4-1BBL has a Possible Role in Mediating Castration-Resistant Conversion of Prostate Cancer via Up-Regulation of Androgen Receptor

Abstract

4-1BB ligand (4-1BBL) was a transmembrane glycoprotein belonging to the tumor necrosis factor family. It was expressed on activated T lymphocytes and function as a co-stimulatory molecule via cross-linking with 4-1BB (a.k.a, CD137). In addition to its role in immune regulation, 4-1BBL transmitted signals into the cells on which it was expressed (reverse signaling). 4-1BBL represented a promising target for enhancing antitumor immune responses. Recent studies indicated that 4-1BBL also expressed in non-immune cells and possessed different functions in various types of cells. Here, we reported that 4-1BBL didn't express in normal prostate tissues and benign prostatic hyperplasia tissues, but it expressed in prostate cancer (PCa) tissues at moderate level. Expression of 4-1BBL was up-regulated during the transition from PCa to castration resistant prostate cancer (CRPC). Increasing expression of 4-1BBL not only promoted expression of androgen receptor (AR), but also augmented proliferation and invasion ability of prostate cancer cells in androgen deprivation environment. These results were further verified by xenograft tumor experiments. Meanwhile, inhibiting AR signal pathway by chemical antagonist was able to significantly reduce 4-1BBL mediated proliferation and invasion of PCa cells. These novel findings indicated that 4-1BBL might mediate prostate cancer progression to castration-resistant prostate cancer via enhancing expression and function of AR.

Keywords: 4-1BBL; androgen receptor; castration resistant prostate cancer; prostate cancer.

Figure 1

Immunohistochemical expression of 4-1BBL in Normal prostate, BPH, ADPC and CRPC tissues (magnification, 400). A, Normal prostate tissue showed no 4-1BBL expression. B, BPH tissue also showed no 4-1BBL expression. C, ADPC tissue showed moderate 4-1BBL expression. D, CRPC tissue showed strong 4-1BBL expression.

Figure 2

Expression of 4-1BBL in normal prostate, BPH, ADPC and CRPC tissues was confirmed at mRNA level by qRT-PCR and was confirmed at protein level by western blot. A, 4-1BBL mRNA expression was not detected in Normal prostate and BPH tissues, but the 4-1BBL mRNA expression was significantly higher in CRPC compared to ADPC tissue (*P<0.01 versus ADPC). B, 4-1BBL mRNA expression was not detected in RWPE-1, but it was higher in CRPC cell lines (PC3, DU145 and C4-2) than in ADPC cell lines (LNCaP). C, 4-1BBL expression was not detected in Normal prostate and BPH tissues, but the 4-1BBL expression was significantly higher in CRPC compared to ADPC tissue (*P<0.01 versus ADPC). D, 4-1BBL expression was not detected in RWPE-1, but it was higher in CRPC cell lines (PC3, DU145 and C4-2) than in ADPC cell lines (LNCaP). In addition, 4-1BBL expression was highest in C4-2 cell line (#P<0.01 versus LNCaP).(N: Normal prostate; BPH: Benign prostate hyperplasia; ADPC: Androgen dependent prostate cancer; CRPC: Castration resistant prostate cancer)

Figure 3

Function of 4-1BBL on proliferation ability and invasion ability of LNCaP cells in androgen deprivation environment. A, LNCaP cells transfected with pCDNA3.1/4-1BBL still demonstrates strong proliferation ability in phenol red free RPMI1640 medium with activated Charcoal/Dextran treated FBS. B, LNCaP cells transfected with pCDNA3.1/4-1BBL demonstrates stronger invasion ability than LNCaP or LNCaP/ pCDNA3.1 cells in phenol red free RPMI1640 medium with activated Charcoal/Dextran treated FBS). (*P<0.05 versus LNCaP or LNCaP/ pCDNA3.1).

Figure 4

Effect of 4-1BBL on AR expression in PCa cells was confirmed by qRT-PCR and by western blot. A, 4-1BBL mRNA expression was significantly higher in LNCap/4-1BBL cells compared to LNCap cells, while 4-1BBL mRNA expression was significantly lower in C4-2/si-4-1BBL cells compared to C4-2 cells (1: LNCap cell; 2: LNCap/4-1BBL cell; 3: C4-2 cell; 4: C4-2/si-4-1BBL cell). B, AR expression was significantly higher in LNCap/4-1BBL cells compared to LNCap cells, while AR expression was significantly lower in C4-2/si-4-1BBL cells compared to C4-2 cells. (*P<0.05 versus LNCap; #P<0.05 versus C4-2). C, Proliferation ability of LNCap/4-1BBL cells influenced by Enzalutamide was assessed by CCK8 assay. Enzalutamide (an inhibitor of AR signaling) decreased proliferation ability of LNCaP cells induced by 4-1BBL. D, Invasion ability of LNCap/4-1BBL cells influenced by Enzalutamide was assessed by Transwell invasion assay. Enzalutamide reduced invasion ability of LNCaP cells induced by 4-1BBL. (Enzalutamide group Vs Vekicle group, P<0.05)

Figure 5

A, Expressions of 4-1BBL and AR in Control group, Castration group and 4-1BBL group were confirmed at protein level by western blot. Both 4-1BBL and AR expressions were significantly up-regulated in the Castration group than in the Control group (P<0.05). In the 4-1BBL group, both 4-1BBL and AR expressions were significantly higher compared to the Control group (P<0.05), and both 4-1BBL and AR expressions were significantly higher than in the Castration group (P<0.05). B, Immunohistochemical expression of KI67 in Control group, Castration group and 4-1BBL group tissues (magnification, 400). The expression of KI67 was significantly higher in the Castration group tissues than in the Control group tissues (P<0.05), furthermore, the expression of KI67 was significantly higher in the 4-1BBL group than in the Castration group (P<0.05).