A Novel Interplay between Rap1 and PKA Regulates Induction of Angiogenesis in Prostate Cancer

<div><p>Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.</p> </div

Schematic depicting interplay between Rap1 and PKA in the tumor microenvironment.

<p>Schematic depicting interplay between Rap1 and PKA in the tumor microenvironment.</p

Effect of SDF-1α on secreted VEGF in prostate cancer cells under hypoxic-like conditions.

<p>Cells were treated as indicated and VEGF levels were measured by ELISA as described in Methods. (A and B) PC3 or PC3-cRap1 cells; *p<0.05, n = 3. (C and D) LNCaP or LNCaP-cRap1 cells; *p<0.05, n = 2.</p

Effect of PKA inhibitor on tumor growth and angiogenesis in PC3-cRap1 xenografts.

<p>(A and B) The PKA inhibitor, H-89 (2.5 µmol), reversed tumor growth inhibition and tumor weight reduction mediated by 8CPT in PC3-cRap1 xenografts. PC3-cRap1 tumors from mice infused with PBS, 8CPT, or H-89 were measured and tumor volume determined as described in Methods; * p<0.05; n = 8 for each treatment group. (C) CD31 and VEGF immunoreactivity in PC3-cRap1 tumors treated as indicated. Immunostaining was measured as described in Methods. <i>Upper Panels</i>: Representative photomicrographs. <i>Lower Panels</i>: Quantification of immunohistochemical staining of tumor slices; *p<0.05; n = 8 for each treatment group; a.u. = arbitrary units.</p

Effect of 8CPT treatment on HIF-1α and VEGF in PC3 and PC3-cRap1 cells under hypoxic-like conditions.

<p>(A) VEGF levels were measured by ELISA in PC3 and PC3-cRap1 cells under hypoxic-like conditions (CoCl₂) as described in Methods; *p<0.05, (n = 3). (B) Immunoblot analysis of nuclear extracts from PC3 and PC3-cRap1 cells treated as indicated. HIF-1α protein levels were determined by immunoblotting with specific antibody; (C) Analysis of VEGF mRNA levels by qPCR in PC3 and PC3-cRap1 cells treated as indicated. Data were normalized to GAPDH levels; *p<0.05 (<i>n</i> = 3).</p

Effect of constitutive Rap1 expression (cRap1) and/or 8CPT treatment on PC3 xenograft growth.

<p>(A and B) <i>Left Panel</i>: Tumor volume (expressed in mm³) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049893#s2" target="_blank">Methods</a>. Day 0 represents the first day of treatment; *p<i><</i>0.05, n = 7 for each group. <i>Right Panel</i>: Images of a representative tumor from each treatment group at the end of the experiment (Day 28). (C) Tumor volume (expressed in mm³) of human PC3 and PC3-cRap1 xenografts treated as indicated was measured as in A; *p<i><</i>0.05, n = 7 for each group. (D) Tumors from panel C were weighed at the end of the experiment (Day 28) and expressed in grams; *p<0.05; (<i>n</i> = 7).</p