Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma

Abstract

Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the antiangiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.

Fig. 1

A dual PI3K/mTOR inhibitor leads to decreased tumor burden and improved survival in GEM and primary orthotopic xenograft models of MYCN-driven neuroblastoma. (A) Viability and proliferation screens: MYCN-amplified cells [human primary culture (SFNB-06) and three cell lines (IMR32, Lan-5, and SK-N-BE2)] were sensitive to NVP-BEZ235 compared with nonamplified cells [two human primary cultures (SFNB-05 and SFNB-07) and cell lines (SK-N-SH, SY5Y, and SHEP)]. *P < 0.05, Student’s t test. (B) Tumor-bearing TH-MYCN mice were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage for 28 days. Kaplan-Meier analysis demonstrates that NVP-BEZ235 enhanced mice survival (n = 5 for each group; P < 0.003, log-rank test). Arrow indicates cessation of therapy at 28 days. (C) A heavily pretreated primary MYCN-amplified human tumor (SFNB-06) was established orthotopically, and mice were treated with NVP-BEZ235 or vehicle, as above, for 28 days. NVP-BEZ235 prolonged survival (n = 10 for each group; P < 0.049, log-rank test). Arrow again indicates cessation of therapy at 28 days. (D and E) Tumor burden: tumor-bearing TH-MYCN mice (D) and orthotopic xenografts of SFNB-06 (E) were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage for 14 days. NVP-BEZ235 treatment led to decreased tumor volume and weight in both models (*P < 0.05, Student’s t test).

Fig. 2

A dual PI3K/mTOR inhibitor suppresses both proliferation of tumor cells and angiogenesis in neuroblastoma. (A to H) Tumors from mice transgenic for TH-MYCN were treated with NVP-BEZ235 (35 mg/kg) or PEG300 vehicle once daily by oral gavage, and mice were killed at 14 days of treatment. Endo-thelial cell density (CD31), pericyte density (α-SMA), proliferation of tumor cells (Ki67), and H&E staining were assessed. Scale bars, 50 μm. n = 3 for each arm. (I) Quantification of (A) to (C) and (E) to (H) using ImageJ (*P < 0.0008, Student’s t test). (J to P) Mice carrying MYCN-amplified human orthotopic xenografts (SFNB-06) were treated with NVP-BEZ235 or vehicle, collected, and analyzed as in (A) to (I). (R) Quantification of (J) to (L) and (N) to (P) with ImageJ (*P < 0.0008, Student’s t test). n = 3 for each arm. In all quantitations, vehicle arms were normalized to 100%; NVP-BEZ235 arms were graphed as percent of vehicle.

Fig. 3

NVP-BEZ235 blocks PI3K/mTOR and destabilizes MYCN in vivo. (A) Tumor-bearing mice transgenic for TH-MYCN were treated daily with NVP-BEZ235 or vehicle for 7 or 14 days (same dosage as in Fig. 2). Western analysis demonstrates decreased levels of MYCN protein in tumor lysates from animals treated with NVP-BEZ235 in comparison with control. (B) Western analysis of lysates from human orthotopic tumors in Fig. 1, I to O, confirms expression of MYCN in primary tumor and in vehicle-treated orthotopic xenografts, with decreased levels of MYCN protein in lysates from two different xenografts, treated with NVP-BEZ235 for 14 days. (C) Real-time qRT-PCR: NVP-BEZ235 (7 and 14 days) had no significant effect on levels of MYCN mRNA in the TH-MYCN [from (A)]. P = 0.56 for 7 days, P = 0.75 for 14 days. (D) Real-time qRT-PCR: primary human SFNB-06 tumors treated with vehicle or NVP-BEZ235 for 14 days [from (B)]. P = 0.62, Student’s t test. Vehicle arms were normalized to 100%. NVP-BEZ235 arms were graphed as percent of vehicle.

