Androgen-induced lncRNA SOCS2-AS1 Promotes ...

JBC Papers in Press. Published on June 24, 2016 as Manuscript M116.718536 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.718536

lncRNA SOCS2-AS1 and prostate cancer

Androgen-induced lncRNA SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells Aya Misawa1, 4, Ken-ichi Takayama1, 2, 4, Tomohiko Urano1, Satoshi Inoue1, 2, 3 1

Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan 2

Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan

3

Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan 4

Both authors contributed equally to this work.

To whom correspondence should be addressed: Prof. Satoshi Inoue, Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-5800-8834; Fax: 81-3-5800-9126; E-mail: [email protected]. Keywords: androgen, androgen receptor, apoptosis, long noncoding RNA (long ncRNA, lncRNA), prostate cancer

(SOCS2-AS1),

ABSTRACT

whose

expression

higher in castration-resistant

Long noncoding RNAs (lncRNA)

was

prostate

have been associated with the development

cancer model cells, long-term androgen

of cancer. However, the interplay between

deprived (LTAD) cells than in parental

lncRNAs and androgen receptor (AR)

androgen-dependent

signaling in prostate cancer is still unclear.

SOCS2-AS1 promoted castration-resistant

Here, we identified lncRNAs induced by

and androgen-dependent cell growth. We

androgen in AR-positive prostate cancer

found

cells, whose induction was abolished by

upregulated genes related to the apoptosis

AR

an

pathway, including tumor necrosis factor

By

super family 10 (TNFSF10), and sensitized

knockdown

anti-androgen,

as

well

bicalutamide.

as

LNCaP

SOCS2-AS1

cancer

cells

cells.

knockdown

combining these data, we identified an

prostate

androgen-regulated lncRNA, suppressor of

treatment.

cytokine signaling 2-antisense transcript 1

demonstrated that SOCS2-AS1 promotes

Moreover,

to

docetaxel we

also

1

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Running title: lncRNA SOCS2-AS1 and prostate cancer

androgen signaling by modulating the

mechanisms for AR activation to improve the

epigenetic control for AR target genes

treatment of CRPC.

including

TNFSF10.

These

Some lncRNAs studied in prostate

findings an

cancer, act in a highly prostate-specific

important role in the development of

manner (13-18). In prostate cancer, the first

castration-resistant prostate cancer by

prominent lncRNA, PCA3, was initially

repressing apoptosis.

described as a novel biomarker of disease

suggest

that

SOCS2-AS1

plays

(19) and subsequently defined as a promising

technologies have greatly enhanced our

urine test for this disease (20). Similarly, the

understanding of the human transcriptome,

lncRNA PCGEM1 has been implicated in

revealing that more than 90% of the human

prostate cancer as a regulator of apoptosis

genome is actively transcribed, although only

(21). PCAT-1 was characterized as a novel

a minority is translated into proteins (1,2).

prostate-specific lncRNA, regulator of cell

Most of the non-coding transcripts more than

proliferation and target of the Polycomb

200

ncRNAs

Repressive Complex 2 (PRC2) (15). HOTAIR

(lncRNAs). (3). Although the number of

binds to AR protein to block the interaction

known human lncRNAs is still evolving,

with E3 ubiquitin ligase MDM2, thereby

some of them have been functionally

preventing protein degradation and AR

characterized and experimentally validated.

activation (22).

nt

are

known

as

long

Androgen receptor (AR) and its

In the previous study, we have

downstream signaling have a crucial role in

analyzed global AR transcriptional network

the development and progression of both

by mapping genome-wide transcriptional stat

localized and advanced metastatic prostate

sites (TTSs) regulated by androgen and AR

cancer (4-6). High-risk localized prostate

binding sites (ARBSs). This integrative

cancer is treated with androgen deprivation

genomic

therapies

and

AR-regulated transcripts from intergenic or

radiotherapy (7-9). However, many prostate

AS regions of genes in prostate cancer cells

cancers inevitably escape from androgen

(23). In addition, we investigated the

dependence leading to castration-resistant

functional

prostate cancer (CRPC) (10). Past studies

androgen-responsive long non-coding RNAs,

have revealed that elevated AR expression

such as a lncRNA located at the AS region of

(4), activation of AR transcription (11) and

C-Terminal Binding Protein 1 (CTBP1) gene,

development of AR variants (12) were

CTBP1-AS, revealing an oncogenic role of

observed in the progression to CRPC,

androgen-regulated lncRNAs in prostate

suggesting the importance of identifying AR

cancer progression (24).

in

addition

to

surgery

study

roles

revealed

of

comprehensive

these

novel

In the present study, to explore

downstream signals and new molecular 2


Recent advances in sequencing

5α-dihydrotestosterone (DHT). In addition,

roles in prostate cancer progression, we

LNCaP and VCaP cells were also treated

performed

sequencing

with DHT plus bicalutamide, or 10 nM short

analysis in androgen-treated prostate cancer

interfering RNA (siRNA) targeting AR

cell lines. Among the androgen-induced

(siAR). After 24 hours, total RNAs were

lncRNAs in LNCaP and VCaP cells, we

extracted, and then, RNA-seq analysis was

focused on suppressor of cytokine signaling

performed. Bioinformatic analysis identified

2-antisense transcript 1 (SOCS2-AS1), whose

lncRNAs that were upregulated more than

expression

and

1.5 fold by DHT treatment, and repressed to

are

less than 0.75 fold by bicalutamide and siAR

cells

treatment in both LNCaP and VCaP cell lines.

derived from LNCaP and VCaP cells

Nine transcripts were common in both cell

(GenBank accession number: NR_038263).

lines using the GENCODE annotation and

SOCS2-AS1 promoted cell growth and

two in the NONCODE annotation (Fig. 1A).

migration, and repressed several genes

We examined these 11 transcripts and found

related to the apoptosis pathway, including

that they correspond to lncRNAs transcribed

TNFSF10, suggesting that androgen-induced

from five different genes (Table 1).

SOCS2-AS1 would play an important role in

SOCS2-AS1 is an androgen-induced lncRNA

the progression of prostate cancer.

highly

high-throughput

was

higher

VCaP-LTAD

in

cells

castration-resistant

prostate

LTAD

which cancer

expressed

in

castration-resistant

prostate cancer cells — Next, we performed RESULTS

qRT-PCR to analyze the expression of five

Identification of androgen-induced lncRNAs

lncRNAs in both LNCaP, VCaP and their

by directional RNA-sequencing — In order to

LTAD cells. We validated their androgen

investigate hormone-regulated lncRNAs in

induction as observed in RNA-seq data (Fig.

prostate cancer, we performed directional

1B and C). Among these five lncRNAs, we

RNA-sequencing (RNA-seq) and identified

found that SOCS2-AS1 was highly expressed

lncRNAs induced by androgen in prostate

in LTAD and VCaP-LTAD compared to

cancer cell lines. For lncRNA analysis, we

parental cell lines by RNA-seq and qRT-PCR

used two databeses, GENCODE V19 (25)

analysis (Fig. 1A-C, Fig. 2A). Therefore, we

and

v4

chose this lncRNA for further study. Our

positive

ChIP-seq data (26, 27) revealed AR binding

prostate cancer cell lines, LNCaP and VCaP,

site (ARBS) in the promoter region (Chr12:

and their corresponding castration-resistant

93,963,629-93,965,465 (hg19), +1.5 kbp -

cell

-291

NONCODE

(http://www.noncode.org/).

lines,

LTAD

AR

(long-term

androgen

bp

from

TSS

of

NR_038263).

deprived) and VCaP-LTAD cell lines (24)

Interestingly,

were treated with vehicle (ethanol) or 10 nM

repressed after 48 hour of androgen treatment 3

while

its

expression

was


more androgen-dependent lncRNAs that have

in LNCaP cells, it was further induced even

SOCS2-AS1 expression level in LTAD cells

after 72 hours of androgen stimulation in

(Fig. 3C). In addition, AR binding to the

LTAD cells (Fig. 2B). Its sense gene, SOCS2,

promoter was observed in the absence of

showed a similar pattern of induction (Fig. 2

DHT and increased by low concentration of

B and C). These data indicates a distinct

DHT (Fig. 3D). These results indicate one

pattern of induction in castration-resistant

possibility that enhanced AR sensitivity in

prostate

LTAD cells induce the overexpression of

cancer

cells

compared

to

SOCS2-AS1.

hormone-naïve prostate cancer.

SOCS2-AS1 induces prostate cancer cell

transcribed from the opposite strand of the

growth — In order to investigate the role of

protein coding SOCS2 gene. SOCS2 is one of

SOCS2-AS1 in prostate cancer cells, we

the eight members of the SOCS family,

designed

which are induced by cytokine stimulation

androgen-induced

through

levels in LNCaP and LTAD prostate cancer

the

Janus

kinase

(JAK/STAT)

two

siRNAs

to

SOCS2-AS1

reduce expression

to

cell lines, as well as a SOCS2-targerting

cytokine inhibition by reducing JAKs or

siRNA (Fig. 4A, B and C). We found that

STATs phosphorylation, inhibiting the same

both SOCS2-AS1 and SOCS2 knockdown

cascade initiating their production through a

decreased cell proliferation in LNCaP and

negative feedback mechanism (28-30). We

LTAD cells (Fig. 4D). In addition, we

identified ARBSs at a common promoter

transfected siSOCS2-AS1 or siSOCS2 to

region of both SOCS2-AS1 and SOCS2, and

LNCaP and LTAD cells, and counted cell

in the intronic region of SOCS2-AS1,

number over time after androgen treatment.

suggesting that their expressions are directly

In line with the results of MTS assays, cell

regulated by AR (Fig. 2A). Consistently, AR

proliferation of siSOCS2-AS1 or siSOCS2

knockdown by siRNA and pre-treatment with

transfected cells was repressed compared to

the anti-androgen bicalutamide repressed

control siRNA transfected cells, supporting

SOCS2-AS1 and SOCS2 mRNA induction by

that SOCS2-AS1 and SOCS2 increase cell

androgen in both LNCaP and VCaP cells (Fig.

proliferation rate (Fig. 4E).

