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. 2015 Feb;75(2):161-74.
doi: 10.1002/pros.22901. Epub 2014 Oct 13.

Androgen receptor splice variants contribute to prostate cancer aggressiveness through induction of EMT and expression of stem cell marker genes

Dejuan Kong  1 , Seema Sethi, Yiwei Li, Wei Chen, Wael A Sakr, Elisabeth Heath, Fazlul H Sarkar
Affiliations

Androgen receptor splice variants contribute to prostate cancer aggressiveness through induction of EMT and expression of stem cell marker genes

Dejuan Kong et al. Prostate. 2015 Feb.

Abstract

Background: The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking.

Methods: Expressions of epithelial-to-mesenchymal transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere-forming capacity was assessed using PCa cells under all experimental conditions and 3,3'-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers.

Results: Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment.

Conclusion: AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa.

Keywords: 3,3′-diindolylmethane; androgen receptor splice variants; cancer stem cells; self-renewal.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Figures

Fig-1
Fig-1. AR and AR3 expression in PCa tissue specimens and PCa cell lines
(A) The expression of AR mRNA was increased in PCa tissue specimens with Gleason grade 6, but not in patients' samples with Gleason score ≥7 compared with adjacent normal tissues. Similar data for AR mRNA levels were reported previously [Kong et at. Am J Transl Res 2012; 4(1): 14-23], and also showed different expression pattern between AR and AR3 in PCa tissue specimens (TumorG6: tumor tissues from patients with Gleason grade 6. N = 93 for normal, N=136 for tumor, N = 42 for Tumor G6, N = 45 for Tumor G7, N = 49 for Tumor G8 and 9). (B) The levels of AR3 were found to be significantly up-regulated in PCa tumor tissue specimens and especially in tumor tissues with Gleason grade≥8 compared with adjacent normal tissues. (N = 91 for normal, N=134 for tumor, N = 42 for Tumor G6, N = 44 for Tumor G7, N = 48 for Tumor G8 and 9). (C) Significant up-regulation in the expression of AR and AR3 mRNA in PCa cell lines compared with immortalized normal prostate epithelial cell lines such as RWPE-1 and PZ-HPV-7. (D) Increased protein levels of AR and AR3 as well as stem cell markers such as Nanog and Oct4, and mesenchymal marker fibronectin (FN) in PCa cell lines compared with normal prostate epithelial cell lines. Expression of PSA was found to be increased in PCa cell lines, which was inversely correlated with AR3 expression.
Fig-2
Fig-2. Overexpression of AR and AR3 resulted in the induction of EMT and stem cell phenotype in PCa cells
(A) and (B) Transfection of LNCaP and DU145 cells with plasmids expressing AR showed increased expression of mesenchymal markers such as fibronectin (FN), ZEB1 and Twist. Transfection of LNCaP and DU145 cells with plasmid expressing AR3 showed not only increased expression of mesenchymal markers such as fibronectin and ZEB1 but also enhanced expression of stem cell marker genes such as Nanog, Lin28B and CD44 (* p<0.05, ** p<0.01). (C) Down-regulation of AR3 but not AR by transfection of 22RV1 cells with siRNA inhibited the expression of Nanog, Oct4 and ZEB1. (D) Overexpression of AR and AR3 by transfection of DU145 and PC3 with plasmids expressing AR and AR3 promoted cell migration. (E) LNCaP cells transfected with plasmids expressing AR (LNCaP-AR), AR3(LNCaP-AR3) or control vector (LNCaP-con) and incubated for 24 hours, then the transfected cells were collected and seeded in 100 mm dishes at density of 2000 cells. After 2 weeks of incubation, the colonies were stained and photographed.
Fig-3
Fig-3. Overexpression of AR3 increased sphere-forming (prostasphere-forming) capacity
(A) and (C) PC3 cells transfected with AR3 but not AR showed increased prostasphere-forming ability. (B) and (D) DU145 cells overexpressing AR3 but not AR showed increased prostasphere numbers and Size (**, p<0.01).
Fig-4
Fig-4. EMT and stem cell signatures were found in PCa cells grew in androgen deprivation condition
