Ruoqian Shen

Taxane-Induced Blockade to Nuclear Accumulation of the Androgen Receptor Predicts Clinical Responses in Metastatic Prostate Cancer

Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired β-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy.

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

Synergy in tumor suppression by direct interaction of Neutral Endopeptidase with PTEN

We show in this study that endogenous NEP and PTEN associate in cells directly through electrostatic interactions between a highly basic residue stretch in the intracellular domain of NEP and the major phosphorylation site in PTENs tail. NEP binds and engages in higher order complexes both phosphorylated and unphosphorylated PTEN. NEP recruits PTEN to the plasma membrane and enhances its stability and phosphatase activity. As a result, an enzymatically inactive NEP mutant preserves the ability to bind PTEN, inactivates the Akt/PKB kinase, and partially suppresses the growth of PC cells. This study demonstrates a molecular cooperation between NEP and PTEN tumor suppressors in which NEP constitutively recruits and activates PTEN to inhibit the PI3K/Akt oncogenic pathway.

Neprilysin inhibits angiogenesis via proteolysis of fibroblast growth factor-2.

Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.01) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis.

Loss of neutral endopeptidase and activation of protein kinase B (Akt) is associated with prostate cancer progression

Neutral endopeptidase (NEP) is a cell‐surface peptidase that can regulate the activation of Akt kinase through catalytic‐dependent and independent mechanisms. NEP expression is absent in approximately 50% of prostate cancers. The authors investigated whether NEP loss in vivo would result in Akt phosphorylation and potentially contribute to prostate cancer progression by examining the interaction of NEP, Akt, and phosphatase and tensin homolog (PTEN) in a prostate xenograft model and in clinical specimens from patients with prostate cancer.

Endothelin-1 enhances the expression of the androgen receptor via activation of the c-myc pathway in prostate cancer cells.

Increasing evidence suggests that androgen independent prostate cancer (PC) maintains a functional androgen receptor (AR) pathway despite the low levels of circulating androgen following androgen withdrawal, the molecular mechanisms of which are not well defined yet. To address this question, we investigated the effects of endothelin‐1 (ET‐1) on AR expression. Western analysis and RT‐PCR revealed that in the presence of ET‐1, levels of AR significantly increased in a time‐ and dose‐dependent manner in LNCaP cells. Pretreatments with inhibitors of Src and phosphoinositide kinase 3 (PI‐3K) suppressed ET‐1‐induced AR expression. As ET‐1 was reported to cause a transient increase in c‐myc mRNA levels, we examined the involvement of c‐myc in ET‐1‐mediated AR expression. Transient transfection of c‐myc siRNA neutralized ET‐1‐induced AR expression, suggesting that AR induction by ET‐1 is c‐myc dependent. AR can regulate the transcription of its own gene via a mechanism in which c‐myc plays a crucial role. Therefore, we assessed if ET‐1‐induced‐c‐myc leads to the enhancement of AR transcription. Reporter gene assays using the previously identified AR gene enhancer containing a c‐myc binding site were conducted in LNCaP cells. We found that ET‐1 induced reporter gene activity from the construct containing the wild‐type but not mutant c‐myc binding site. Chromatin immunoprecipitation assays confirmed that ET‐1 increased interaction between c‐myc and c‐myc binding sites in AR enhancer, suggesting that ET‐1‐induced AR transcription occurs via c‐myc‐mediated AR transcription. Together, these data support the notion that ET‐1, via Src/PI‐3K signaling, augments c‐myc expression leading to enhanced AR expression in PC.

Inactivation of the NF2 tumor suppressor protein merlin in DU145 prostate cancer cells.

The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact‐dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21‐activated kinase (PAK) inactivates merlins growth suppressing function, and is regulated by cell‐culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells.

Interaction of retinoic acid and interferon in renal cancer cell lines.

Retinoic acid (RA) can potentiate the antitumor effect of interferons (IFN) in a variety of tumor types, including renal cell carcinoma (RCC). The mechanisms by which RA and IFN increase the antitumor effects in RCC are unknown. We used growth assays and mobility shift assays to examine the effects of combining 13-cis-retinoic acid (CRA) and IFN-alpha (plus IFN-gamma) on proliferation and on the expression of the IFN-specific transcription factor IFN-stimulated gene factor 3 (ISGF3) in RCC cell lines. Combining CRA and IFN-alpha resulted in a significant increase in growth inhibition in four cell lines compared with IFN-alpha or CRA alone. Binding of nuclear extracts from RCC cells to an IFN-stimulated response element (ISRE) oligonucleotide probe following incubation with IFN-alpha was not increased by CRA but was significantly increased by pretreatment by IFN-gamma in a time-dependent fashion. Proliferation assays showed that sequential addition of IFN-gamma and IFN-alpha significantly increased growth inhibition. IFN-alpha but not IFN-gamma or CRA increased the cellular levels Stat2 and p48 but not Statl. IFN-gamma pretreatment enhanced the upregulation of p48 levels by IFN-alpha. Combining RA and IFN results in additive growth inhibition on RCC cell lines. This increase in growth inhibition is not mediated by increased ISGF3 expression.

Neutral endopeptidase inhibits prostate cancer tumorigenesis by reducing FGF-2-mediated angiogenesis.

Neutral endopeptidase (NEP) is a cell surface peptidase that catalytically inactivates a variety of physiologically active peptides including basic fibroblast growth factor (FGF-2). We investigated the effect of using lentivirus to overexpress NEP in NEP-deficient DU145 prostate cancer cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP), catalytically inactive mutant NEP (L-NEPmu), and green fluorescent protein (L-GFP) were stably introduced into DU145 cells. FGF-2 levels in cell culture supernatants decreased by 80% in L-NEP-infected DU145 cells compared to cells infected with L-NEPmu or L-GFP (P<0.05) while levels of other angiogenic factors were not altered. In vitro tubulogenesis of human vascular endothelial cells induced by conditioned media from DU145 cells infected with L-NEP was significantly reduced compared with that from DU145 cells infected with L-GFP (P<0.05). Tumor xenografts from L-NEP-infected DU145 cells were significantly smaller compared to control cell xenografts and vascularity within these tumors was decreased (P<0.05). Our data suggest that stable expression of NEP in DU145 cells inhibits prostate cancer tumorigenicity by inhibiting angiogenesis, with a probable mechanism being proteolytic inactivation of FGF-2.

Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein–protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2±4.9 and 17.2±5.3 pmol/μg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.