3 A and B).

We have previously reported

In addition, we established LNCaP cells

the enhancement of AR expression and

overexpressing SOCS2-AS1 or SOCS2 stably

sensitivity in LTAD cells compared with

(Fig. 5A and B). SOCS2-AS1 is located on

parental cells (31). We analyzed whether

chromosome

high sensitivity of AR to low concentration

(hg19)

of DHT is involved in the induction of

overlapping the promoter region of SOCS2

SOCS2-AS1 in these cell lines. We observed

(Fig. 2A). Given this close proximity, we

that low concentration of DHT increased the

hypothesized that SOCS2-AS1 may regulate

signaling.

SOCSs

genes

contribute

4

12

reverse

(93,959,404-93,965,174 strand,

NR_038263)


SOCS2-AS1 is an antisense lncRNA

SOCS2. However, SOCS2 mRNA and

potential of prostate cancer cells.

protein

with

SOCS2-AS1 regulates the expression of

SOCS2-AS1 overexpression (Fig. 5B) and the

apoptosis associated genes such as TNFSF10

negative

— We performed microarray analysis to

levels

were

regulation

expression

by

not of

altered

SOCS2

antisense

protein

transcript

investigate

was

trans-regulatory

effects

of

marginally detected but not evident (Fig. 5C).

SOCS2-AS1 in LNCaP cells by comparing

We performed cell proliferation

the gene expression profiles in cells treated

assays using normal culture medium and

with siSOCS2-AS1, siSOCS2 or negative

androgen-deprived medium. We found that

control siRNA (siNC) (Fig. 7A). We then

increase

found SOCS2-AS1 and SOCS2 have distinct

of

cell

proliferation

rate

of

downstreams,

of SOCS2 stable cells, not only in the normal

biological responses by both genes were

medium

induced by such different gene regulations.

(Fig.

5D),

but

also

in

suggesting

different

androgen-deprived medium (Fig. 5E). These

Genes

results suggest that SOCS2-AS1 plays a

siSOCS2-AS1 transfection in DHT treated

central

both

cells were analyzed using the DAVID

hormone-dependent and castration-resistant

bioinformatics platform (32,33). Go-term

prostate cancer cell growth.

analysis showed an enrichment of genes

SOCS2-AS1 induces prostate cancer cell

involved in apoptosis (P = 4.4E-09) for 594

migration — Next, we performed migration with siSOCS2-AS1 or negative control siRNA.

upregulated by siSOCS2-AS1 transfection compared with Control siRNA in LNCaP cells (Fig. 7B).

Both cell lines were cultivated for two days

Interestingly, Tumor necrosis factor super

in androgen-deprived medium, transfected

family 10 (TNFSF10), which is involved in

with siRNA for 24 hours, transferred into

the apoptosis signaling was the most

8-µm pore seize inserts. The number of cells

upregulated

migrated among SOCS2-AS1 knockdown

transfection. Moreover, we also found genes

cells was lower compared to control cells

related with cell proliferation or cell cycle are

(Fig. 6A and B). We also performed

major signaling of SOCS2-AS1 downstreams.

migration assay using LNCaP cells stably

We further performed microarray analysis

expressing SOCS2-AS1, SOCS2 and control

using siSOCS2-AS1 transfected LTAD cells

cells. The number of cells migrated among

(Fig. 7C). We identified genes regulated by

SOCS2-AS1 overexpressing cells was higher

SOCS2-AS1 in LTAD cells (We obtained 301

than SOCS2 overexpressing cells or control

upregulated genes and 413 downregulated by

cells (Fig. 6C and D). These data suggest that

SOCS2-AS1 knockdown in the presence of

SOCS2-AS1 may enhance the metastatic

DHT). Interestingly, 37% of upregulated and

role

in

promoting

differentially

the

expressed

with

genes

assay in LNCaP and LTAD cells transfected

5

gene

by

siSOCS2-AS1


SOCS2-AS1 stable cells was higher than that

81% of downregulated genes were found to

upregulated TNFSF10 expression in the

be LTAD specific SOCS2-AS1 targets (Fig.

presence of DHT in VCaP cells (Fig. 8C).

7D). GO-analysis of these genes indicates

Interestingly, TNFSF10 expression was

that SOCS2-AS1 regulates more variety of

totally repressed in LTAD cells (Fig. 8D, E)

signals

and

such

as

cell

migration

and

it

was

induced with

SOCS2-AS1

knockdown (Fig. 8F), suggesting the role of

apoptosis, cell cycle and cell proliferation

SOCS2-AS1 in repressing TNFSF10 in CRPC.

(Fig. 7E). We also found LTAD specific

Interestingly, most of other apoptosis-related

target genes associated with apoptosis such

genes in LNCaP cells (Fig. 8A) are also

as apoptosis-inducing factor 2 (AIF2) or

repressed in LTAD compared with LNCaP

checkpoint kinase 2 (CHEK2), suggesting the

cells and upregulated by siSOCS2-AS1 in

role of other signaling for apoptosis in LTAD

LTAD cells (Fig. 8G), suggesting the role of

cells.

SOCS2-AS1

upregulation

in

the

anti-apoptotic ability in CRPC cells.

TNFSF10 belongs to a small subset of pro-apoptotic protein ligands in the Tumor

SOCS2-AS1 inhibits apoptosis — In order to

necrosis factor (TNF) superfamily. TNF

examine whether SOCS2-AS1 is involved in

superfamily consists of proteins involved in

apoptosis, we performed cell proliferation

proliferation, differentiation and apoptosis,

assay in LNCaP and LTAD cells treated with

which also include TNF and Fas Ligand

docetaxel or DMSO, previously transfected

(FasL) (34,35). We also found that TRAIL

with siSOCS2-AS1 or siSOCS2. LNCaP cells

receptor 1 (DR4 or TNFRSF10A) as well as

were treated with 1 nM docetaxel, and LTAD

the cell death surface receptor Fas were also

with 2 nM docetaxel for 24 hours. In addition,

upregulated by siSOCS2-AS1 (Fig. 8A),

we showed further repression of SOCS2-AS1

indicating that SOCS2-AS1 may repress the

levels after 7 days of siRNA transfection (Fig.

apoptotic pathways. We confirmed that

9A). Cell viability was suppressed in the

TNFSF10 expression in LNCaP cells stably

presence of docetaxel in SOCS2-AS1 or

overexpressing SOCS2-AS1 was lower than

SOCS2

in control cells (Fig. 8B). In contrast,

SOCS2-AS1 knockdown LTAD cells (Fig 9B).

TNFSF10 expression was higher in LNCaP

Next, we performed Tunnel Assay with

cells treated with siSOCS2-AS1 knockdown

LNCaP and LTAD cells transfected with

cells (Fig. 8B). We confirmed by qRT-PCR

siSOCS2-AS1, siSOCS2 or siNC. Green

that TRAIL receptor 1 and Fas were

fluorescence-stained cells, representing cells

upregulated, supporting that SOCS2-AS1 may

undergoing apoptosis were observed in both

repress the apoptotic pathway by modulating

siSOCS2-AS1

the expression of these genes (Fig. 8B). We

LNCaP cells and siSOCS2-AS1 transfected

also

LTAD cells, whereas apoptotic cells were not

observed

siSOCS2-AS1

treatment 6

knockdown

and

LNCaP

siSOCS2

cells

and

transfected


transcriptional regulation in addition to

significantly observed in control cells (Fig.

(35%) genes were induced (> 1.4 fold). This

9C, D). We further examined the role of

repression of androgen signaling was not

SOCS2-AS1 in apoptosis using VCaP cells

evident in SOCS2 knockdown samples. By

treated with DMSO or 2 nM docetaxel and

qPCR

obtained similar results (Fig. 9E, F).

androgen-dependent induction of ACSL3 or

Conversely,

analysis,

TMPRSS2,

SOCS2-AS1-overexpressing

both

we are

observed representative

cells showed resistance to docetaxel-induced

androgen-regulated genes, were enhanced in

inhibition of cell proliferation compared with

LNCaP cells overexpressing SOCS2-AS1 (Fig.

control cells (Fig. 10A). In addition, Tunnel

11B). In addition, we also confirmed that

Assay

SOCS2-AS1

using

SOCS2-AS1

and

SOCS2

knockdown

inhibits

these

androgen-mediated gene inductions in both

docetaxel

LNCaP and VCaP cells (Fig. 11C, D).

for

48h

showed

that

cells

overexpressing SOCS2-AS1 and SOCS2 were

Next, we further investigated whether AR

more resistant to apoptosis (Fig. 10B and C).

function is regulated by SOCS2-AS1. Among

Furthermore,

genes

western

blotting

analysis

upregulated

by

SOCS2-AS1

showed that the expression of cleaved-form

knockdown, we found AR-binding genes

PARP

in

were significantly enriched compared with

SOCS2-AS1 stably expressing cells compared

background by analyzing ChIP-seq data (Fig.

to control cells, when treated with 1 nM

12A). Then we explored the regulatory

docetaxel for 48 hours (Fig. 10D). Thus,

mechanism of AR signaling by SOCS2-AS1.

these data suggest that SOCS2-AS1 as well as

We found 8 genes out of 14 representative

SOCS2

genes

was

significantly

inhibit

apoptosis

reduced

and

promote

related

with

apoptosis

including

prostate cancer cell survival.

TNFSF10 have ARBSs in the vicinity. In the

SOCS2-AS1

androgen-mediated

intron region of TNFSF10, we found a strong

gene expression — We further analyzed

AR binding region and recruitment of CtBP2,

whether SOCS2-AS1 regulates AR activity

a cofactor important in prostate cancer

because we found more than half of

progression (28), to the ARBS (Fig 12B). In

upregulated

in

addition, androgen-dependent repression of

LNCaP cells were repressed by androgen

TNFSF10 was reversed by AR knockdown,

treatment and TNFSF10 is also repressed by

indicating the regulation of TNFSF10 by AR

androgen (Fig. 7A). We selected 559

(Fig. 12C). However, by western blot

androgen-induced (> 1.5 fold) genes. Among

analysis,

them, 267 (48%) genes were repressed (< 0.8

phosphorylated

fold) by siSOCS2-AS1 in the presence of

modification for AR binding to genome (36),

DHT (Fig. 11A). In addition, among 521

were not affected by siSOCS2-AS1 (Fig.

androgen-repressed genes (< 0.7 fold), 186

12D). In this ARBS, we observed histone

regulates

genes

by

siSOCS2-AS1

7

protein AR

levels

of

(ser81),

AR

or

important


overexpressing cells treated with 2 nM

deacetylation and recruitment of HDAC to

TNFSF10 is repressed by androgen, whose

this region was inhibited by SOCS2-AS1

expression may be modulated by the action

knockdown (Fig. 12E, F), suggesting that

of SOCS2-AS1 on AR. In order to evaluate the importance of

SOCS2-AS1 is involved in AR activity by

SOCS2-AS1 in the clinical setting, we

modulating the epigenetic function. SOCS2-AS1

in

investigated the expression of SOCS2-AS1 as

cofactor

recruitments

—

well as its potential target, TNFSF10, in

Therefore, we investigated how SOCS2-AS1

clinical samples (38). SOCS2-AS1 was

regulates AR epigenetic function by ChIP

upregulated in some metastatic prostate

assay in three ARBSs (TNFSF10, ACSL3 and

cancer compared to clinically localized

TMPRSS2). Interestingly, androgen-mediated

primary prostate cancer, while TNFSF10

recruitment of CtBP2 was repressed by

inversely

SOCS2-AS1

AR

expression, supporting the oncogenic role of

bindings were not influenced (Fig. 13A).

