Depletion of SAG/RBX2 E3 ubiquitin ligase suppresses prostate tumorigenesis via inactivation of the PI3K/AKT/mTOR axis

Abstract

Background: SAG (Sensitive to Apoptosis Gene), also known as RBX2, ROC2 or RNF7, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our recent study showed that SAG/RBX2 co-operated with Kras to promote lung tumorigenesis, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. However, it is totally unknown whether and how Sag would play in prostate tumorigenesis, triggered by Pten loss.

Methods: Sag and Pten double conditional knockout mice were generated and prostate specific deletion of Sag and Pten was achieved by PB4-Cre, and their effect on prostate tumorigenesis was evaluated by H&E staining. The methods of immunohistochemistry (IHC) staining and Western blotting were utilized to examine expression of various proteins in prostate cancer tissues or cell lines. The effect of SAG knockdown in proliferation, survival and migration was evaluated in two prostate cancer cell lines. The poly-ubiquitylation of PHLPP1 and DEPTOR was evaluated by both in vivo and in vitro ubiquitylation assays.

Results: SAG is overexpressed progressively from early-to-late stage of human prostate cancer with the highest expression seen in metastatic lesion. Sag deletion inhibits prostate tumorigenesis triggered by Pten loss in a mouse model as a result of suppressed proliferation. SAG knockdown in human prostate cancer cells inhibits a) proliferation in monolayer and soft agar, b) clonogenic survival, and c) migration. SAG is an E3 ligase that promotes ubiquitylation and degradation of PHLPP1 and DEPTOR, leading to activation of the PI3K/AKT/mTOR axis, whereas SAG knockdown caused their accumulation. Importantly, growth suppression triggered by SAG knockdown was partially rescued by simultaneous knockdown of PHLPP1 or DEPTOR, suggesting their causal role. Accumulation of Phlpp1 and Deptor with corresponding inactivation of Akt/mTOR was also detected in Sag-null prostate cancer tissues.

Conclusions: Sag is an oncogenic cooperator of Pten-loss for prostate tumorigenesis. Targeting SAG E3 ligase may, therefore, have therapeutic value for the treatment of prostate cancer associated with Pten loss.

Keywords: DEPTOR; PHLPP1; Prostate tumorigenesis; Pten; SAG KO; SAG-SCF E3; Ubiquitin ligase.

Fig. 1

SAG is crucial for progression of prostate cancer in human and mice. (a & b) Expression of SAG is assessed in a human prostate cancer TMA. SAG staining indexes in cohorts of normal prostate epithelial cells (n = 24) and prostate tumors (n = 58) are shown as stacked columns. (c & d) PB4-Cre inactivates Pten and Sag in prostate epithelium cells. Prostate tissues from mice at age of 6 months with indicated genotypes were fixed in 10% formalin, embedded, sectioned and stained with Pten and Sag Abs. (e) Western blotting for Pten and Sag in prostate tissues (four independent samples with two genotypes). (f) Haematoxylin and eosin (h & e) staining of the prostate tissues from mice at age of 6 months with indicated genotypes. Scale bar represents 100 μm. (g) Pie graphs show prostate tumor progressions in Pten ^PC-/-;Sag ^+/+ and Pten ^PC-/-;Sag ^PC-/- mice at age of 6 months (n = 10). HGPIN, high-grade PIN; LGPIN, low-grade PIN. The quantitative results of tumor progression are from two randomly selected slides of each mouse in a total 10 pairs of the entire animal cohort. (h) Mice with indicated genotypes were sacrificed at ages of 3, 6, and 9 months, respectively. The weight of prostate vs. whole body were weighed, the % weight was calculated and plotted (n = 6). * p < 0.05. (i & j) Prostate tissues with indicated genotypes were stained for Ki67 with representative images shown (i). Positive cells were counted from at least 3 randomly selected microscopic fields with % positivity calculated (j). * p < 0.05. Scale bar: 100 μm

Fig. 2

SAG Knockdown suppresses growth, survival and migration of human prostate cancer cells via inactivation of the PI3K/AKT/mTOR axis. DU145 human prostate cancer cells were infected with Lenti-SAG (Lt-SAG), along with Lenti-GFP (Lt-Con) as a control for 72 hrs. Cell proliferation was measured by ATP-lite assay (a), clonogenic survival (b), soft agar assay (c), and Boyden chamber migration assay (d), as well as western blotting assay using indicated Abs (e). Shown are mean ± SEM from three independent experiments (a-d). * p ; ** p < 0.01. Scale bar represents 50 μm

