Role of IGF-1/IGF-1R in regulation of invasion in DU145 prostate cancer cells

Abstract

Background: Prostate cancer progression to androgen independence is the primary cause of mortality by this tumor type. The IGF-1/IGF-1R axis is well known to contribute to prostate cancer initiation, but its contribution to invasiveness and the downstream signalling mechanisms that are involved are unclear at present.

Results: We examined the invasive response of androgen independent DU145 prostate carcinoma cells to IGF-1 stimulation using Matrigel assays. We then examined the signaling mechanisms and protease activities that are associated with this response. IGF-1 significantly increased the invasive capacity of DU145 cells in vitro, and this increase was inhibited by blocking IGF-1R. We further demonstrated that specific inhibitors of the MAPK and PI3-K pathways decrease IGF-1-mediated invasion. To determine potential molecular mechanisms for this change in invasive capacity, we examined changes in expression and activity of matrix metalloproteinases. We observed that IGF-1 increases the enzymatic activity of MMP-2 and MMP-9 in DU145 cells. These changes in activity are due to differences in expression in the case of MMP-9 but not in the case of MMP-2. This observation is corroborated by the fact that correlated changes of expression in a regulator of MMP-2, TIMP-2, were also seen.

Conclusion: This work identifies a specific effect of IGF-1 on the invasive capacity of DU145 prostate cancer cells, and furthermore delineates mechanisms that contribute to this effect.

Figure 1

IGF-1 stimulates the in vitro invasion of DU145 cells through the IGF-1R, via both the PI3-K and MAPK pathways. A) Representative experiment showing number of cells invading a Matrigel-coated membrane relative to surface area. Serum-deprived DU145 cells were treated for 24 hours with indicated concentrations of IGF-1 after which 5 × 10⁴ cells were allowed to invade through the Matrigel for 24 hours. IGF-1 treatment induces a dose-responsive increase in the invasive potential of DU145 cells through Matrigel compared to invasion in mock-treated cells that were administered a volume of 1× PBS similar to the 200 ng/ml condition. (*p < 0.05; Fisher exact T-test). B) Number of cells invading a Matrigel-coated membrane relative to surface area. Serum-deprived DU145 cells were pretreated for 24 hours with a neutralizing IGF-1R antibody (IGF-1Rab), then treated with 200 ng/ml IGF-1 for 24 hours. 5 × 10⁴ cells were allowed to invade through the Matrigel for 24 hours. The increase in Matrigel invasion of DU145 cells stimulated by IGF-1 is significantly decreased in the presence of the IGF-1R neutralizing antibody (*p < 0.05; Fisher exact T-test) The control consisted of adding a similar amount of the vehicle (1× PBS) for each addition in the test conditions. C,D,E,F) Serum-deprived DU145 cells were pre-treated in the presence or absence of the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059 for 1 hour, then treated with 200 ng/ml IGF-1 for 1 hour, maintaining previous inhibitor conditions. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. C) Immunoblotting shows an increase in Akt phosphorylation in DU145 cells treated with IGF-1, but not in the presence of wortmannin. D) Immunoblotting shows an increase in the phosphorylation of p42/44 MAPK in DU145 cells treated with IGF-1, but not in the presence of PD98059. E,F) Representative experiments showing number of cells invading a Matrigel-coated membrane relative to surface area. At the end of the respective treatments, 5 × 10⁴ cells were added to each invasion chamber and allowed to invade through the Matrigel for 24 hours. E) Each condition was performed on three separate occasions. The relative rate of inhibition by wortmannin of the IGF-1 effect on invasion was consistent across experiments, with a mean of 0.79 +/- 0.083. F) Each condition was performed on four separate occasions. The relative rate of inhibition by PD98059 of the IGF-1 effect on invasion was consistent across experiments, with a mean of 0.37 +/- 0.096.

Figure 2

IGF-1 induces the activity of MMP-9 and MMP-2 via both PI3-K and MAPK pathways. Serum-deprived DU145 cells were pre-treated for 1 hour with either the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059, then treated with IGF-1 for 24 hours in the same conditions of inhibitor use. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. Conditioned media was concentrated, normalized to cell number and used for gelatin zymography. Protease activity is seen as clear digested bands at 92 kDa for MMP-9, at 72 kDa for MMP-2 and at 52 kDa for MMP-1 (A). Densitometric quantification indicates that MMP-9 activity is increased by IGF-1, with this increase prevented by either wortmannin or PD98059 (B). MMP-2 activity is also induced by IGF-1; presence of either inhibitor completely abrogates this activation (C). The activity of MMP-1 is unaffected by IGF-1 both in the absence and presence of wortmannin or PD98059 (D).

Figure 3

IGF-1 regulates the activity of MMP-9 and MMP-2 by different mechanisms. A, B) DU145 cells were treated with IGF-1 for varying amounts of time and both cell lysate and conditioned media were collected. A) Immunoblot of cell lysates indicates that IGF-1 induces an increase in the cellular protein expression of MMP-9 in a time-dependent manner at 8 and 24 hours of treatment, then a decrease at 32 and 48 hours of treatment. B) Immunoblot of conditioned media indicates that IGF-1 induces an increase in the secreted protein expression of MMP-9 in a time-dependent manner seen after 32 hours of treatment. The amount of protein in each lane is normalized to cell number. C, D)DU145 cells were pre-treated with the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059, then treated with IGF-1 for 8 hours and both cell lysate and conditioned media were collected. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. C) Immunoblot of lysates indicates that the IGF-1-induced increase in the cellular protein expression of MMP-9 is attenuated in the presence of PD98059 and even more so in the presence of wortmannin. No change in MMP-2 expression is seen in any of the conditions. D) Protein expression of MMP-2 in conditioned media is unaltered compared to mock-treated control cells, whereas that of MMP-9 follows the same pattern seen in cell lysates. The amount of protein in each lane is normalized to cell number. All blots are representative of three separate experiments in which a similar trend was observed.

Figure 4

IGF-1 regulates secreted TIMP levels. DU145 cells were pre-treated for 1 hour with either the PI3-K inhibitor wortmannin or the MEK inhibitor PD98059, then treated with IGF-1 for 24 hours in the same conditions of inhibitor use. The control consisted of adding a similar amount of the vehicle (1× PBS for IGF-1, DMSO for wortmannin or PD98059) for each addition in the test conditions. Conditioned media from these treatments were concentrated and probed with (A) anti-TIMP-2 antibody or (B) anti-TIMP-1 antibody. The amount of protein in each lane is normalized to cell number. Blots are representative of three separate experiments in which a similar trend was observed.