SOCS2-AS1 (Fig. 14A and B). Taken together,

SRC1 is associated with gene induction by

our study provides a link between an

enhancing histone acetylation (37). We

androgen-induced lncRNA, SOCS2-AS1, and

observed recruitment of SRC1 only to

the apoptosis pathway, which may be

ARBSs of androgen-induced genes and

mediated by TNFSF10 (Fig. 14C).

involvement

AR-mediated

repressed

knockdown,

by

although

siSOCS2-AS1

correlated

with

SOCS2-AS1

treatment.

Androgen dependent histone acetylation was

DISCUSSION Previous studies have reported that several

also inhibited by siSOCS2-AS1 (Fig. 13B), SOCS2-AS1-mediated

lncRNAs are certainly vital for the molecular

epigenetic control in AR signaling could

chain of events that leads to prostate cancer

affect both AR-mediated gene induction and

development and progression. Therefore,

repression. Moreover, we analyzed whether

RNA-based therapy could be potential

SOCS2-AS1 is involved in AR activity by

strategy for CRPC. In the present study, we

interacting with AR. By qRT-PCR analysis,

investigated lncRNAs induced by androgen

we showed that SOCS2-AS1 is enriched in

in two AR-positive prostate cancer cells

the nuleus of LNCaP cells (Fig. 13C). By RIP

using directional RNA-seq method. Among

assay using nuclear lysates, we observed that

these lncRNAs, we found the expression

AR associates with SOCS2-AS1 in the

level of SOCS2-AS1 was higher in CRPC

presence of androgen (Fig. 13D). These

model

results suggest that SOCS2-AS1 interacts with

androgen-dependent LNCaP cells. Socs2

AR in the nucleus and modulates AR activity

knockout mice as well as transgenic mice

by

for

have been reported to display gigantism (39,

epigenetic controls. As an AR target gene,

40), suggesting a dual role for SOCS2 in

suggesting

regulating

that

cofactor

recruitment

8

LTAD

cells

than

in

parental


of

The

we

and the effect of knockdown is relatively

SOCS2-AS1

small for inducing apoptosis in LTAD or

overexpression promoted castration-resistant

VCaP Tunel assay (Fig. 9D, E). Although

and androgen-dependent cell growth and

SOCS2-AS1 and SOCS2 are induced by

migration. On the other hand, SOCS2-AS1

androgen and knockdown of both decreased

knockdown inhibited cell growth of CRPC

cell proliferation rate, they affect different

model cells, as well as parental prostate

genes and pathways as suggested in the

cancer cells. Thus, our results indicate that

present study (Fig. 7A). These results support

SOCS2-AS1 may be a useful target for

that both, SOCS2-AS1 and SOCS2 confers

treating CRPC.

specific biological response to cell growth

growth

regulation.

demonstrated

Several

In that

studies of

have

study

addressed

bidirectional

and

the

apoptosis

by

modulating

different

downstream signaling.

transcription

(41-46). The expression of two genes from a

SOCS2-AS1 works as a regulator at

bidirectional promoter suggests a shared

the transcriptional level to affect the gene

upstream transcriptional regulator-mediated

expression profiles in several pathways

control of both genes, and implies that their

linked

functions might contribute to the same

apoptosis.

cellular response independently. Here we

apoptosis in cancer cells leaving normal cells

identified SOCS2-AS1 and SOCS2 as a pair

intact (47, 48), was found as a candidate of

of transcripts whose expression may be

SOCS2-AS1 target. TNFSF10 expression was

regulated by the same promoter/enhancer, in

repressed in CRPC cells and repression of

the opposite direction. Although our western

SOCS2-AS1 sensitized prostate cancer cells

blot analysis of SOCS2 in LNCaP cells

to

transfected with siSOCS2-AS1 suggests the

findings suggest that SOCS2-AS1 plays a key

putative negative sense/antisense regulation,

role in the development of CRPC by

further

repressing

experiments

about

stability

of

to

cancer

progression

TNFSF10,

docetaxel-induced

such

which

mediates

apoptosis.

TNFSF10

and

as

These

apoptosis.

transcript, for instance, are necessary to show

Furthermore, siSOCS2-AS1 downregulated

the relationship. Importantly, SOCS2-AS1

several known oncogenic genes known such

induction by androgen was concordant with

as Forkhead box protein M1 (FOXM1)

its sense strand gene, SOCS2, and both

and its target gene, Centromere Protein F

transcripts

the

(CENPF) which are highly expressed in

siAR

prostate cancer (49). FOXM1 encodes a

were

anti-androgen

repressed

bicalutamide

by and

forkhead

treatment. In

contrast

overexpression

to did

SOCS2-AS1, not

transcription

factor

involved in the regulation of cell growth,

SOCS2

enhance

domain

cell

proliferation and migration (Fig. 5, 6, 10A) 9

DNA damage,

genomic

resistance

metastasis.

and

stability, In

drug

addition,


regulation

this

CENPF

12C). TNFSF10 was the representative gene,

activates PI3K and MAPK signal pathways

with a marked ARBS in the intronic region.

(50, 51). Together, these data support a role

We

for

cancer

SOCS2-AS1 on the AR pathway by analyzing

progression and provides a link of this

the effects of SOCS2-AS1 knockdown on the

lncRNA with cancer-specific genes.

expression of androgen-regulated genes.

coexpression

of

SOCS2-AS1

FOXM1

in

and

prostate

further

determined

the

effects

of

Microarray data indicate that SOCS2-AS1

been controversial (52-55). In prostate cancer,

depletion inhibited the androgen-mediated

it was reported that SOCS2 protein levels are

gene regulations (Fig. 11A). We further

reduced in CRPC, and that SOCS2 inhibits

analyzed the role of SOCS2-AS1 in AR

the oncogenic events caused by growth

binding and epigenetic regulation by ChIP

hormone (GH) such as cell proliferation and

assay.

invasion (56). In contrast, another report

modulate the cofactor recruitments and

revealed that SOCS2 protein expression was

histone modifications in TNFSF10 ARBS.

increased in prostate tumor tissues compared

Interestingly,

to benign tissues, and that SOCS2 has a

SOCS2-AS1 associated with AR by RIP assay

growth-promoting role in vitro and in vivo

in the nuleus. Thus, the transcriptional

(57). Our present study is in line with the

regulations by SOCS2-AS1 may be caused by

latter report, supporting the oncogenic role of

modulating AR epigenetic action. These

SOCS2 in prostate cancer progression, in

results suggest that high AR signaling in

concert with SOCS2-AS1. SOCS2 knockdown

castration-resistant prostate cancer induce

also repressed or induced genes related to the

strong SOCS2-AS1 expression, which can

apoptosis (data not shown), suggesting that

activate high levels of anti-apoptosis signals,

both transcripts may cooperate to inhibit

cell growth and lead to metastasis by

apoptosis and promote prostate cancer cell

enhancing AR signaling. Further studies will

survival.

be necessary to address the mechanisms of

We

demonstrated

we

also

SOCS2-AS1

observed

that

SOCS2-AS1 for transcriptional regulation.

Moreover, our microarray data

Recent

suggest that SOCS2-AS1 confers oncogenic

studies

have

signaling by transcriptional regulation of

several

genes associated with apoptosis, cell cycle,

whose

proliferation and migration. We found that

propensity of a tumor to metastasize. In this

genes related to apoptosis and upregulated by

study,

siSOCS2-AS1 transfection in DHT treated

potential of prostate cancer cells. A possible

LNCaP cells have ARBSs in the vicinity by

mechanism for such migration and cell

ChIP-seq analysis, suggesting that AR may

growth promoting ability of SOCS2-AS1 may

modulate the expression of these genes (Fig.

be the modulation of AR signaling by 10

metastases-associated

identified

overexpression SOCS2-AS1

can

lncRNAs, signify

enhanced

the

migration


The role of SOCS2 in cancer has

SOCS2-AS1. As like several lncRNAs such as

androgen-dependent LNCaP cells, promotes

HOTAIR (22) and PCGEM1 (58), Our ChIP

castration-resistant and androgen-dependent

analysis indicated that SOCS2-AS1 may be

cell growth and cell migration. SOCS2-AS1

involved in the modification of AR for

modulated genes related to the apoptosis

recruiting

without

pathway, including TNFSF10, suggesting that

changing AR protein level or binding to

it may play a key role in the development of

genome. However, TNFSF10 is upregulated

CRPC by repressing apoptosis.

epigenetic

modifiers

even in the absence of DHT by SOCS2-AS1 EXPERIMENTAL PROCEDURES

possible

for

Cell culture and Reagents — LNCaP cells

SOCS2-AS1 such as the recruitment of other

were grown in RPMI medium supplemented

transcriptional factors to the chromatin site,

with 10% fetal bovine serum (FBS). VCaP

like CTBP1-AS (24), or the coactivation of

cells were grown in DMEM medium

cancer-related genes such as c-myc, like

supplemented with 10% FBS. LTAD cells

PCGEM1

were grown in phenol red-free RPMI

mechanisms

(59).

of

The

functions

investigation

of

associated transcription factors or epigenetic

medium

factors will be required to clarify how

charcoal-dextran–stripped FBS. For androgen

SOCS2-AS1

and

deprivation, cells were cultured for 3 days in

modulates AR signaling in prostate cancer.

phenol red-free RPMI medium (Nacalai

To show more molecular mechanisms of

Tesque,

SOCS2-AS1,

interaction

charcoal-dextran –stripped FBS. All the cells

partners by mass spectrometry would be

were maintained at 37oC in 10% O2 and 5%

interesting. Additionally, by comparing the

CO2. LNCaP and VCaP cells were purchased

results

from American Type Culture Collection.

of

interferes

with

investigating

microarray,

we

AR

identified

supplemented

Kyoto,

Japan)

with

10%

with

LTAD-specific target genes of SOCS2-AS1. A

DHT

previous study (6) has demonstrated that

bicalutamide (Casodex) were purchased from

different AR binding sites in CRPC model

Sigma (St. Louis, MO, USA).

cells from hormone naïve cells. Therefore,

RNA sequencing (RNA-seq) — RNA library

changes of AR or other transcription factor

preparation was performed according to the

binding sites in CRPC cells may influence on

manufacturer’s

the action of SOCS2-AS1, which interacts

OP-Illumina-Directional_RNA_seq-single_e

with AR for transcriptional regulation.

nd-v1.0 (Illumina, San Diego, CA, USA).

