Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium

Abstract

GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.

Fig. 1.

Homozygous deletion of Grp78 does not affect prostate development. (A) Schematic representations for the Grp78 cDNA, the wild type allele (+), the f allele, and the knockout allele (−). The exons encoding the ATPase domain and peptide-binding domain of GRP78 and the loxP sites (arrow head) are indicated. The position and length of the PCR product for each allele are indicated. (B) PCR analysis of tail DNA from Grp78^f/f, cGrp78^f/f and cGrp78^+/+ mice. (C) The gross anatomy of the prostate of Grp78^+/+ and cGrp78^f/f mice at 20 wk. AP, anterior prostate; DLP, dorsolateral prostate; B, bladder; SV, seminal vesicle. (D) From left to right, images of the gross anatomy of the prostate of Grp78^+/+ and cGrp78^f/f mice at 30 wk; immunohistochemical staining of GRP78 (depicted in brown) of DLP section, H&E staining of the DLP sections in different magnifications.

Fig. 2.

Prostate specific homozygous deletion of Grp78 blocks prostate tumorigenesis resulting from PTEN inactivation. (A) The cPten^f/fGrp78^f/+, cPten^f/fGrp78^f/f, and cPten^f/fGrp78^f/− male mice were genotyped by PCR analysis. (B) The gross anatomy of the prostate of the mice with the indicated genotypes at 20 wk. All lobes of cPten^f/fGrp78^+/+ mice formed solid tumor mass. In cPten^f/fGrp78^f/+ mice, only AP developed solid tumor mass. The cPten^f/fGrp78^f/f and cPten^f/fGrp78^f/− mice are normal and cancer free. AP, anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate; B, bladder; SV, seminal vesicle. (C) Histological comparison of H&E staining prostate sections of the mice in B. The cPten^f/fGrp78^+/+ mice developed invasive adenocarcinoma in all prostate lobes. In cPten^f/fGrp78^f/+ mice, AP had extensive invasive adenocarcinoma, whereas DLP and VP only developed diffusive prostate intraepithelial neoplasia (PIN). All lobes of the cPten^f/fGrp78^f/f, and cPten^f/fGrp78^f/− mouse prostates were normal. (D) The distribution of prostate lobes in 3 histological states, normal, PIN, and adenocarcinoma, for each genotype: Pten^f/fGrp78^f/f (I), cPten^f/fGrp78^+/+ (II), cPten^f/fGrp78^f/+ (III), cPten^f/fGrp78^f/f (IV), and cPten^f/fGrp78^f/− (V).

Fig. 3.

Knockout of GRP78 Inhibits the AKT activation. (A) Immunofluorescence staining of PTEN and GRP78 in the DLP of mice with the indicated genotypes. The stromal cells in mutant prostates remained PTEN positive (white arrow), which confirmed the PB-Cre4 mediated deletion is prostate epithelium specific. (B) Immunofluorescence staining of p-AKT (Ser-473) and the double staining of p-AKT and PTEN of prostate sections of the mice in A. Staining for p-AKT was strong in the prostate epithelium of cPten^f/fGrp78^+/+ mice, decreased in the cPten^f/fGrp78^f/+ mice, and further decreased in the cPten^f/fGrp78^f/f and cPten^f/fGrp78^f/− mice. (C) GRP78 knockdown inhibits AKT phosphorylation. Human prostate cancer cell line PC3 was transfected with siRNA against GRP78 (siGrp78) or control siRNA (siCtrl), and then treated with 300-nM Tg. Cells were harvested at the time points (in hours) as indicated and subjected to Western blotting. (D) The ratio of p-AKT to total AKT level in C was quantitated. The ratio at the 0 h time point in cells transfected with siCtrl was set as 1.