Fig. 3

SAG interacts with PHLPP1 or DEPTOR and shortens their protein half-lives. (a-e) SAG interacts with PHLPP1 or DEPTOR: DU145 cells were co-transfected with indicated plasmids, cell lysates were prepared and IP with anti-HA Ab, IB with anti-FLAG Ab (a), or IP with anti-FLAG Ab, IB with anti-HA Ab (b). Sub-confluent DU145 cells were lysed and subjected to IP with anti-SAG (c), anti-PHLPP1 (d) or anti-DEPTOR Ab (e), along with IgG control, followed by IB with indicated Abs. (f & g) SAG overexpression shortens protein half-lives of PHLPP1 and DEPTOR: DU145 cells were transfected with HA-PHLPP11or FLAG-DEPTOR, along with the vector control or FLAG-SAG for 48 hrs. Cells were treated with cycloheximide (CHX; 100 μg/ml) to block new protein synthesis for the indicated time periods, followed by IB analysis. (h & i) SAG knockdown extends the protein half-lives of PHLPP1 and DEPTOR: DU145 cells were infected with Lt-SAG, along with Lt-Con for 72 hrs. Cells were then treated with CHX (100 μg/ml) for indicated time periods and subjected to IB analysis. Densitometry quantification was performed using ImageJ software with β-actin as the loading control and plotted (bottom panels)

Fig. 4

SAG-SCF^β-TrCP promotes poly-ubiquitylation of PHLPP1 and DEPTOR: a & c The in vivo ubiquitylation assay: The 293 cells were transfected with indicated plasmids and lysed under denatured condition (6 M guanidinium solution), followed by Ni-bead pull-down. Washed beads were boiled and subjected to IB for PHLPP1 (a) or DEPTOR (c). b & d The in vitro ubiquitylation assay: SAG-CUL1 E3 was prepared by FLAG-conjugated beads IP using 293 cells transfected with SAG, along with CUL1. PHLPP1 or PHLPP1(4A) was prepared by transfecting HA-PHLPP1 or HA-PHLPP1(4A) into 293 cells, followed by HA-conjugated beads IP and 1× HA peptide elution (b) or DEPTOR or DEPTOR(3A) was transfected into 293 cells, followed by FLAG-conjugated beads and 3× FLAG peptide elution (d). SCF E3 and substrates (PHLPP1 or DEPTOR) or their mutants were added, respectively, into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by constant mixing for 60 min. The reaction mixture was boiled with loading buffer and then loaded onto SDS-PAGE gel for IB using PHLPP1 (b) or DEPTOR (d) Ab

Fig. 5

Knockdown of PHLPP1 or DEPTOR partially rescues growth suppression triggered by SAG deletion. DU145 cells were first infected with Lt-SAG to knockdown SAG along with the control (Lt-Con), followed by transfection with PHLPP1 or DEPTOR siRNA oligonucleotides. A portion of cells were harvested for immunoblotting a; the other portions for monolayer growth for 4 days, followed by ATP-lite proliferation assay b; clonogenic assay for survival c; soft agar assay for anchorage-independent growth d, or Boyden chamber migration assay e. Shown are mean ± SEM from three independent experiments b-e. * P < 0.05; ** P < 0.01

Fig. 6

Sag-SCF^βTrCP modulates the PI3K/AKT/mTOR axis during mouse prostate tumorigenesis driven by Pten loss. a-c Accumulation of Phlpp1 and Deptor, and reduction of pAkt, pS6 and p4Ebp1: (a & b) Prostate tissues from mice at age of 6 month with indicated genotypes were stained with indicated Abs. Shown are representative areas of stained tissues (left panel), and the staining was analyzed using 3D Histech software and plotted (right panel). All values represent means ± SD; n ≥ 5 for each group. *p ≤ 0.05; **p ≤ 0.01. Scale bars: 100 μm. c Cell lysates from prostate tissues with indicated genotypes (4 independent samples from two genotypes, respectively) were analyzed by Western blotting with indicated Abs. d Working Model: Sag deletion blocks prostate tumorigenesis triggered by Pten loss. The PI-3 K/AKT/mTOR axis was activated upon Pten loss to trigger prostate tumorigenesis, whereas Sag deletion caused accumulation of Phlpp1 to inactivate Akt, and of Deptor to inactivate mTOR, leading the suppression of prostate tumorigenesis