EURURO Vol. 72 No. 5

(10 mg/kg body weight every 2 wk) as a control (

= 4).

During the entire experimental duration, Enz (30 mg/kg

body weight every 24 h) was administered to all groups of

mice. Mouse tumor growthwas measuredweekly andmice

sacrificed after 4 wk. The results revealed that both

Malat1

siRNA and ASC-J9

significantly suppressed EnzR tumors

( Fig. 5 C

and D).

We also examined the AR-v7 and AR expression in vivo

through immunohistochemistry staining and results indi-

cated a significant reduction of AR-v7 expression in mice

treated with

Malat1

-siRNA and ASC-J9

while

Malat1

siRNA had little effect on AR

( Fig. 5 E

Discussion

Among several mechanisms involved in the development of

Enz resistance in CRPC, including induction of AR-v7

[4] ,

ARF876L mutation

[16–18] ,

and altered glucocorticoid

receptor signals

[19]

, the induction of AR-v7

[4]

has the

strongest clinical data support derived from a clinical study

showing CRPC patients with detectable AR-v7 in CTCs had

poor responses to ADT-Enz

[4] .

Furthermore, AR-v7 might

be linked to bone metastases in CRPC

[20] .

These clinical

data point to the possible reason why ADT-Enz may always

fail after an initial clinical response. Therefore, it is

important to develop new approaches to suppress AR

function beyond antiandrogens since induced AR-v7 lacks

the ligand-binding domain and can be activated in the

castration condition

[21–24] .

In this study, we found that

Malat1

expression and SF2 activity are upregulated in EnzR

C4-2 cells, which contributed to AR-v7 production and led

to Enz resistance

( Fig. 5

F). Our finding is consistent with

previous work showing that SF2 could recognize and bind to

the intron between exon 3 and exon 4 of the AR transcript to

facilitate AR-v7 production

[25]

The linkage of Enz resistance to the induction of the

Malat1/

SF2

AR-v7 axis strongly suggests a new and better

therapy to further suppress CRPC during or after develop-

ment of Enz resistance, via targeting the

Malat1

, SF2,

and

[(Fig._4)TD$FIG]

Fig. 4 – Androgen receptor splicing variant 7 (AR-v7) and

Malat1

confer enzalutamide (Enz)-resistance to prostate cancer cells. (A) AR-v7 stably

transfected C4-2 cell line displays Enz insensitivity compared with the control. Equal numbers of cells were seeded and subjected to various

concentrations of Enz. After 4 d, cell growth/viability was determined by MTT assay. (B) Knockdown of AR-v7 in EnzR cell line (R1) makes cells more

sensitive to Enz treatment. (C)

Malat1

-expressing cells show Enz insensitivity compared with control cells. Equal numbers of cells were seeded and

treated as indicated for MTT. (D) The deficiency of

Malat1

in EnzR cell line makes cells sensitive to Enz treatment. (E)

Malat1

-mediated Enz-resistance

can be reversed by short hairpin (sh)AR-v7. After two rounds of virus infection, equal numbers of cells were seeded and treated as indicated for MTT.

(F)

Malat1

-mediated Enz-resistance can be reversed by shRNA-serine/arginine rich splicing factor 1 (SF2) in cells seeded and treated as indicated for

MTT. For A-D the statistical analysis was made between

Malat1

-expressing cells and controls cells. For E-F ANOVA t test was performed among groups.

siCtr = si-control.

< 0.05

;

< 0.05.

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