Inhibition of androgen-independent prostate cancer cell growth is enhanced by combination therapy targeting Hedgehog and ErbB signalling

Abstract

Background: Prostate cancer is a leading cause of male cancer specific mortality. When cure by radical prostatectomy is not possible the next line of prostate cancer treatment is androgen deprivation. However prolonged androgen deprivation often results in relapse and androgen-independent prostate cancer that is inevitably fatal despite optimal chemotherapy. The Hedgehog signalling pathway has recently been implicated in prostate cancer development and metastasis. EGFR or ErbB2 expression has been also correlated with androgen independence, shorter survival and metastasis.

Results: We determined that the Hedgehog and ErbB signalling pathways are active in circulating tumour cells isolated from androgen-independent prostate cancer patients and in the androgen-independent prostate cancer cell line LNCaP C4-2B. As a basis for synergistic chemotherapy protocols combinations of the Hedgehog specific inhibitor cyclopamine and the ErbB signalling inhibitors gefitinib or lapatinib were tested in this study. Androgen-independent prostate cancer cell growth was inhibited by a SMO inhibitor (cyclopamine) which blocks Hedgehog signalling and by ErbB inhibitors (gefitinib and lapatinib). The isobologram and combination index method of Chou and Talalay was used to evaluate drug interactions. Synergistic antiproliferation effects were observed when the Hedgehog and ErbB inhibitors were combined.

Conclusion: Androgen-independent prostate cancer cell proliferation was associated with activity of the Hedgehog and ErbB signalling pathways. Cyclopamine, gefitinib or lapatinib treatment significantly decreased the proliferation of androgen-independent prostate cancer cells. The Hedgehog pathway therefore represents a promising new therapeutic target in androgen-independent prostate cancer. Synergistic effects were observed when Hedgehog and ErbB inhibitors were used together. This study may have clinical implications for improving the treatment of advanced prostate cancer.

Figure 1

Circulating tumour cells isolated from AIPC patients are EpCAM, PTCH, ErbB2 and EGFR positive. (A) EpCAM, PTCH, ErbB2 and EGFR immunofluorescence in circulating tumour cells isolated from AIPC patients. (B) Relative PTCH, EGFR, ErbB2 and DD3^PCA3 RNA expression in circulating tumour cells isolated from normal subjects and androgen-independent prostate cancer patients.

Figure 2

Expression of PTCH, GLI1, ErbB2, EGFR, PSA and AR RNA in LNCaP C4-2B cells.

Figure 3

Effect of (A) cyclopamine, (B) gefitinib and (C) lapatinib on growth of androgen-independent prostate cancer cells.

Figure 4

(A) Cyclopamine inhibits expression of PTCH and GLI1 RNA in LNCaP C4-2B cells: control cells (lanes 1 and 3); 24 hrs 14 nM cyclopamine treatment (lanes 2 and 4). Relative expression determined by QPCR (error bars show standard deviation). (B) Immunoblot showing inhibitory effect of 168 nM gefitinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr gefitinib; lane 3: 48 hr gefitinib). (C) Immunoblot showing inhibitory effect of 102 nM lapatinib on EGFR phosphorylation following EGF treatment in AIPC cells (lane 1: control; lane 2: 24 hr lapatinib; lane 3: 48 hr lapatinib).

Figure 5

Concentration effect curves of (A) single agent cyclopamine and gefitinib and their combinations, (B) single agent cyclopamine and lapatinib and their combinations.

Figure 6

Analysis of cyclopamine and (A-B) gefitinib or (C-D) lapatinib in LNCaP C4-2B cells. (A and C) combination index plot for the drug combinations. (B and D) Isobologram for the combination of gefitinib or (C-D) lapatinib and cyclopamine for effect level (F_a = 0.5). Note the combination data points fall on the lower left of the hypotenuse for F_a = 0.5 (shown), F_a = 0.75 and F_a = 0.9 indicating synergism.