Inhibition of STAT3 with orally active JAK inhibitor, AZD1480, decreases tumor growth in Neuroblastoma and Pediatric Sarcomas In vitro and In vivo

Abstract

The IL-6/JAK/STAT pathway is a key signal transduction pathway implicated in the pathogenesis of many human cancers, suggesting that kinase inhibitors targeting JAK/STAT3 may have a broad spectrum of antitumor activity. AZD1480, a pharmacological JAK1/2 inhibitor, exhibits anti-tumor potency in multiple adult malignancies. To evaluate the efficacy of inhibition of JAK/STAT3 signal transduction pathway we assessed the activity of AZD1480 in pediatric malignancies using preclinical models of three highly malignant pediatric solid tumors: neuroblastoma (NB), rhabdomyosarcoma (RMS) and the Ewing Sarcoma Family Tumors (ESFT). In this study, we employed panels of biomedical and biological experiments to evaluate the in vitro and in vivo activity of AZD1480 in NB, RMS and ESFT. Our data indicate that AZD1480 blocks endogenous as well as IL-6 induced STAT3 activation. AZD1480 decreases cell viability in 7/7NB, 7/7RMS and 2/2 ESFT cell lines (median EC50 is 1.5 μM, ranging from 0.36-5.37 μM). AZD1480 induces cell growth inhibition and caspase-dependent apoptosis in vitro and decreases expression of STAT3 target genes, including cell cycle regulators CyclinD1, 3 and CDC25A, anti-apoptotic genes Bcl-2 and survivin, the metastasis-related factor TIMP-1 and c-Myc. In vivo studies showed AZD1480 significantly decreased tumor growth and prolonged overall survival in tumor-bearing mice. Tumors from AZD1480-treated mice showed inhibition of activated STAT3 as well as decreased expression of STAT3 downstream targets. Our study provides strong evidence of the anti-tumor growth potency of JAK inhibitor AZD1480 in pediatric solid tumors, providing proof-of principle that inhibition of the JAK/STAT3 signal transduction could be a promising therapeutic target for high-risk pediatric solid tumors.

Figure 1. The effects of AZD1480 on high-risk pediatric tumor cell lines in vitro

A) Typical growth inhibition curves determined by MTS assays for the NB, RMS and ESFT tumor cell lines compared with normal control ARPE19 and HEK293 cell lines. B) The median EC₅₀ ratio graph shows the relative EC₅₀ values for all the cell lines tested. Each bar represents the ratio of the panel EC₅₀ to the EC₅₀ value of the indicated cell line. Bars to the right represent cell lines with less sensitivity, while bars to the left indicate cell lines with higher sensitivity to AZD1480. C) Effect of AZD1480 on the cell cycle of pediatric solid tumors. Two NB cell lines (KCNR and SY5Y), one RMS cell line (Rh18), one ESFT cell line TC32 and the normal ARPE19 cell line were treated with various concentrations of AZD1480 (0, 0.5, 1 and 2.5 μM) for 72 hours. Cells were stained with propidium iodide and analyzed by flow cytometer. Results are tabulated within the graph and graphically shown in histograms (left). The bars represent the percentages of cells different phases of the cell cycle; sub-G1 (black), G1 (light grey) and the S + G2/M phase (dark grey).

Figure 2. AZD1480 induced caspase3/7-dependent apoptosis

A) Induction of caspase-3/7 activity by AZD1480 in pediatric NB and sarcoma cell lines. Tumor cell lines (KCNR, SY5Y, Rh18 and TC32) and normal cell lines (ARPE19 and HEK293) were treated with different concentrations of AZD1480 (0, 0.5, 1.0 and 2.5 μM) for 24 hours. Caspase-3/7 activity was determined using a Caspase-Glo3/7 Assay Kit. A representative graph of 3 independent experiments is depicted. Data represent mean±SD, of triplicates within 1 experiment (t-test). *, P<0.05 for AZD1480-treated cells vs. vehicle-control cells. B) Rescue of AZD1480-induced cell growth arrest by Z-VAD-FMK. KCNR, SY5Y, Rh18 and TC32 cells were pretreated with Z-VAD-FMK (50 μM) for 3 hours, followed by AZD1480-treatment (0.5 μM for KCNR and SY5Y, 2.5 μM for Rh18 and TC32) for additional 72 hours, or treated with AZD1480 or Z-VAD-FMK alone for 72 hours. MTS assay was used to assess cell survival. Data represent mean±SD of triplicate samples from a representative experiment of 3 independent experiments performed. P values were indicated for AZD1480/ Z-VAD-FMK-co-treated cells vs. AZD1480-treated cells (t-test).