In summary, we show that an

(5α-Dihydrotestosterone)

2.5%

protocol

and

for

the

Sequencing libraries was constructed starting

SOCS2-AS1,

from 2 μg of total RNA. Sample DNA for

which is highly expressed in CRPC model

cluster generation was prepared according to

LTAD

the

androgen-induced cells

lncRNA, than

in

parental 11

manufacturer’s

protocol

for

the


knockdown (Fig. 8A), suggesting other

genes using RefSeq Genes to determine the

RIP (RNA immunoprecipitation) assay —

exons genomic locations of known transcripts

was generated using PrimeScript RT reagent

Confluent LNCaP cells in 15-cm dishes were harvested and nuclear fractions were lysed in RIP buffer as described before (24). Specific antibody was added to the supernatant, and the mixture incubated overnight with rotation at 4°C. Thirty microliters of protein G beads was added, followed by incubation at 4°C for 2 h with gentle rotation. The beads were washed 3 times with lysis buffer and resuspended in 1 ml of ISOGEN (Nippon Gene). Co-precipitated RNA was isolated and used for qRT-PCR analysis.

kit (Takara, Kyoto, Japan). Expression levels

siRNA transfection — siRNAs were designed

were quantified by qPCR using KAPA SYBR

using

FAST ABI Prism 2X qPCR Master Mix and

(http://sidirect2.rnai.jp/) and purchased from

ABI StepOne system (Life Technologies,

Sigma

Gaithersburg, MD, USA). Relative mRNA

Cells were transfected with siRNAs using

levels were determined by normalization to

Lipofectamine

glyceralde- hyde-3-phosphate dehydrogenase

reagent (Thermo Fisher Scientific, Rockford,

(GAPDH) mRNA level. Primer sequences

IL, USA) at a final concentration of 20 nM,

are listed in Table 2.

according to the manufacturer’s protocol.

The

RNA-seq readings were analyzed using whole transcriptome software tools from Illumina.

Matching

locations

were

subsequently used to generate counts for

or coverage files (wiggle format). Finally, genes expression was determined as the number of reads per kilobase of exon model per million mapped reads (RPKM). RNA-seq data

was

uploaded

to

GEO

database

(GSE82225). Integrative Genomics Viewer version

2.2

(IGV

browser,

https://www.broadinstitute.org /igv/) was used for visualization. qRT-PCR — Total RNA was isolated using RNeasy Kit (QIAGEN). First strand cDNA

ChIP

(Chromatin

immunoprecipitation)

siDirect Genosys

ver.

2.0

(http://www.genosys.jp/). RNAiMax

transfection

siRNA sequences are listed in Table 3.

assay — ChIP was performed as described

MTS assay — Cells were plated at 2 × 103

before (23, 24). The fold enrichment

cells per well in 96-well plates. For RNA

relative to the GAPDH locus was quantified by qPCR using SYBR Green PCR master mix and the ABI StepOne System (Life Technologies, Carlsbad,

interference

experiments,

cells

were

transfected with 20 nM siRNA 24 h after plating. Cell viability was assessed at different time points using CellTiter 96 12


annotated features, exons, transcripts or

CA). We used Myoglobin locus as a negative control (NC). Antibodies for ChIP have been described before. The primer sequences for ChIP qPCR have been described before (23, 24) or listed in Table 2.

OP-HiSeq2000-sequencing-cBot-v3.0.

AQueous One Solution Cell Proliferation

Tokyo, Japan).

Assay kit (Promega, Madison, WI, USA).

Migration Assay — Cell migration assays

Plates were incubated at 37°C for 50 min and

were performed using a 24-well plate

optical density was measured at 490 nm

matrigel-coated

using

microplate-spectrophotometer

Biosciences, San Jose, CA, USA). Briefly, 5

(Benchmark Plus, BioRad, Richmond, CA,

× 104 cells were suspended in serum-free

USA). Relative cell survival (mean ± SD of

RPMI

triplicate determinations) was calculated by

suspended 8 µm pore inserts. RPMI medium

standardizing the non-treated cell survival to

supplemented with 10% FBS was placed at

100%.

the bottom of the wells. After 24 h of

a

Cell proliferation assay — Cells were plated

medium

invasion

and

chambers

transferred

(BD

into

culturing, cells migrated to the opposite side

4

of the membrane were fixed with methanol

The following day, cells were transfected

and stained with Giemsa. The number of five

with 20 nM siRNA and cell number was

random fields were counted using Olimpus

counted at different time points as described

CKX41 microscope (×10 objective) and the

(24).

average number of cells per field was

Western blotting — Whole cell lysates were

calculated.

prepared using NP40 lysis buffer. Protein

Microarray analysis — For expression

concentration was measured by BCA assay

microarrays, a GeneChip Human Exon 1.0

(Pierce, Rockford, IL, USA). Cell lysates

ST Array (Affymetrix, Santa Clara, CA) was

were fractionated on 8% or 12% SDS–PAGE

used

gels, and transferred onto Immobilon-P

protocol as described (24). Data analysis was

Transfer Membrane (Millipore, Billerica, MA,

performed using the Affymetrix Microarray

USA). Membranes were incubated with

Suite

o

according

software.

to

the

To

manufacturer’s

compare

arrays,

primary antibodies at 4 C overnight and with

normalization was performed on data from

secondary antibodies at room temperature for

all probe sets. GO term and Pathway analysis

1 h, using Blocking One buffer (Nacalai).

was

Antibody-antigen complexes were detected

Microarray data was uploaded to GEO

with ECL Plus detection system (GE

database (GSE82225).

Healthcare,

Plasmid construction — To overexpress

Piscataway,

NJ,

USA).

performed

DAVID

SOCS2-AS1,

rabbit antibody #2779S (Cell Signaling,

specific primers which are listed in Table 3.

Danvers, MA USA), PARP rabbit antibody

cDNA synthesized using total RNA extracted

46D11

Signaling),

from androgen treated LTAD was used as

cleaved-PARP rabbit antibody D214 #9541S

template. We chose the most abundant PCR

(Cell signaling) and β-Actin #A5316 (Sigma,

product which corresponded to the length of

(Cell

13

performed

(25,26).

Antibodies used in this study are SOCS2

#9532S

we

using

PCR

with


at 5 × 10 cells per well in 24-well plates.

SOCS2-AS1 variant 2 (ENST00000499137.2)

Tunnel

and cloned it into pcDNA3 expression vector.

DeadEnd™ Fluorometric TUNEL System

SOCS2 expressing vector was constructed by

(Promega). Briefly, slides were fixed with

transferring its protein coding sequence from

4% paraformaldehyde, permeabilized with

pDNR-LIB-SOCS2 vector (cDNA clone

0.2% Triton-X, equilibrated with buffer and

MGC:13697

to

stained with the reaction mix. The reaction

were

was stopped with 2 x SSC and cells were

Flag-tagged

IMAGE:4277425) pcDNA3.

Constructs

assay

was

performed

stained

1

mg/mL

Analyzer (Life Technologies).

4′,6-diamidino-2-phenylindole

(DAPI)

Establishment of SOCS2 and SOCS2-AS1

(Nacalai Tesque). Slides were mounted onto

overexpressing cells — SOCS2-AS1, SOCS2

glass slides and positively stained cells were

expressing vectors and empty vector were

counted with Olympus FluoView (FV10i)

transfected into LNCaP cells using FuGENE

microscope (×60 objective). Images were

HD (Promega) according to manufacturer’s

obtained through FluoView software (ver

protocol. Transfected cells were treated with

2.0)

500 µg/mL G418 (geneticin disulphate

Statistical

solution, Life Technologies) for selection of

proliferation assay, we analyzed 4 wells. For

stably transfected cells. Surviving colonies

the growth assay using stable cell lines, we

were picked up and grown as stable

performed two-way analysis of variance

transfected cells.

(ANOVA) at each time point. For other cell

Tunnel Assay — For RNA interference

line experiments, statistical differences (P

experiments, 5 × 104 LNCaP, LTAD or VCaP

values) among groups were obtained using a

cells were reverse transfected with 20 nM

two-sided Student’s t-test. All experiments

siSOCS2-AS1 (#1, #2), siSOCS2 or siNC

were performed at least twice and similar

(negative

results were obtained. Chi-square test was

and

suspended

on

analysis

to

—

analyze

For

the

the

ratios

cell

poly-L-lysine coated slides placed in each

performed

of

well of a 24-well plate. For SOCS2-AS1 and

AR-binding. P values less than 0.05 were

SOCS2 stably expressing cells, 5 × 104 cells

considered to be statistically significant.

were suspended on poly-L-lysine coated

Statistical procedures were performed using

slides placed in each well of a 24-well plate.

GraphPad Prism 5 software (GraphPad

24 h after plating, cells were treated with 1 or

Software, San Diego, CA, USA) or MS

2 nM docetaxel and cultured for another 24 h.

Excel.

Acknowledgements: The authors thank RIKEN for sequencing our samples. We are grateful to N. Sasaki and T. Oishi for technical assistance (microarray and bioinformatics analyses). We also thank to Dr. K. Ikeda (Saitama Medical University) for technical advice in siRNA design. 14


sequenced using ABI PRISM 310 Genetic

control)

with

using

This work was supported by grants of the Cell innovation Program and the P-DIRECT from Ministry of Education, Culture, Sports, Science and Technology, Japan (SI); grants from Japan Society for the Promotion of Science, Japan (KT, grant 15K15581; SI, grant 15K15353); a grant of the Program for Promotion of Fundamental Studies in Health Sciences from NIBIO, Japan (SI); Grants-in-Aid (S.I.) from the MHLW, Japan; by Grants from Takeda Science Foundation (S.I. and K.T.); Grants from Mochida Memorial Research Foundation (K.T), Japan, the Yasuda Memorial Foundation (K.T.) and Princess Takamatsu Cancer Research Fund (K.T.)

