EURURO Vol. 72 No. 5

836 860

1. Introduction
Castration-resistant prostate cancer (CRPC) is characterized
by its continuous androgen receptor (AR) function and
resistance to androgen-deprivation therapy (ADT) . The
recently developed antiandrogen, enzalutamide (Enz, also
called MDV3100), was approved recently by the Food and
Drug Administration after a demonstration that it could
prolong survival in men with metastatic CRPC However,
Enz resistance eventually occurs in most patients due to a
variety of AR-dependent and AR-independent mechanisms.
Relevant in this setting is the recent demonstration that Enz
resistance is strongly associated with the expression of the
most common AR splicing variant 7 (AR-v7, also called AR3)
in circulating tumor cells (CTCs) from CRPC patients.
Importantly, AR-v7 was also expressed higher in the tumors
of Enz-treated patients compared with tumors of Enz-naı¨ve
patients . The detailed mechanism(s) of how AR-v7 is
induced and its linkage to Enz resistance, however, remain
unclear. Here, we show a long noncoding RNA (lncRNA),
Malat1, is required for Enz-induced AR-v7 production, and
targeting the Malat1/AR-v7 axis could overcome Enz
resistance and may become a future therapeutic choice to
better suppress the Enz-resistant (EnzR) CRPC progression.
2. Materials and methods
2.1. Generation of Enz-resistant (R1 and R2) cell lines
R1 cells were generated by culturing C4-2 cells under increasing Enz
concentrations from 10 mM to 40 mM (every 20 d) for 3 mo (Supple-
mentary Fig. 1). For R2, cells were cultured at 10 mM Enz for 3 mo before
experiments. After generation, both R1 and R2 were maintained inmedia
with 10 mM Enz.
2.2. In vivo xenograft mouse model
EnzR cells (R1), 1 106, were injected subcutaneously into 6-wk-old
nude mice 1:1 with Matrigel. After 4 wk, tumor bearing mice were
randomly grouped and injected with scramble RNA (10 mg/kg), Malat1-
short interfering RNA (siRNA; 10 mg/kg), or ASC-J91 (75 mg/kg) for
2 wk. Invivofectamine 2.0 kit (#1377501; Invitrogen, Carslbad, CA, USA)
was used to deliver the Malat1-siRNA. Briefly, 50-m l 3-mg/ml Malat1-
siRNA was diluted with 50-ml complexation buffer, then mixed 1:1 with
invivofectamine 2.0 for several seconds. The mixture was injected into
the peritumor region. Enz (30 mg/kg) was diluted with corn oil and
intraperitoneal injected every other day during the therapies. Tumors
were measured by caliper every week, then mice sacrificed and tumors
removed for final measurement and immunohistochemistry studies.
2.3. Analysis of CTCs
Blood collection, processing, and CTC isolation procedures were
described previously CTC samples used for study included 113 frozen
samples, each representing a unique blood draw from men with
metastatic CRPC who signed consent forms for blood draw before,
during, and after standard-of-care treatment with abiraterone, Enz, or
taxane chemotherapies. Samples were processed and data generated
while blinded to treatment status. From this dataset, 10 pairs of pre- and
post-Enz treatment Malat1 expression data were avaialble following
unblinding. Expression data was normalized to a control gene (RPL13A),
and normalized data presented for each pretreatment and post-
treatment pairs. Expression levels of full-length AR (AR-FL), AR-v7,
and Malat1 were quantified by quantitative polymerase chain reaction
(qPCR) using specific primers listed in Supplementary Table 2. Other
materials and methods used are described in Supplementary data.
3. Results
3.1. Enhanced expression of AR-v7 and Malat1 in EnzR-PCa
cells
Recent clinical data suggested that CRPC patients with
higher AR-v7 expression in their CTCs responded poorly to
Enz therapy However, the mechanism(s) how AR-v7
was generated and its linkage to the development of Enz
resistance remain largely unclear. Here we established two
EnzR-PCa C4-2 cell lines (R1 and R2) using different Enz
treatment strategies (Supplementary Fig. 1A), and con-
firmed their resistance phenotype (Supplementary Fig. 1B–
D).
Since lncRNAs have been reported to play essential roles
in cancer development and drug resistance, we speculated
that some selective lncRNAs might be altered after
development of Enz resistance. We focused on 32 lncRNAs
whose expression was either relatively specific in the
prostate or have higher expression in PCa, and were
intrigued by two of them, PCGEM1 and Malat1 (Supplemen-
tary Table 1). PCGEM1 has been identified as an AR/AR-v7
signaling regulatory lncRNA , and Malat1 has been
reported to be upregulated in CRPC and may contribute
to RNA splicing via binding to the serine/arginine rich
splicing factor 1 (SF2) complex .
Since AR-v7 is the predominant splicing variant of AR
and could confer Enz resistance to PCa cells (Supplementary
Fig. 1E), we were interested to see the potential linkage of
the increased Malat1 expression in EnzR cell lines to the AR-
v7 expression. The results revealed that AR-v7 and Malat1
expression at both protein ) and messenger RNA
levels ) was increased in those EnzR-PCa cells
compared with their parental Enz-sensitive cells. Also, AR-
FL was also elevated in the EnzR-PCa cells A). Since the
R1 cells have a relatively higher expression of AR-v7, we
focused on this EnzR cell line for subsequent experiments.
To prove higher Malat1 expression may lead to increased
AR-v7 expression, we manipulated Malat1 expression by
transducing two individual transcription activator-like
effector (TALE)-based transcription factors into the up-
stream promoter region of Malat1 C), and found this
induction of Malat1 expression D) increased the AR-
v7 expression at the mRNA/protein levels in C4-2 cells
D and E) with little impact on other AR variants
), suggesting the specific role of Malat1 on the
induction of AR-v7 production.
Importantly, the forced expression of Malat1 by two
TALE-based Malat1-inducers also altered the expressions of
22 AR-v7-regulated downstream genes ), and
adding the Malat1-siRNA into these two inducer-harboring
cells led to blocking/reversing the Malat1-induced AR-v7
target gene expressions ).
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