Figure 3. Inhibition of JAK/STAT3 signaling by AZD1480 in pediatric cells in vitro

A and B) AZD1480 blocks endogenous constitutive (A) and IL-6-induced (B) activation of JAK2 and STAT3. Cells were treated with indicated doses of AZD1480 alone for 24 hours (A), or pretreated with IL-6 (10 ng/ml) for 15 minutes, washed with PBS 3 times and then followed by treatment with indicated doses of AZD1480 for 24 hours (B), then lysed and immunoblotted with indicated antibodies. Ratios of p-STAT3/T-STAT3 and p-JAK2/T-JAK2 shown under the representative blots were normalized to that of untreated-control (normalized as “1”) in each cell line. C and D) AZD1480 decreased STAT3-downstream target gene expression. Cells were treated with indicated doses of AZD1480 for 24 hours. C. Real-time PCR was performed for indicated STAT3 target genes. Data represent mean±SD, of 3 independent experiments. *, P<0.05 was indicated for AZD1480-treated cells vs. control cells (t-test). D. Immunoblots were performed as described in Materials and Methods for indicated STAT3-downstream targets. Ratios of the detected target/GAPDH shown under the representative blots was normalized to that of untreated-control (normalized as “1”) in each cell line.

Figure 4. AZD1480 inhibited STAT3 activity and growth of NB, RMS and ESFT xenografts in vivo

Subcutaneous xenografts of NB were established by injection of 2×10⁶ of NB cells (KCNR and SY5Y) into the right flank of 4-6 week-old female nude mice. Orthotopic xenografts of RMS and ESFT were established by injecting 2×10⁷ Rh18 or TC32 cells into the left gastrocnemius muscle of SCID/Beige mice. AZD1480 treatment were initiated when tumors reaches the size at 100-200 mm³. AZD1480 and vehicle were administered daily for up to 3 weeks (30 mg/kg QD for KCNR and SY5Y, 30 mg/kg BID for Rh18 and TC32) by oral garage. Tumor sizes were measured three times a week. A) The graph represents a comparison of mean tumor volumes between control and AZD1480-treated groups in each cell line during the course of AZD1480 or placebo treatment. Data represent mean ± SD. P value between control and AZD1480-treated group in each cell line was determined by a two-way ANOVA. B) Effect of AZD1480 on STAT3 phosphorylation and its downstream targets in tumor xenografts in vivo. Two mice in each group (C=Control group, A=AZD1480-treated group) were killed after 9 doses of AZD1480-treatment in KCNR, SY5Y, Rh18 and TC32, respectively. Tumors were excised and proteins were extracted. Total proteins (15 μg) were analyzed for phosphorylated (p) -STAT3 ^Tyr705, total (T)-STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH by immunoblotting.

Figure 5. Beneficial effect of AZD1480 on the survival of tumor-bearing mice

To evaluate survival in the cohort of mice treated in Fig. 4, the treatments were halted at 3 weeks and tumor xenograft growth was monitored in mice. When tumor xenografts reached a maximal diameter of 2 cm, the mice were euthanized. A) Survival curve was plotted by Kaplan-Meier analysis. P value was calculated using a two-sided long-rank test. B) After mice were sacrificed, tumor samples were frozen for immunoblotting to evaluate the late inhibition effect of AZD1480 on STAT3 activation and its downstream targets.

Figure 6. Histological evaluation by hematoxylin-eosin (HE) staining on SY5Y, KCNR, Rh18 and TC32 xenografts

Tumor tissues from euthanized mice were fixed in formalin and sectioned for histological evaluation by HE staining. The magnification of the representative pictures is ×20 (scale bar =100 μm).