Conflicts of interests: The authors declare no competing financial interests Author contributions: KT designed the study and performed RNA-seq. AM and KT performed manuscript.

15


experiments and analyzed the data. SI supervised the study. AM, KT, TU and SI wrote the

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24

FIGURE LEGENDS Figure 1. Analysis of androgen-induced lncRNAs in prostate cancer and identification of SOCS2-AS1 as a lncRNA highly expressed in castration-resistant prostate cancer. (A) Flow diagram for the identification of SOCS2-AS1 as an AR-targeted lncRNA upregulated in LTAD cells.

Venn

diagram

representing

overlap

of

lncRNAs

induced

by

androgen

(10

nM

5α-dihydrotestosterone, DHT) and repressed by 1mM bicalutamide and 10 nM siAR treatment for 24 h in LNCaP and VCaP cell lines. (B) qRT-PCR Analysis of androgen-induced lncRNAs by qRT-PCR in LNCaP cells. (C) qRT-PCR Analysis of androgen-induced lncRNAs by qRT-PCR in VCaP cells. Cells were treated with 100 nM DHT or ethanol (Et) for 24 h. Expression level of each lncRNA was determined by qRT-PCR. N = 3. Expression levels are

Figure 2. SOCS2-AS1 is induced by androgen in LNCAP cells and is highly expressed in castration-resistant prostate cancer model LTAD cells. (A) RNA-sequencing (RNA-seq) and ChIP-sequencing (ChIP-seq) analysis of SOCS2-AS1 and SOCS2 in LNCaP and VCaP cell lines treated with 10 nM DHT or ethanol (Et) for 24 h. (B) Time course analysis of SOCS2-AS1 and SOCS2 mRNA after DHT treatment in LNCaP and LTAD cell lines determined by qRT-PCR. (C) Western blotting analysis of SOCS2 after 10 nM DHT treatment at indicated time points. β-Actin was used as an internal control. IB: immunoblot. Values represent mean ±s.d.; **, P < 0.01. Figure 3. AR knockdown and bicalutamide treatment represses SOCS2-AS1 expression. (A) qRT-PCR analysis of SOCS2-AS1, SOCS2 mRNA and AR mRNA levels following 10 nM siAR treatment and subsequent androgen treatment (10 nM DHT for 18 h) in LNCaP and VCaP cell lines (N = 3). (B) SOCS2-AS1 and SOCS2 mRNA levels following 1μM bicalutamide or dimethyl sulfoxide (DMSO) and 10 nM DHT or Et treatment for 24 h in LNCaP and VCaP cell lines by qRT-PCR (N = 3). RNA expression levels are presented relative to the value of GAPDH as reference gene. (C) qRT-PCR analysis of SOCS2-AS1 mRNA level after low dose of androgen treatment (0, 0.1, 1 nM DHT for 18 h) in LNCaP and LTAD cell lines (N = 3). (D) ChIP analysis of AR recruitments to the ARBS in the SOCS2-AS1 promoter region by low level of androgen. LNCaP and LTAD cells were treated with Et or 0.1 nM DHT for 24 h. 10 nM DHT was used as a positive control. Fold enrichment over control region (GAPDH) was measured by qPCR. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 4. SOCS2-AS1 knockdown inhibits LNCaP and LTAD cell growth. (A) Knockdown 25


presented relative to the value of GAPDH as reference gene. Values represent mean ±s.d.

efficiency of SOCS2-AS1 by siRNA. LNCaP and LTAD cells were transfected with siNC:control siRNA or siSOCS2-AS1 #1 and #2 for 48 h and then treated with 10 nM DHT or Et for 18 h. Expression level of SOCS2-AS1 was analyzed by qRT-PCR (N = 3). (B) Knockdown efficiency of SOCS2 by siRNA, analyzed by qRT-PCR (N = 3) in LNCaP and LTAD cells treated with 10 nM DHT for 18 h. (C) Knockdown efficiency of SOCS2 by siRNA, analyzed by western blotting in LNCaP and LTAD cells, treated with 10 nM DHT or ethanol (Et) for 18 h. IB: immunoblot. (D) MTS assay in LNCaP and LTAD cell lines transfected with 20 nM siNC, siSOCS2-AS1 or 20 nM siSOCS2 for 24 h, following androgen treatment. (E) Cell proliferation assay in LNCaP and LTAD cell lines transfected with 20 nM siNC, siSOCS2-AS1 or 20 nM siSOCS2 for 24 h, following androgen (10 nM DHT) treatment. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01.

stable cells analyzed by qRT-PCR (N = 3). (B) qRT-PCR (N = 3) and western blotting analysis of SOCS2 expression in stable cell lines. IB: immunoblot. (C) SOCS2 protein expression analyzed by western blotting in LNCaP cells transfected with 20 nM siSOCS2-AS1 or siNC for 24 h, following androgen (10 nM DHT) or ethanol treatment for 18 h. IB: immunoblot. (D, E) MTS assays of stable cells cultured with normal medium (D) or phenol-free RPMI supplemented with 10% charcoal stripped FBS (E). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 6. SOCS2-AS1 overexpression enhances cell migration. (A) Migration assay of siSOCS2-AS1 (#1, #2) and control siRNA transfected cells in 10 nM DHT medium. (B) Quantification of migrated cells. The number of four random fields were counted using a microscope and the average number of cells per field was calculated. (C) Migration assay of SOCS2 (#1, #2), SOCS2-AS1 (#1, #2) and control (#1, #2) stable cells. (D) Quantification of migrated cells. The number of four random fields were counted using a microscope and the average number of cells per field was calculated. Data represent the average of four different views. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 7. SOCS2-AS1 inhibits the apoptosis pathway. (A) Microarray analysis of LNCaP cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h. Cells were treated with Et or DHT for 18 h. Genes repressed or induced more than 2-fold by siSOCS2-AS1 transfection in the presence of androgen were selected as SOCS2-AS1 downstream signals. Expression values displayed in gradient of red or green are the ratio between log2-transformed gene expression values in siSOCS2-AS1, siSOCS2 and siNC transfected cells. (B) GO-term analysis of 26


Figure 5. SOCS2-AS1 overexpression enhances cell growth. (A) SOCS2-AS1 expression in

upregulated or downregulated SOCS2-AS1 signals. Most significantly enriched functions were presented. (C) Microarray analysis of LTAD cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h. Cells were treated with Et or DHT for 18 h. Genes repressed less than 0.7 fold or induced more than 1.6-fold by siSOCS2-AS1 transfection in the presence of androgen were selected as SOCS2-AS1 downstream signals. (D) Venn diagram showing the overlap of SOCS2-AS1 target genes in LNCaP and LTAD cells. (E) GO-term analysis of upregulated or downregulated SOCS2-AS1 signals in LTAD cells. Most significantly enriched functions were presented.

Figure 8. SOCS2-AS1 modulates TNFSF10 expression. (A) Expression levels of Representative genes, TNFSF10, FAS, TRAILR1 are shown by arrows. (B) TNFSF10 mRNA expression in SOCS2-AS1 stable cells analyzed by qRT-PCR. TNFSF10, Fas and TRAILR1 mRNA expression in 20 nM siSOCS2-AS1 (siSOCS2-AS1#2) or siNC transfected cells for 24 h in LNCaP cells. (C) TNFSF10 mRNA expression in 20 nM siSOCS2-AS1 (siSOCS2-AS1#2) or siNC transfected cells for 24 h in VCaP cells. (D) TNFSF10 mRNA expression in LNCaP and LTAD cell lines analyzed by directional RNA-seq using Integrative Genomics Viewer (IGV browser). (E) TNFSF10 mRNA expression in LNCaP and LTAD cell lines analyzed by qRT-PCR. (F) TNFSF10 mRNA expression in siSOCS2-AS1 transfected LTAD cells. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. (G) (Left) Expression levels of apoptosis related genes (Fig. 8A) in LTAD cells were compared with LNCaP cells using microarray data. (Right) We compared expression levels of these genes in LTAD cells treated with siSOCS2-AS1with those in siNC. Figure 9. SOCS2-AS1 knockdown sensitizes cells to docetaxel treatment and increases apoptosis. (A) Knockdown efficiency of SOCS2-AS1 by siRNA, analyzed by qRT-PCR (N = 3) in LNCaP and LTAD cells treated with docetaxel for 7 days. (B) MTS assay of LNCaP and LTAD cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h, following 1 nM docetaxel or DMSO treatment for indicated time points. (C, D) Tunel assay in LNCaP and LTAD cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h following 1 or 2 nM docetaxel treatment for 24 h. (C) Panels represent 4′,6-diamidino-2-phenylindole (DAPI) (upper panels) and Alexa Fluor 488 (lower panels) signals detected in LNCaP cells treated with docetaxel. (D) Graph represents quantification of apoptotic cells in LNCaP and LTAD cells. (E) Tunel assay in VCaP cells. VCaP cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h following 2 nM docetaxel treatment for 24 h. (F) MTS assay of VCaP cells 27


apoptosis-related genes included in SOCS2-AS1 regulated genes in LNCaP microarray data.

transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h, following 2 nM docetaxel or DMSO treatment for indicated time points. Data represent the average of three different views. (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01 (vs siNC). Figure 10. SOCS2-AS1 overexpressing cells are more resistant to docetaxel treatment and apoptosis. (A) MTS assay of LNCaP stable cells treated with 2 nM docetaxel or DMSO for indicated time points. (B) Tunel assay in LNCaP stable cells treated with 2 nM docetaxel for 48 h. Panels represent 4′,6-diamidino-2-phenylindole (DAPI) (upper panels) and Alexa Fluor 488 (lower panels) stained docetaxel treated stable cells. (C) Quantification of apoptotic cells of Tunel assay. Data represent the average of three different views. (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. (D) Docetaxel-induced apoptosis is inhibited in SOCS2-AS1 expressing SOCS2-AS1 or SOCS2 and control cells, treated with 1 nM docetaxel for 48 hours. PARP and cleaved-PARP antibodies were used. PARP proenzyme (116 kD) and cleaved subunit, c-PARP, (89 kD) are indicated on the left by arrows. β-Actin was used as a loading control. IB: immunoblot. Figure 11. SOCS2-AS1 promotes androgen-mediated gene regulation. (A) Global effect of SOCS2-AS1 knockdown on androgen-mediated gene induction and repression. Microarray data from LNCaP cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h was analyzed. Among DHT-induced genes (> 1.5 fold), those repressed (< 0.8 fold) with siSOCS2-AS1 (#2) or siSOCS2 transfection are showed as the gray fraction (upper). Similarly, among DHT-repressed genes (< 0.7 fold), those induced (> 1.4 fold) with siSOCS2-AS1 (#2) or siSOCS2 transfection are showed as the black fraction (lower). (B) LNCaP cells overexpressing SOCS2-AS1 or vector control cells were treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were measured by qPCR. Fold inductions over vehicle (Et) control are shown. (C) LNCaP cells were treated with siNC or siSOCS2-AS1#1 for 24 h and then treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were were measured by qPCR. (D) VCaP cells were treated with siNC, siSOCS2-AS1 or siSOCS2 for 48 h and then treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were measured by qPCR. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 12. Androgen-dependent epigenetic control at TNFSF10 ARBS is modified by SOCS2-AS1. (A) AR binding genes were significantly enriched in SOCS2-AS1 downstream signals identified by microarray analysis (Fig. 7A). AR binding genes were determined based on 28


stably expressing cells. PARP cleavage was analyzed by western blotting in LNCaP cells stably

our AR ChIP-seq data in LNCaP cells (28). Among genes upregulated or downregulated by siSOCS2-AS1 treatment in LNCaP cells, the number of AR binding genes was counted. Chi-square test was performed to determine the P-value. (B) Identification of ARBSs in the vicinity of apoptosis-related genes. Results of AR ChIP-seq are shown in the genomic loci of representative genes, TNFSF10, TNFSF18 and TNFAIP3. In TNFSF10, the result of CtBP2 ChIP-seq data (31) is also shown. (C) qRT-PCR analysis of TNFSF10 mRNA following 10 nM siAR treatment and subsequent androgen treatment (10 nM DHT for 18 h) in LNCaP cell lines (N = 3). (D) LNCaP cells were transfected with siSOCS2-AS1 #1 or siNC for 48 h and then treated with Et or DHT for 24 h. Protein levels of phosphorylated AR (ser81) and total AR were analyzed by western blotting. IB: immunoblot. β-Actin was used as a loading control. (E) ChIP analysis of histone acetylation (AcH3) was performed in LNCaP cells. Cells were transfected over GAPDH exon locus were calculated by qPCR (N = 3). (F) ChIP analysis of histone deacetylase 1 (HDAC1) was also performed. NC: myoglobin locus (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 13. SOCS2-AS1 interacts with AR and controls the association of AR cofactors for histone modification. (A) ChIP analysis of AR, CtBP2 and SRC1 was performed in LNCaP cells. Cells were transfected with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments over GAPDH exon locus were calculated by qPCR. NC: myoglobin locus (N = 3). (B) ChIP analysis of histone H3 acetylation (AcH3) was performed in LNCaP cells. Cells were transfected with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments over Et control were calculated by qPCR (N = 3). (C) LNCaP cells were treated with Et or DHT for 18 h. Total RNA was extracted from nuclear and cytosolic fractions. Expression level of SOCS2-AS1 was determined by qRT-PCR (N = 3). (D) RIP analysis of AR was performed in LNCaP cells. Cells were treated with Et or DHT for 18 h. Nuclear lysates were obtained and then immunoprecipitation using AR antibody was performed (N = 3). Associated RNA was isolated and then RNA level of SOCS2-AS1 was determined by qRT-PCR. MB: Myoglobin. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 14. SOCS2-AS1 and TNFSF10 expression in clinical samples. (A, B) SOCS2-AS1 and TNFSF10 expression in publicly available datasets (GEO accession # GSE3325). SOCS2-AS1 (A) and TNFSF10 (B) microarray data was comparatively plotted in clinically localized primary prostate cancer and metastatic prostate cancer. Box plots are shown with lower and upper bounds of the boxes representing the 25th and 75th quartiles, respectively. Values represent normalized RNA expression levels relative to the value of GAPDH as reference gene. *, P < 29


with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments

0.05; **, P < 0.01. (C) Schematic model of SOCS2-AS1 androgen induction and cancer progression in prostate cancer.


30


Database

RP1-288H2.2

FAM13A-AS1-002

FAM13A-AS1-001

SOCS2-AS1-005

SOCS2-AS1-004

SOCS2-AS1-003

SOCS2-AS1-002

SOCS2-AS1-001

TCONS_00029024

CTB-167B5.2

CTB-167B5.2

RP1-288H2.2

FAM13A-AS1-002

FAM13A-AS1-001

SOCS2-AS1-005

SOCS2-AS1-004

SOCS2-AS1-003

SOCS2-AS1-002

SOCS2-AS1-001

Transcript Name

ENST00000585152.1

ENST00000547538.1

ENST00000500765.1

ENST00000511543.1

ENST00000549723.1

ENST00000551626.1

ENST00000547845.1

ENST00000500986.1

ENST00000499137.2

n377170

n341270

ENST00000585152.1

ENST00000547538.1

ENST00000500765.1

ENST00000511543.1

ENST00000549723.1

ENST00000551626.1

ENST00000547845.1

ENST00000500986.1

ENST00000499137.2

ID

chr7:87900206-87903065

chr7:87900207-87903065

chr12:52486269-52498081

chr4:89630940-89651254

chr4:89642866-89649837

chr12:93960515-93965066

chr12:93959360-93965174

chr12:93945209-93960955

chr12:93936240-93965544

chr12:93936239-93965544

chr21:42953358-42954625

chr7:87900206-87903065

chr7:87900207-87903065

chr12:52486269-52498081

chr4:89630940-89651254

chr4:89642866-89649837

chr12:93960515-93965066

chr12:93959360-93965174

chr12:93945209-93960955

chr12:93936240-93965544

chr12:93936239-93965544

Locus

+

-

-

-

+

+

-

-

-

-

-

+

-

-

-

+

+

-

-

-

-

-

Strand

-

-

-

-

FAM13A

FAM13A

SOCS2

SOCS2

SOCS2

SOCS2

SOCS2

-

-

-

-

FAM13A

FAM13A

SOCS2

SOCS2

SOCS2

SOCS2

SOCS2

Sense Strand Gene

1182

2859

2859

512

3210

2453

354

618

555

1655

3325

1182

2859

2859

512

3210

2453

354

618

555

1655

3325

0.1693

0.2248

0.2248

5.6580

0.0988

0.1149

0.3409

0.3796

0.1528

0.0760

0.5654

0.0000

0.0000

0.0000

0.6635

0.0887

0.1160

2.1786

1.2480

1.3896

0.6158

0.3369

Et

29.9912

30.7869

30.7869

65.0695

0.5959

0.3946

30.6916

17.6279

19.7518

7.9838

4.0296

0.8558

0.4622

0.4622

1.5663

0.7211

0.7876

9.2162

5.2792

5.8784

2.1971

1.1402

DHT

8.8549

10.6454

10.6454

22.7286

0.1918

0.0858

9.6933

5.5525

6.1828

2.4825

1.2633

0.2429

0.0223

0.0223

0.2907

0.4074

0.3676

2.3425

1.3418

1.4941

0.6424

0.3453

DHT Bic

9.6232

7.2389

7.2389

18.6027

0.2155

1.1556

7.2389

10.0214

5.7733

6.4103

2.3647

0.0000

0.0443

0.0443

0.0825

0.1217

0.0818

2.2895

3.9970

2.5494

0.9953

0.5240

DHT siAR

RPKM

Genecode

CTB-167B5.2

n341270

chr21:42953358-42954625

Noncode

Genecode

Noncode

CTB-167B5.2

n377170

Length

Table 1. List of androgen-induced lncRNAs. lncRNAs induced by more than 1.5-fold with 10 nM DHT compared to ethanol (Et) treatment, repressed to less than 0.75-fold by bicalutamide (Bic) and siRNA targeting androgen receptor (siAR) in LNCaP and VCaP cell lines are listed. RPKM: reads per kilobase per million mapped reads.

LNCaP

VCaP

TCONS_00029024

Table 2. List of siRNAs and PCR primers used for RNA knockdown, qRT-PCR, ChIP-qPCR analysis and SOCS2-AS1 expression vector construction. siRNAs siSOCS2-AS1

#1

siSOCS2-AS1

#2

siSOCS2

SOCS2 TNFSF10 GAPDH

Forward Reverse Forward Reverse Forward Reverse Forward Reverse

CCATACAGGTCAACTTTTCCACCAC CCAACCTCAGCTCTGCTCTCTT AACCGCTCTACACGTCAGCA TGGTAAAGGCAGTCCCCAGA AGACCTGCGTGCTGATCGTG CAAGCAATGCCACTTTTGGA GGTGGTCTCCTCTGACTTCAACA GTGGTCGTTGAGGGCAATC

PCR primers for cloning of SOCS2-AS1 Forward CTCAAGCTTAATTTGGGAACGAGATGCCCGGGGA Reverse CCCTCTAGATAGAAAGCTCCAGGTGTGTTTTATTTTA ChIP qPCR Primers SOCS2-AS1 ARBS TNFSF10 ARBS

Forward Reverse Forward Reverse

TGGGACCGACGTTTAACTCT GGGGTATCCTGAGGCTGTG TCCGTACAGCTTGTGTGGAG TTGGTGGTTAGGGATCAAGC


PCR Primers SOCS2-AS1

UUAUCACUCAUCAUUUCAGAA CUGAAAUGAUGAGUGAUAAUG GACCUGUAUGGUCAUUAUCACUC GUGAUAAUGACCAUACAGGUCAA UAUAUUCUUCCAAGUAAUCUU GAUUACUUGGAAGAAUAUAAA

GENCODE

Cell lines: LNCaP, VCaP

A

Treatment: DHT 10 nM 24h DHT + Bicalutamide DHT + siAR

LNCaP

58

Directional RNA-seq analysis Mapping: GENCODE, NONCODE Identification of AR-targeted lncRNAs

VCaP

9

72

NONCODE VCaP

Androgen induction Fold > 1.5 Repressed by bicalutamide or siAR

LNCaP

2

11 candidates (5 genes) of AR-targeted lncRNAs 53 Upregulation in LTAD, VCaP-LTAD cells Compared with parental cells By RNA-seq and qRT-PCR

114

AR-regulated lncRNAs in LNCaP cells.

