer patients

A, Kaplan-Meier plots showing probability of brain metastasis-free survival (BrMFS)

in EMC192 and EMC286 based on the expression level of c-MYC transcript. c-MYC

high and low groups were stratified based on the median expression value of c-MYC

transcript. M indicates the number of patients with brain metastasis in each group.

Total number of patients at risk at each follow-up time is also shown (bottom).

B, Similar analysis as in A but with using c-MYC-Brain Metastasis Gene Set (MYC-

BrMGS). BrMGS-positive and negative groups were stratified based on a hierarchical

clustering algorithm and probability of BrMFS was analyzed.

P-values: Log-Rank test.

Figure 6. Increased c-MYC activity in BrM-BCCs sensitizes them to TRAIL-

induced apoptosis

A, Comparison of cell viability between 231-Par, BrM, and LM cells upon TRAIL

treatment. Cell viability was assessed by the Cell Titer Glo system 24 h post

treatment. n = 3. P-values: Two-way ANOVA, 231-Par vs BrM.

B, The indicated cells were treated with 10 ng/ml of TRAIL for 0, 4, or 6 h and

cleavage of the indicated proteins was monitored by immunoblotting analysis.

C, BAX oligomerization in 231-Par and -BrM cells upon TRAIL treatment (10 ng/ml, 6

h). Mitochondria was isolated and treated with cross-linking agent BMH for 30

minutes, followed by western blotting analysis. 1x, 2x, 3x, and 4x indicate mono-, di-,




33

tri-, and tetramers, respectively.

D, TRAIL sensitivity (left) and relative c-MYC protein levels (right) of the indicated

231-BrM. Cell viability was assessed 24 h post treatment. P-values: Two-way ANOVA

(cntr vs shMYC and shMYC vs c-MYC rescue). n = 3.

E, Similar experiments as in (B) with the indicated 231-BrM cells.

F, Relative DR5 and FLIP protein levels of the indicated 231-BrM cells.

G, Similar experiments as in (C) with the indicated 231-BrM cells.

H, Comparison of brain metastasis in mice treated with water or TRAIL. 231-BrM

cells were intracardiacally introduced into the immunocompromised mice. After 8

days, mice were treated with water or APO2/TRAIL (500ug per injection) everyday for

2 weeks. Brains were extracted at day 28 post injection and metastatic colonization

was quantified by BLI (top). Representative brain images on the bottom. P-values:

One-tailed Mann Whitney test.

All data represent mean ± SEM. *, P < 0.05; **, P < 0.01, ***, P < 0.001, ****, P <

0.0001.






















Published OnlineFirst September 28, 2018.Mol Cancer Res Ho Yeon Lee, Junghwa Cha, Seon Kyu Kim, et al. Promotes Synthetic Lethality with TRAIL.c-MYC Drives Breast Cancer Metastasis to the Brain, but

Updated version

10.1158/1541-7786.MCR-18-0630doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2018/09/28/1541-7786.MCR-18-0630.DC1

Access the most recent supplemental material at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/early/2018/09/28/1541-7786.MCR-18-0630To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-18-0630

http://mcr.aacrjournals.org/content/suppl/2018/09/28/1541-7786.MCR-18-0630.DC1

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/early/2018/09/28/1541-7786.MCR-18-0630


c-MYC Drives Breast Cancer Metastasis to the Brain, but ...€¦ · 28/9/2018 · Brain metastasis in breast cancer is particularly deadly, but effective treatments remain out of

Documents