(A) C4-2B cells grew in charcoal stripped fetal bovine serum (CS-FBS), at least 2 weeks, showed increased expression of AR and AR3 mRNA, inhibited the expression of PSA, and also up-regulated the expression of stem cell marker genes such as Lin28B, Nanog and Sox2, and EMT markers such as ZEB1, Twist, N-cadherin and vimentin. (B) C4-2B (upper panel) and VCaP cells (lower panel) grew in CS-FBS for 3 days and then treated with or without 10 nM DHT and incubated for another 24 hours. DHT treatment inhibited mRNA expression of AR and AR3, concomitant with decreased mRNA expression of Lin28B, Nanog, ZEB1, Twist and vimentin in C4-2B cells, and depressed mRNA expression of Sox2, N-cadherin and vimentin in VCaP cells. (C) C4-2B cells grew in CS-FBS for, at least 2 weeks, promoted protein expression of AR, AR3 and mesenchymal marker such as vimentin, whereas C4-2B cells grew in CS-FBS for 4 weeks showed increased protein expression of stem cell marker such as Nanog. (D) DHT treatment for 24 hours reversed protein expression of AR, AR3, PSA, Lin28B, vimentin and N-cadherin in C4-2B and VCaP cells (*p<0.05; **p<0.01).
Fig-5
Fig-5. Lin28B was increased in PCa tissue specimens, which was positively correlated with AR3 expression, and abrogated by BR-DIM treatment in a phase II clinical trial
Total RNA was obtained from formalin-fixed paraffin-embedded (FFPE) tissue specimens of PCa patients and BR-DIM clinical trial samples with matched tumor Gleason grade, tumor stage and patient age as control group. BR-DIM was given to patients for 2-4 weeks prior to surgery. (A) The levels of Lin28B mRNA were found to be significantly increased in patients' tumors with higher Gleason grade (Gleason score ≥7) compared with adjacent normal tissues (N = 75 for normal, N = 38 for Tumor G6, N = 46 for Tumor G7, N = 34 for Tumor G8 and 9). (B) Immunohistochemical staining showed increased expression of Lin28B localized in the cytoplasm. (C) Lin28B expression was positively correlated with expression of AR3 in patients' tumors with Gleason grade ≥8 (N = 36). (D) BR-DIM treatment led to decreased expression of Lin28B (N = 67 for patients, N = 7 for patients with BR-DIM treatment).
Fig-6
Fig-6. BR-DIM treatment inhibited the expression of AR, AR variants and stem cell markers
Single cell suspensions of LNCaP and 22RV1 cells were plated in 6-well plates with ultra-low attachment surface at 2000 cells/well and incubated for 3 days. The cells subsequently received fresh medium with or without BR-DIM, and then incubated for another 4 days. Prostasphere cells were collected for preparation of cell lysates or isolation of RNA. (A) Increased expression of Lin28B, Nanog, Oct4 and Sox2 mRNA were found in sphere-forming cells from LNCaP. (B) The mRNA expression of AR, AR3, EMT markers such as ZEB1 and vimentin, and stem cell markers such as Lin28B, Nanog, Sox2 and CD44 were found to be increased in sphere-forming cells from 22RV1 cells. (C) BR-DIM treatment decreased the expression of AR variants (AR-Vs) and PSA in LNCaP and LNCaP sphere-forming cells. (D) BR-DIM treatment inhibited the expression of stem cell marker: Nanog in LNCaP and LNCaP sphere cells. (E) BR-DIM treatment also repressed the mRNA expression of stem cell markers such as Lin28B, Nanog, CD44, Sox2 and Oct4 in 22RV1 sphere cells. (F) Expression of full length AR (AR-FL), AR variants and AR3 was reduced by BR-DIM treatment in 22RV1 cells and 22RV1 sphere-forming cells (*p<0.05; **p<0.01).
Fig-7
Fig-7. BR-DIM treatment inhibited cell migration and prostasphere-forming ability
(A) BR-DIM treatment for 24 hours down-regulated the expression of AR, AR variants, AR3 and PSA as well as fibronectin (FN) in LNCaP and C4-2B cells. (B) BR-DIM treatment for 2 days and 3 days inhibited the expression of AR, AR variants, AR3, PSA, fibronectin and Nanog in LNCaP and C4-2B cells. (C) BR-DIM treatment significantly reduced prostasphere numbers and size (**p<0.01). (D) BR-DIM treatment significantly inhibited C4-2B cell migration after 24h.
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