Article Figures & Data
Figures
- Figure 1.
Inhibition of Raf1 does not interfere with prostate cancer growth inhibition mediated by targeting microtubules. A and B, Effect on the growth of androgen-independent PC3-M prostate cancer cells in vitro. Data are the mean ± SEM (N = 3 replicates) of cell viability in a 3-day growth assay in the presence of 10 μmol/L KBU2046 or vehicle (control) treated with docetaxel or cabazitaxel. C, Effect on the growth of androgen-independent prostate cancer cells in vivo. Mice received subcutaneous implants of PC3-M cells, were monitored until tumors were detectable (250 mm3), and then treated daily with oral 80 mg KBU2046/kg (46), intraperitoneal docetaxel 20 mg/kg weekly (doc), the combination (doc+46), or with oral and intraperitoneal vehicle (CO), beginning on day 1, and tumor size measured. Data are mean ± SEM, with N = 20 mice per cohort.
- Figure 2.
Combined Raf1 and microtubule targeting exhibits increased antimetastatic efficacy. Cohorts of N = 10 mice bearing orthotopic implants of PC3-M-luc cells were treated daily with 150 mg/kg KUB2046 (46), intraperitoneal docetaxel 7.5 mg/kg weekly (doc), the combination (doc+46), or with oral and intraperitoneal vehicle (CO). KBU2046 treatment began 3 days before implantation, and docetaxel began 1 week after. A, Lung metastasis measured by qPCR for human Alu sequences, expressed as percent control. B, Weekly IVIS imaging of tumor. C, Tumor weight. *, P < 0.05 for differences between cohorts denoted by bars. All data are mean ± SEM.
- Figure 3.
Raf1 inhibition does not affect androgen signaling or therapeutic targeting. LNCaP or VCaP cells cultured in hormone-free media were treated with vehicle control (CO), R1881 (R), KBU2046 (46), and/or enzalutamide (E). A and B, Effects on AR-responsive gene expression. PSA expression was measured by qRT/PCR, normalized to GAPDH, and expressed as a percent of control cells (N = 3 replicates); *, P < 0.05 for groups separated by bar. C and D, Effects on cell growth. Three-day growth assays were performed. Data are the number of viable cells, expressed as percent of control (N = 3 replicates); *, P < 0.05 compared with control. E, Effects on AR activation. Western blots of nuclear (N) and cytoplasmic (C) preparations of cell extracts were probed for AR, laminin B1, or α-tubulin. F and G, Effects on tumor outgrowth after ADT. LNCaP/AR-luc cells were implanted subcutaneously (N = 6 mice/cohort) or orthotopic (N = 15 mice/cohort were implanted; N = 5 yielded tumors) into castrate mice, treatment with KBU2046 began as noted, and weekly IVIS imaging performed. Data are tumor size, expressed as luminescence intensity. All data are expressed as mean ± SEM.
- Figure 4.
KBU2046 (46) inhibits osteoclast function. A, Effects on Raf1 activation. RAW 267.4 cells were treated with RANKL for 4 days, treated with 10 μmol/L KBU2046 for the indicated time periods, and Western blot for the denoted proteins performed. B, Effects on cell morphology. RAW 267.4 cells were treated with RANKL and with KBU2046 for 4 days, or not, as denoted, and stained for actin (green) and DAPI (blue); white arrow denotes an actin ring, yellow arrow a resorptive cavity. Representative immunofluorescence images are depicted. C, TRAP expression. Cells were treated as in B, stained for TRAP (denoted by presence of red), and representative images from light microscopy depicted. D, Bone degradation. RAW 267.4 cells were plated onto Osteo Assay plates, treated with RANKL for 6 days and with 1 or 10 μmol/L KBU2046 (or vehicle for controls, CO) as indicated, and bone surface area quantified. Data are the mean ± SEM (N = 3 replicates) of bone surface area, expressed as the percentage of control cells; *, P < 0.05 compared with control.
- Figure 5.
Raf1 inhibition combined with modulation of the bone microenvironment has improved systemic efficacy. A, Bone degradation. Osteo assays were performed as in Fig. 4D. Cells were treated with KBU2046 (46), ZA at 2, 10, or 20 μmol/L, with the indicated combinations, or vehicle (control; CO). Data are the mean ± SEM (N = 3 replicates); *, P < 0.05 compared with control, or between conditions denoted by bars. B, Representative photomicrographs of Osteo assay wells. Black color denotes presence of bone matrix; RL: RANKL. C–E, Cell viability, cytotoxicity, and apoptosis. RAW 267.4 cells were treated with RANKL, at day 4 they were treated with KBU2046, ZA at 1 or 10 μmol/L (ZA1 or ZA10), the indicated combination, or vehicle (CO), and Triplex assay performed 24, 48, or 72 hours later, per Materials and Methods. Data are the mean ± SEM (N = 4 replicates); ¥, *, and # denotes P < 0.05 compared with 24, 48, and 72 hour timepoint controls, respectively. F–H, In vivo efficacy. Four cohorts of mice, N = 15/cohort, were given intracardiac injections of PC3-luc cells, yielding the following successful injections: N = 12 control, N = 12 ZA, N = 13 KBU2046, and N = 13 ZA+KBU2046. Treatment was oral 150 mg KBU2046/kg daily (starting 3 days before intracardiac injection), intraperitoneal ZA 100 μg/kg weekly (starting 1 week before intacardiac injection), with oral and intraperitoneal vehicle controls given as indicated. F, Representative CT images. Image types are side view and coronal sections of skull, and axil and sagittal sections of the femurs. Red arrows denote areas of bone destruction. G and H, Quantification of bone lesions. The macroscopic lesions in the jaw were quantified by direct measurement from CT images, and expressed as the mean ± SEM percent of control. Bone density of femurs was measured from CT images, and expressed as the mean ± SEM percent of control. *, P < 0.05 compared with control, and for groups indicated by bars.
Additional Files
Supplementary Data
- Supplementary Fig S1 - Supplementary Fig S1 shows that KBU2046 does not interfere with chemotherapy efficacy
- Supplementary Fig. S2 - Supplementary Fig. S2 shows that docetaxel inhibits human prostate cancer growth in mice in a dose-responsive fashion
- Supplementary Fig. S3 - Supplementary Fig. S3 shows that PC3-M-luc cells can be quantified in mouse lung by qPCR
- Supplementary Fig. S4` - Supplementary Fig. S4 shows that KBU2046 inhibits Raf1 signaling in osteoclasts
- Supplementary Fig. S5 - Supplementary Fig. S5 shows that small molecule Raf1 inhibitors inhibit osteoclast function.
- Supplementary Fig. S6 - Supplementary Fig. S6 shows weekly IVIS signals in mice bearing human prostate cancer and treated with zoledronic acid and/or KBU2046
Volume 19, Issue 1