Fig. 4

MYCN drives paracrine signaling between tumor vascular cells. (A to C) SHEP cells were stably transduced with MYCN^WT or MYCN^T58A. (A) Cycloheximide (CHX) pulse-chase assay: cycloheximide (50 μg/ml) with NVP-BEZ235. MYCN^T58A protein showed a prolonged half-life. (B) Western blot: MYCN^T58A protein was resistant to NVP-BEZ235. (C) Vehicle-treated MYCN^WT and MYCN^T58A were normalized to 100%. NVP-BEZ235–treated arms were graphed as percent change of vehicle (raw data in fig. S3, F to H). Proliferation and viability assays: MYCN^T58A conferred resistance to NVP-BEZ235 versus MYCN^WT (*P < 0.026; **P < 0.05). VEGF ELISA: NVP-BEZ235 suppressed levels of VEGF in MYCN^WT cells, with blunting response in MYCN^T58A cells (***P < 0.002). HUVEC migration: HUVECs (upper chamber) with MYCN^WT or MYCN^T58A cells (bottom chamber). NVP-BEZ235 reduced migration was blunted in MYCN^T58A cocultures (****P < 0.008). (D to F) MYCN-amplified Kelly cells were transduced with shRNA MYCN (Sh-MYCN) and shRNA control vector (Sh-Control). (D and E) Western blot: Sh-MYCN (#3) was more efficient than NVP-BEZ235, without altering p-Akt/p-rpS6. (F) Proliferation and viability: Sh-MYCN recapitulated the effects of NVP-BEZ235 (*P < 0.001; **P < 0.007, Sh-MYCN versus shRNA vehicle; ^#P < 0.04, Sh-MYCN versus NVP-BEZ235). VEGF ELISA: Sh-MYCN and NVP-BEZ235 similarly reduced VEGF (***P < 0.005, Sh-MYCN versus shRNA control; ^##P < 0.04, Sh-MYCN versus NVP-BEZ235). HUVEC migration: Sh-MYCN and NVP-BEZ235 blocked migration (****P < 0.002, Sh-MYCN versus shRNA control; ⁺P < 0.0001, all assays with Sh-MYCN + NVP-BEZ235 versus Sh-Control + vehicle).

Fig. 5

Stabilization of MYCN protein blocks the efficacy of NVP-BEZ235. (A) Kelly MYCN-amplified neuroblastoma cells were stably transduced with three different HUWE1 shRNAs or shRNA control. Western analysis demonstrates that shRNA in lane 2 (Sh-1) knocked down HUWE1 without affecting the PI3K target p-Akt or mTOR target p-rpS6. Sh-1 was used for subsequent experiments. (B) Western blot: HUWE1 knockdown promoted partial resistance to NVP-BEZ235 (lane 4 versus lane 2). (C) Cycloheximide pulse-chase assay: cycloheximide was added over a 2-hour time course. HUWE1 knockdown promoted longer half-life of MYCN versus control. (D) Vehicle-treated arms from both Sh-Control and HUWE1 knockdown lines were normalized to 100%, and NVP-BEZ235–treated arms were graphed as percent change compared to vehicle-treated (original graphs in fig. S9, C to E). Proliferation and viability: with NVP-BEZ235, HUWE1 knockdown cells were resistant versus Sh-Control cells (*P < 0.003; **P < 0.003). VEGF ELISA: NVP-BEZ235 blocks VEGF in Sh-Control, with significant resistance mediated by HUWE1 knockdown (***P < 0.03). HUVEC migration: HUWE1 or Sh-Control cells (lower chamber) with HUVECs (upper chamber). With NVP-BEZ235 treatment, HUWE1 knockdown cells were significantly resistant versus Sh-Control cells (****P < 0.003).

Fig. 6

Tumors derived from HUWE1 knockdown cells were resistant to treatment with NVP-BEZ235 in vivo. (A to S) HUWE1 knockdown (Sh-1) or shRNA control Kelly lines were transplanted orthotopically into renal capsules of nude mice. Daily treatment with NVP-BEZ235 (35 mg/kg) or vehicle was initiated on day 14. Tumors were collected for analysis after 2 weeks of treatment. n = 5 for each arm. Graphs were normalized as in Fig. 4C. (A) Tumor burden (weight and volume): NVP-BEZ235 blocked shRNA control tumors, whereas HUWE1 knockdown tumors showed significant growth despite treatment with NVP-BEZ235 (*P < 0.003, Student’s t test, for difference). (B) Representative images of whole tumors under each condition. White ovals indicate kidney in each sample. Scale bar, 5 mm. (C to R) Histological analysis: CD31 (endothelial cells), Ki67 (proliferating cells), DAPI (nuclei), and H&E. Scale bars, 50 μm. (S) Quantification of data from (C) to (N). Vascular density (CD31) and proliferation (Ki67) in HUWE1 knockdown tumors were resistant to treatment with NVP-BEZ235 relative to shRNA control tumors (**P < 0.003, Student’s t test). n = 3 mice in each arm. Three representative images per mouse were quantified using ImageJ.