Targeting IGF-IR with ganitumab inhibits tumorigenesis and increases durability of response to androgen-deprivation therapy in VCaP prostate cancer xenografts

Abstract

Prostate cancer is the most commonly diagnosed malignancy in men. While tumors initially respond to androgen-deprivation therapy, the standard care for advanced or metastatic disease, tumors eventually recur as castration-resistant prostate cancer (CRPC). Upregulation of the insulin-like growth factor receptor type I (IGF-IR) signaling axis drives growth and progression of prostate cancer by promoting proliferation, survival, and angiogenesis. Ganitumab (formerly AMG 479) is a fully human antibody that inhibits binding of IGF-I and IGF-II to IGF-IR. We evaluated the therapeutic value of ganitumab in several preclinical settings including androgen-dependent prostate cancer, CRPC, and in combination with androgen-deprivation therapy. Ganitumab inhibited IGF-I-induced phosphorylation of the downstream effector AKT and reduced proliferation of multiple androgen-dependent and castration-resistant human prostate cancer cell lines in vitro. Ganitumab inhibited androgen-dependent VCaP xenograft growth and increased tumor-doubling time from 2.3 ± 0.4 weeks to 6.4 ± 0.4 weeks. Ganitumab blocked growth of castration-resistant VCaP xenografts for over 11.5 weeks of treatment. In contrast, ganitumab did not have appreciable effects on the castration-resistant CWR-22Rv1 xenograft model. Ganitumab was most potent against VCaP xenografts when combined with complete androgen-deprivation therapy (castration). Tumor volume was reduced by 72% after 4 weeks of treatment and growth suppression was maintained over 16 weeks of treatment. These data suggest that judicious use of ganitumab particularly in conjunction with androgen-deprivation therapy may be beneficial in the treatment of prostate cancer.

Figure 1. Analysis of ganitumab effects on IGF-1R signaling and prostate cancer cell proliferation in vitro.

VCaP and 22Rv1 were cultured in serum deprived media for 24 hours, and treated with ganitumab [0 nM, 10 nM, 100 nM, 1000 nM] for 90 minutes prior to treatment with 1nM IGF-1 for 30 minutes. Lysates were probed by western blot for phospho-AKT and total-AKT in VCaP (A) and 22Rv1 (B). The effects of seven day ganitumab treatment (0–1000 nM) on prostate cancer cell proliferation in 2% FBS containing media [(C), 22Rv1 (D), CWR-R1 (E), and LNCaP (F)] were determined. (G) VCaP, 22Rv1, CWR-R1 and LNCaP were cultured in medium containing 2%FBS for 72 hours with 500 nM ganitumab or control. Lysates were immunoblotted for cleaved PARP and actin. (C–E) are representative of at least 3 experiments performed in triplicate. (F) Represents 3 combined experiments. (G) is representative of 2 independent experiments. All data are shown ± SD. (*p<.05, **<.01, ***<.001, Two-tailed Student’s t-test)

Figure 2. Ganitumab inhibits the growth of androgen-dependent VCaP xenografts

VCaP xenografts were established in intact (androgen-replete) mice. When tumors reached an average volume of 260–320 mm³, treatment with ganitumab or control antibody was initiated. (A) Tumor volumes (mm³) are shown relative to treatment initiation (n=3–4 for control; n=4–8 for ganitumab). (B) Tumor doubling times for control and ganitumab treated mice are shown (n=4 for control; n=8 for ganitumab). (C) Serum PSA doubling times are shown (n=4 for control; n=8 for ganitumab). (D) Xenograft lysates were immunoblotted for IGF-1R, INSR, and actin for control-treated and ganitumab treated tumors. Western blots were quantified by densitometry and relative IGF-1R (E) and relative INSR (F) normalized to actin are shown. All data are displayed ± SEM. (*p<.05, **<.01, Two-tailed Student’s t-test)

Figure 3. Ganitumab inhibits the growth of castration-resistant VCaP xenografts

Castration-resistant VCaP xenografts were established and mice were treated with control antibody beginning one week after castration. Five weeks after castration with an average tumor volume of 538 mm³, mice were randomized into 2 groups and treatment with ganitumab was initiated (Control → Ganitumab) or control antibody was continued (Control → Control). (A) Tumor volumes (mm³) are shown relative to time of castration (n=4–5 for control; n=3–4 for ganitumab). (B) Serum PSA (ng/mL) levels are shown (n=18 for pre-treatment; n=3–4 for control; n=3 for ganitumab). (C) Xenograft lysates were immunoblotted for IGF-1R, INSR, and actin for Control → Control and Control → Ganitumab tumors. Blots were quantified by densitometry and relative IGF-1R (D) and relative INSR (E) normalized to actin are shown. All data are displayed ± SEM. (*p<.05, Two-tailed Student’s t-test)

Figure 4. Ganitumab does not affect the growth of well-established castration-resistant 22Rv1 xenografts

22Rv1 xenografts were established in castrated nude mice. When tumors reached an average volume of 260–320 mm³ treatment with ganitumab or control antibody was initiated. (A) Tumor volumes are shown for ganitumab and control treated mice (n=4–14 for control, n=3–14 for ganitumab). (B) Xenograft lysates were immunoblotted for IGF-1R, INSR, and actin. (C) Blots were quantifed by densitometry and relative IGF-1R levels (C) and relative INSR levels (D) normalized to actin are shown. All data are displayed ± SEM. (*p<.05, Two-tailed Student’s t-test)

Figure 5. Combining ganitumab with androgen-deprivation therapy most effectively inhibits VCaP xenografts

VCaP xenografts were established in intact mice, and mice were castrated when tumors reached an average volume of 260–320 mm³. One week after castration mice were randomized into 2 groups and treatment with ganitumab or control antibody was initiated. (A) Tumor volumes (mm³) are shown relative to time of castration for control and ganitumab treated mice (n=4–14 for control; n=5–16 for ganitumab) (control-treated tumor volumes are those from figure 3a). (B) Weeks observed without recurrences were calculated and are shown above (n=11 for control; n=5 for ganitumab) (C) Serum PSA (ng/mL) is shown (n=3–13 for control; n=5–11 for ganitumab). Relative IGF-1R (D) INSR (E) protein levels detected by MSD multiplex analysis are shown for intact (pre-castrate baseline), control-treated, and ganitumab-treated mice relative to time of castration (n=3~5 per group). All data are displayed ± SEM. (*p<.05, ***<.001, Two-tailed Student’s t-test)

Figure 6. Evaluation of durability and reversibility of combined ganitumab and androgen-deprivation therapy on VCaP xenografts

VCaP xenografts were established in intact mice, and mice were castrated when tumors reached an average volume of 260–320 mm³. Beginning one week after castration mice were treated with ganitumab for four weeks. Mice were randomized into 2 groups and treatment with control antibody was initiated or ganitumab was continued. (A) Discontinuation of ganitumab after 4 weeks (5 weeks post castration) resulted in recurrence of all VCaP tumors (n=4) († – deceased mouse due to causes unrelated to treatment). (B) Long-term treatment with ganitumab resulted in recurrence in 2 of 5 tumors. (Average tumor volume is reflected in figure 5a.) (C) Quantification of recurrence after ganitumab discontinuation (reversibility) and long-term ganitumab treatment (durability) is shown.