SOCS2-AS1

AR-regulated lncRNAs in VCaP cells.

B 40

RP1-288H2.2

30 20 10 DHT

Et

DHT

0 Et

DHT Relative expression

DHT

Et

0

Et

25

DHT

50

5 4 3 2 1 0 Et


75

Et


0 Et

Et

DHT

Et

0

6

DHT

10

12

n341270

n377170

FAM13A-AS1 18

Et

20


SOCS2-AS1

LNCaP LTAD

LNCaP LTAD

LNCaP LTAD

LNCaP LTAD

LNCaP LTAD

SOCS2-AS1

FAM13A-AS1

n377170

n341270

RP1-288H2.2

qRT-PCR

2 0 DHT

Et

DHT

0

4

Et

10

6

DHT

20

8

Et

30

RP1-288H2.2 DHT Relative expression

n341270 40

Et


Et

n377170

250 200 150 100 50 0

DHT

DHT

Et

Et

0

DHT

20

Et

40

FAM13A-AS1 50 40 30 20 10 0

Et

SOCS2-AS1 DHT Relative expression

60

DHT

Relative expression

C

30

Et

Relative expression

qRT-PCR

VCaP VCaPLTAD

VCaP VCaPLTAD

VCaP VCaPLTAD

VCaP VCaPLTAD

VCaP VCaPLTAD

SOCS2-AS1

FAM13A-AS1

n377170

n341270

RP1-288H2.2

Figure 1

A

ChIP-Sequencing and Directional RNA-Sequencing

Chr 12

93,940 kb

93,950 kb

35 kb

93,960 kb

SOCS2-AS1

ARBS K4Me3 For

DHT Rev Et DHT

VCaP

Et DHT

VCAP -LTAD

LTAD

LNCaP LNCaP ChIP-seq

SOCS2

DHT

Et

Et

For Rev For Rev For Rev For Rev For Rev For Rev For Rev

C Western blotting

B 3

SOCS2-AS1 ∗∗

2 ∗∗

1

∗∗ ∗∗

∗∗

∗∗

∗∗

0 0 6 18 24 48 72 0 6 18 24 48 72

Relative expression

Relative expression

qRT-PCR 12

∗∗

SOCS2 ∗∗

8 4

∗∗ ∗∗

∗∗

∗∗

LNCaP IB: SOCS2

0

24 48

22 KDa ∗∗

β-actin 42 KDa

0 0 6 18 24 48 72 0 6 18 24 48 72

hours after induction




LNCaP

LTAD

LNCaP

LTAD

LTAD IB: SOCS2 22 KDa

β-actin 42 KDa

Figure 2

DHT 10 nM (h)

DHT 10 nM (h) 0

24 48

qRT-PCR

120 80 40 0 siNC

AR

SOCS2

∗∗

Relative expression

Relative expression

LNCaP SOCS2-AS1

siAR

120

Relative expression

A

∗∗

80 40 0 siNC

siAR

120

∗∗

80 40 0 siNC siAR

VCaP

B

80 40 0 DHT siAR DHT siNC siCtr siAR

∗∗

∗∗

120

Relative expression

∗∗

120

AR

SOCS2 Relative expression

Relative expression

SOCS2-AS1

80 40 0 DHT DHT siNC siAR siNC siAR

120 80 40 0 DHT DHT siAR siNC siCtr siAR

C

qRT-PCR

qRT-PCR SOCS2-AS1

Relative expression

12

∗∗

8 4 DHT Bic

DHT DMSO

Et DMSO

0 Et

DHT Bic

DHT DMSO

Et DMSO

SOCS2

1.6

∗∗

∗∗∗

∗∗∗

1.2 ∗∗∗

0.8

∗∗

0.4

0 DHT (nM)

0

0.1

1

0

0.1

1

0

siControl LNCaP

VCaP

Figure 3

20

LTAD

AR ChIP

5

10 5

VCaP

DHT Bic

DHT DMSO

Et DMSO

0

Fold enrichment

∗∗

15

Et

DHT DMSO

Et DMSO

Et

25 20 15 10 5 0

D

SOCS2

∗∗

Relative expression

SOCS2-AS1

∗∗

4

∗∗

3 2 1

0 DHT (nM)

0

0.1

LTAD

0

0.1

siAR

LNC P

DHT Bic

VCaP Relative expression

∗

16 12 8 4 0

Relative expression

SOCS2-AS1

Et

Relative expression

LNCaP

0.1 LNCaP

10

#1

Relative expression

120 100 80 60 40 20 0

#2

SOCS2-AS1 ∗∗ ∗∗

120 100 80 60 40 20 0

#1

siNC siNC siSOCS2-AS1

LTAD

LNCaP

LTAD

SOCS2-AS1 ∗ ∗∗

qRT-PCR SOCS2 120 100 80 60 40 20 0

Relative expression

LNCaP

Relative expression

B

qRT-PCR

siNC siSOCS2

Western Blotting

C

#2

SOCS2

∗∗

Relative expression

A

siNC siNC siSOCS2-AS1

IB:

∗∗

120 100 80 60 40 20 0 siNC siSOCS2

LNCaP

LTAD

Et DHT DHT

Et DHT DHT

SOCS2 22 KDa β-actin

D

MTS Assay LNCaP

Day 0

1.6 1.4

LTAD

Day 3

∗∗

1.2 1 0.8 0.6

∗

∗∗

1.8

Day 5

∗∗

Day 0 Day 3 Day 5

Fold Change

Fold Change

∗∗

∗∗

42 KDa

1.2

0.6

0.4 0.2 0 siNC si-NC

si-SOCS2-AS1 siSOCS2-AS1

#1 siSOCS2 si-SOCS2

siNC si-NC

Cell Proliferation Assay LNCaP 80 Et

x 104 cells / well

E

0

#2

60

DHT ∗∗

∗∗

40 20 0

#1

#2

#1

∗∗

x 104 cells / well

#1

#2

siNC siSOCS2siSOCS2-AS1siNCsiSOCS2-AS1 siSOCS2-AS1 siNCsiSOCS2-AS1 siSOCS2 siNC siSOCS2siSOCS2 -AS1 -AS1 Day 1

Figure 4

Day 5

15

#2


LTAD

si-SOCS2 siSOCS2

Et DHT

10 ∗∗

∗∗

#1

#2

∗∗

5 0

#1

#2

siNC siSOCS2siSOCS2-AS1siNCsiSOCS2-AS1 siSOCS2-AS1 siNCsiSOCS2-AS1 siSOCS2 siNC siSOCS2 siSOCS2 -AS1 -AS1 Day 1

Day 5

A

B qRT-PCR

qRT-PCR

SOCS2

D

Relative expression

Relative expression

SOCS2-AS1 2.5 2 1.5 1 0.5 0 #1

#2

#1

LNCaPLNCaP Vec -Vec

#2

#1

#2

IB:

2000

SOCS2

22 KDa

β-actin

42 KDa

1500 1000 500

C

0 #1

#2

#1

#2

#1

E

MTS Assay **

DHT

Et

#2

LNCaP- LNCaP- LNCaPVec SOCS2 LNCaP LNCaP SOCS2LNCaP AS1 -SOCS2 -SOCS2-Vec AS1

LNCaP- LNCaPLNCaP SOCS2LNCaP SOCS2 -SOCS2 -SOCS2AS1 AS1

10

#1 #2 #1 #2 #1 #2

2500

siSOCS2-AS1 IB:

siNC siNC #1

SOCS2

22 KDa

β-actin

42 KDa

MTS Assay

#2

(Androgen deprived) ∗∗

2

8

Day 2

6

Day 5 4

Day 7

Fold change

Fold change

1.5

Day 2 1

Day 5 Day 7

0.5

2

0

0

#1

#2

LNCaP LNCaP-Vec -Vec

Figure 5

#1

#2

LNCaPLNCaP SOCS2 -SOCS2

#1

#2

LNCaP LNCaP-SOCS2SOCS2-AS1 AS1

#1

#2

LNCaP-Vec LNCaP -Vec

#1

#2

LNCaPLNCaP SOCS2 -SOCS2

#1

#2

LNCaPLNCaP -SOCS2SOCS2-AS1 AS1

A

B

Migration Assay

∗∗

siSOCS2-AS1 #2

LTAD

LNCaP

#1

Migrated Cell Number

siNC

∗∗

80

∗∗

∗∗

60 40 20 0 #1 si-NC siNC

#2

#1

si-SOCS2si-NC siSOCS2- siNC AS1 AS1 LNCaP LNCaP

LNCaP Vector -Vec

#1

#2

LNCaP SOCS2 -SOCS2

LNCaP SOCS2-AS1 -SOCS2-AS1

∗∗

120 100 80 60 40 20 0 #1

#2

#1

#2

Vec SOCS2 LNCaP LNCaP LNCaP LNCaP -Vec -SOCS2 -SOCS2 -Vec

Figure 6

si-SOCS2siSOCS2AS1 AS1 LTAD LTAD

D

Migration Assay

Migrated Cell Number

C

#2

#1

#2

SOCS2-AS1 LNCaP -SOCS2AS1

A LNCaP Microarray

B

Downregulated genes siSOCS2 siNC -AS1#2 siSOCS2

GO:0006508~proteolysis

Upregulated genes siSOCS2 siNC -AS1#2 siSOCS2

GO:0006357~regulation of transcription GO:0007242~intracellular signaling cascade GO:0042981~regulation of apoptosis GO:0042127~regulation of cell proliferation

Et DHT Et DHT Et DHT

Et DHT Et DHT Et DHT repressed

GO:0006955~immune response

0

10

20

30

-Log10 P- value Go analysis of upregulated genes by siSOCS2-AS1 in LNCaP cells

Induced

GO:0065003~macromolecular complex assembly

C

GO:0051276~chromosome organization

LTAD Microarray Downregulated genes siSOCS2 siNC -AS1#2 siSOCS2 Et DHT Et DHT Et DHT

