Activation of Rap1 promotes prostate cancer metastasis

Abstract

Elucidating the mechanisms of prostate cancer (CaP) survival and metastasis are critical to the discovery of novel therapeutic targets. The monomeric G protein Rap1 has been implicated in cancer tumorigenesis. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting Rap1 may promote several processes associated with cancer cell metastasis. Examination of CaP cell lines revealed cells with a high metastatic ability exhibited increased Rap1 activity and reduced expression of the negative regulator Rap1GAP. Rap1 can be further stimulated in these cells by stromal-derived factor (SDF-1), an agonist known to regulate tumor cell metastasis and tropism to bone. Activation of Rap1 increased CaP cell migration and invasion, and inhibition of Rap1A activity via RNAi-mediated knockdown or ectopic expression of Rap1GAP markedly impaired CaP cell migration and invasion. Additional studies implicate integrins alpha4, beta3, and alphavbeta3 in the mechanism of Rap1-mediated CaP migration and invasion. Extending the effect of Rap1 activity in CaP metastasis in vivo, introduction of activated Rap1 into CaP cells dramatically enhanced the rate and incidence of CaP metastasis in a xenograft mouse model. These studies provide compelling evidence to support a role for aberrant Rap1 activation in CaP progression, and suggest that targeting Rap1 signaling could provide a means to control metastatic progression of this cancer.

Figure 1. Rap1 activity is increased in progressively metastatic human prostate cancer cell lines

Top panel: Cell lysates (roughly in order of increasing metastatic potential, from left to right) were analyzed in a pull-down assay using a GST fusion of the activated Rap1-binding domain of RalGDS. The levels of precipitated Rap1 were determined by immunoblot analysis using an anti-Rap1 antibody. Levels of total Rap1, Rap1GAP, and actin (loading control) were also determined. Data is from a single experiment that is representative of three separate experiments.

Figure 2. Modulation of Rap1 activity impacts CaP cell migration and invasion

Panels A and B. PC3 cells were transduced with retrovirus to establish stable expression of the indicated constructs--vector, Rap1-63E, or Rap1GAP. A, PC3 cells stably expressing the indicated constructs were lysed and levels of Rap1-GTP, total Rap1 and Rap1GAP determined by immunoblot analysis (as described in legend to Fig 1)). B, Migration (left panel) and invasion (right panel) of PC3 cells stably expressing the indicated constructs. For the migration assays, cells migrated toward a lower chamber containing conditioned media from osteosarcoma cells and were assessed after 4 h. For the invasion assays, cells invaded through a Matrigel layer toward a lower chamber containing 10% serum and were assessed after 24 h. Panels C and D. siRNAs targeting Rap1A or control siRNA, were introduced into PC3 cells and parameters assessed 72 h later. C, Levels of Rap1 and actin (loading control) were determined by immunoblot analysis. D, Migration (left panel) and invasion (right panel) assays of siRNA treated PC3 cells. The data represents that pooled from 3 (B) or 2 (D) independent experiments with at least 2 replicates each, and is plotted as the ratio of the number of migrated or invaded cells vs. that of control cells (B, vector; D, scrambled siRNA) that were determined in parallel. ***, p<0.001; **, p<0.005

Figure 3. SDF-1 stimulation of CaP cell migration and invasion requires Rap1 function

In all panels SDF-1 treatment was at 200ng/ml. A, PC3 cells stably expressing vector control or Rap1GAP were treated with SDF-1 or 10% FBS (control) for the time indicated. Cell lysates were analyzed for Rap1 activation as described in the legend to Fig. 1. The levels of Rap1-GTP, total Rap1, and Rap1GAP were determined by immunoblot analysis. B, Migration of PC3 cells stably expressing the indicated constructs and treated with SDF-1 for 4 h. C, Invasion of PC3 cells stably expressing the indicated constructs and treated with SDF-1 for 24 h. D, Invasion of PC3 cells treated with siRNA to Rap1A for 72 h and treated with SDF-1 for 24 h. Migration and invasion of the cells was evaluated as described in the legend to Fig 2. Data represents that pooled from two independent experiments with at least 2 replicates each, and is plotted as the ratio of the number of migrated or invaded cells vs. that of vehicle-treated control cells (B,C vector; D, scrambled siRNA) determined in parallel. ***, p<0.001; **, p<0.005; *, p<0.05.

Figure 4. Rap1 promotes CaP migration and invasion through integrins

In all panels, PC3 cells stably expressing the indicated constructs were treated with specific blocking antibodies (20 µg/ml) for integrins αvβ3, β3, and α4, and migration and invasion of the cells evaluated as described in the legend to Fig 2. Panels A and B. Migration of vector control cells (A) or Rap1-63E expressing cells (B) treated with antibodies for 4 h. Panels C and D. Invasion of vector control cells (C) or Rap1-63E expressing cells (D) treated with antibodies for 24 h. For all panels, data represents that pooled from two independent experiments with at least two replicates each and is plotted as ratio of the number of the migrated or invaded cells vs. the number of migrated or invaded vehicle-treated control cells (labeled none) determined in parallel. ***, p<0.001; **, p<0.005.

Figure 5. Activation of Rap1 promotes CaP cell metastasis in vivo

The metastasis assay was performed by intracardiac injection of 1 × 10⁵ PC3-Luc cells expressing either vector control or Rap1-63E into the left cardiac ventricle of anesthetized 4 to 6-week-old athymic male mice. At approximately weekly intervals, the mice were IP injected with luciferin, and imaged to visualize the metastatic dissemination of the PC3-Luc cells A, Mice day 22 post-intracardiac injection of PC3Luc-vector (left panel) or PC3Luc-Rap1-63E cells (right panel). Bone metastasis of the cells was detectable by bioluminescence in the tibia, mandible, and thoracic cavity of the mice. Images are pseudo-color representations of the mean bioluminescence detected. Graphic representation of the B, percentage of mice that were metastasis free. C, average number of metastatic events observed per day. D, number of metastatic lesions per mouse at the conclusion of the study (day 30 post-intracardiac injection). Figure represents vector control mice (N=5) and Rap1-63E mice (N=6). ***, p<0.001; *, p<0.05.