Upregulated genes siSOCS2 siNC -AS1#2 siSOCS2

GO:0006259~DNA metabolic process GO:0007049~cell cycle


GO:0000279~M phase

repressed

0

5

10

15

20

25

-Log10 P- value Go analysis of downregulated genes by siSOCS2-AS1 in LNCaP cells Induced

E

D Fold > 2.0

Fold > 1.6

LNCaP

LTAD

GO:0008629~induction of apoptosis GO:0048545~response to steroid hormone GO:0007243~protein kinase cascade

GO:0043933~macrom olecular complex subunit organization GO:0006259~DNA metabolic process

404

GO:0048870~cell motility

110

190

GO:0008285~negative regulation of cell proliferation

GO:0051276~chromos ome organization

GO:0016477~cell migration

Fold < 0.7 LTAD

Fold < 0.5 LNCaP

344

75

327

GO:0000279~M phase

GO:0006928~cell motion GO:0051090~regulation of transcription factor activity GO:0006955~immune response

GO:0007049~cell cycle

0

10 -Log10 P- value

Go analysis of downregulated genes by siSOCS2-AS1 in LTAD cells

Figure 7

20

0

5

-Log10 P- value Go analysis of upregulated genes by siSOCS2-AS1 in LTAD cells

10

15

20

B

Apoptosis related genes siSOCS2 siNC -AS1#2 siSOCS2

LNCaP qRT-PCR TNFSF10

TNFSF10 Relative expression

TNFSF10 /TRAIL

80 60 40 20 0 Et

DHT

Et

repressed

Fas

TNFRSF10A /TRAILR1

0.05 0 #1

#2

LNCaP-Vec LNCaP

-Vec

Et

DHT

siNC siNC

Relative expression

VCaP qRT-PCR

D TNFSF10

∗∗

1.5

Et

0.5

LNCaPLNCaP SOCS2-AS1 -SOCS2-

AS1

Et

DHT

siSOCS2siSOCS2AS1(3) AS1

Et

DHT

siNC siNC

DHT


21 kb 10 kb

LNCaP LTAD

DHT

Et

Directional RNA-Seq Chr 3

∗∗

1

#2

2.5 2 1.5 1 0.5 0

LNCaP Microarray

C

#1

TRAILR1 3 2.5 2 1.5 1 0.5 0

Relative expression

Induced

0.1


siNC siNC

FAS

DHT

∗

0.15

Relative expression


Relative expression

A

0 siNC

siSOCS2AS1 TNFSF10

E

LTAD

∗∗

Relative expression

Relative expression

TNFSF10

200

qRT-PCR

F

qRT-PCR

150 100 50 0

100

Figure 8

LTAD

Expression levels of apoptosis-related genes

∗∗

80

up down

60 ∗∗

40 20

LTAD vs LNCaP

0 #1

Et DHT Et DHT LNCaP

G

TNFSF10

si-NC siNC

#2

si-SOCS2-AS1 siSOCS2-

AS1

up down siSOCS2-AS1 vs siNC in LTAD cells

A 200 ∗∗

150

∗∗

C

∗∗

LNCaP TUNNEL Assay

∗∗

siSOCS2-AS1

siNC

#1 DAPI

100 50 0 siNC siNC

#1 #2 siNC siSOCS2siSOCS2- siNC AS1 AS1

LNCaP LNCaP

#1 #2 siSOCS2siSOCS2AS1 AS1

LTAD LTAD

7 days post-transfection

B MTS Assay

LNCaP

2.5

day1

day3

∗∗ Relative apoptotic cells

1.5 1

∗

∗∗

∗

0.5 0 #1 siNC

#2

#1

siSOCS2-AS1 siSOCS2-AS1

siSOCS2

siNC

∗∗

50

∗∗

40 30 20 10 0 #1

#2


DMSO

LNCaP TUNNEL Assay

D

day5

2 Fold change

siSOCS2

#2

Alexa Fluor 488

Relative expression

SOCS2-AS1

si-NC siNC

#2

sisiSOCS2 SOCS2 Docetaxel 1 nM 24h Docetaxel 1 nM 24 h

siSOCS2

Docetaxel


LTAD day1

day3

LTAD TUNNEL Assay

day5 Relative apoptotic cells

Fold change

2.5 2 1.5 1

∗∗

∗ 0.5 0 #1

#2


siNC

#1 siSOCS2

DMSO

F

∗∗ ∗∗

40

Fold change

Relative apoptotic cells

VCaP TUNNEL Assay

30 20 10

2

∗∗

∗∗

20 15 10 5 0 #1

#2

siNC siSOCS2-AS1siSOCS2-AS1 siSOCS2 siNC siSOCS2-AS1

siSOCS2

Docetaxel

E 50

25

#2


siNC

30

Docetaxel 2 nM 24 h

VCaP MTS assay day1

1.5

day3

day5

∗

∗

1

∗

∗∗

∗∗

0.5

0 #1 siNC siNC

#2


Docetaxel 2 nM 24 h

0 siSOCS2siSOCS2 AS1

Docetaxel 2 nM 24h

Figure 9

#1 siNC

#2

siSOCS2-AS1 DMSO

#1 siSOCS2

siNC

#2

siSOCS2-AS1 siSOCS2-AS1 Docetaxel

siSOCS2

A

Docetaxel 2 nM

MTS Assay 5

Fold change

Fold change

4 3 2

1.5 day0

1

day3 day5

0.5

1 0

0 #1

#2

LNCaP -Vec

B

∗∗

2

DMSO

TUNNEL Assay

#1

#2

#1

#2

LNCaP -SOCS2AS1

LNCaP -SOCS2

#2

LNCaP -Vec

#2

LNCaP-SOCS2 #1 #2

**

D

LNCaP-Vec

#1

#2

LNCaP -SOCS2

#1 #2 LNCaP -SOCS2AS1

LNCaP-SOCS2-AS1 #1 #2

Alexa Fluor 488

DAPI

#1

#1

Relative apoptotic cells

C

**

80 60

IB:

#1 #2

40 PARP

20 c-PARP

0 #1

#2

#1

#2

#1

#2

LNCaPLNCaP- LNCaPLNCaP LNCaP LNCaP Vec SOCS2 -SOCS2SOCS2-Vec -SOCS2 AS1 AS1 Docetaxel 2 nM 48 h

Figure 10

116 KDa 116 KDa 89 KDa

β-actin

#1 #2

#1 #2

Androgen-induced genes siSOCS2 siSOCS2-AS1

LNCaP stable cells

B

Up down Others Androgen-repressed genes siSOCS2-AS1 siSOCS2 Up down

Fold induction by androgen

A

∗∗ ∗∗

4.5 3

ACSL3 1.5

TMPRSS2

0 #1

#2

#1

LNCaP -SOCS2AS1

LNCaP -Vec

Others

#2

C TMPRSS2

ACSL3

10 ∗∗

5 0 siNC

siSOCS2-AS1#1

Relative expression

Relative expression

LNCaP

15

∗

10

Et

5

DHT

0 siSOCS2-AS1#1

siNC

TMPRSS2 40 N.S

30

∗∗

∗∗

20 10 0 siNC

#1

#2

siSOCS2-AS1

Figure 11

siSOCS2

Relative expression

Relative expression

D VCaP ACSL3 3

∗∗

∗

∗

Et

2

DHT

1 0 siNC

#1

#2

siSOCS2-AS1

siSOCS2

A

B

ChIP-seq

Chr 3: TNFSF10

173720 kb

173710 kb

Signal ratio

% of genes

Rate of AR-binding genes ∗∗ 0.3 0.2 0.1

10 4 80

CtBP2 AR

0

0

TSS

Chr 6: TNFAIP3

Chr 1: TNFSF18 171280 kb

171290 kb

138230 kb AR

AR

TSS

TSS MACS peaks

Relative expression

C

138240 kb

MACS peaks

2 1.5

Et

D

qRT-PCR

LNCaP

TNFSF10 ∗

∗

DHT

IB: P-AR

1 0.5 AR 0 siNC

siAR

Et

β-actin

DHT

E

F

HDAC1 ChIP

AcH3 ChIP TNFSF10 ARBS Et

DHT

N.S

4 ∗∗ 2

0 siControl siNC

Figure 12

∗∗

2.5 Fold enrichment

Fold enrichment

6

siSOCS2-AS1#1

2

Et siNC DHT siSOCS2 Et DHT -AS1#1

1.5 N.S.

1 0.5 0 NC

TNFSF10

A Fold enrichment

AR ChIP 150 Vehicle

100

siNC

DHT 50

Vehicle siSOCS2-AS1#l

DHT 0 NC

ACSL3 TMPRSS2 TNFSF10

SRC1 ChIP Fold enrichment

∗∗

30 20

∗∗

∗∗

10 N.S.

N.S.

ACSL3

∗∗ ∗

2

N.S. N.S. N.S.

1 0

TMPRSS2 TNFSF10

B

NC

ACSL3

C qRT-PCR Relative expression

Fold enrichment

AcH3 ChIP

ACSL3 6 ∗∗

4 2 0

Fold enrichment

TMPRSS2 8 4 2 0 Vehicle siNC

DHT

Vehicle

DHT

siSOCS2-AS1

∗∗

3 1.5 0 Et

DHT

Cytoplasmic fraction 10 8 6

Et

DHT

Nuclear fraction

RIP assay MB SOCS2-AS1

∗∗

4 2 0 Et

DHT IgG-IP

Figure 13

TMPRSS2 TNFSF10

SOCS2-AS1

4.5

D

∗∗

6

N.S.

N.S.

0 NC

3

Relative expression

Fold enrichment

CtBP2 ChIP

Et

DHT AR-IP

A

B SOCS2-AS1 2.00

*

**

Relative RNA levels

Relative RNA levels

4.00

TNFSF10

3.00 2.00 1.00 0.00

1.00

0.00

Primary Metastatic

Primary

Metastatic

Cytoplasmic

C Nucleus

AR

DHT

DHT

lncRNA SOCS2-AS1

AR antisense sense

Epigenetic regulation TNFSF10 AR

Genes for cell proliferation and migration

Proliferation ↑↑ Migration ↑ Anti-apoptosis ↑↑

Figure 14

SOCS2

(protein)

Proliferation ↑ Anti-apoptosis ↑

Androgen-induced lncRNA SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells Aya Misawa, Ken-ichi Takayama, Tomohiko Urano and Satoshi Inoue J. Biol. Chem. published online June 24, 2016

Access the most updated version of this article at doi: 10.1074/jbc.M116.718536 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts


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