Chemotherapy-induced apoptosis in a transgenic model of neuroblastoma proceeds through p53 induction

Abstract

Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apoptosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM) induced a complete remission in p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induction of the Bcl-2 homology domain-3-only proteins PUMA and Bim, leading to the activation of Bax and cleavage of caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53. Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors. This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apoptosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.

Figure 1

p53 heterozygosity enhances tumorigenesis in TH-MYCN neuroblastoma. TH-MYCN mice were introduced into a p53+/- background by sequential breeding against p53+/- FVBN animals, and cohorts of mice were followed for tumor formation. (A) Kaplan-Meier survival analysis of animal cohorts showing increased tumor penetrance (85% in p53+/-, 60% in p53+/+ animals) and shorter time to tumor onset (70 days of life in p53+/+, 50 days of life in p53+/-). (B) Early-stage focal tumor (T) of adrenal origin (a) on the superior pole of the kidney (K). (C, D) Histologic appearance of representative, large, late-stage p53+/+ and p53+/- TH-MYCN tumors showing a reduction of apoptotic cells with hyperchromatic nuclei on pale backgrounds and necrotic cells with pyknotic nuclei with hyaline staining (white arrows) in p53+/- tumor (D) versus p53+/+ tumor (C), white arrows. The p53+/- tumor is also relatively more monomorphous with densely packed neuroblasts (white arrows).

Figure 2

CPM induces durable remission in mice transgenic for TH-MYCN. Cohorts of transgene-positive animals (seven per arm) were randomly assigned to receive treatment with either control (saline) or CPM (150 mg/kg per day injected intraperitoneally three times per week). Animals were treated at approximately 60 days of life, when tumors were palpable. (A) Survival after treatment (day of life, 60). All treated animals survived to >100 days of life. Saline-treated animals required euthanasia owing to signs of advanced disease at or before 90 days of life. (B) Tumor-free survival after treatment on day of life 60. All CPM-treated animals remained tumor-free, whereas saline-treated animals developed tumors 10 days after treatment (70 days of life). The proportion of saline-treated animals remaining tumor-free is shown on the Y axis, plotted against tumor-free days after treatment on the X axis. (C) At treatment initiation, animals harbored focal tumors (white arrow), with strong E₂F₁-driven luciferase bioluminescence of up to 6 x 10⁵ photons/sec per square centimeters. At autopsy, animals in the control group harbored large tumors (not shown). (D) No tumors or luciferase bioluminescence were detected in treated animals at 100 days of life (white dashed outline).

Figure 3

Proliferation blockade and apoptosis induction in CPM-treated murine neuroblastoma. Tumor-bearing animals were treated with CPM and killed, and tumors were fixed in paraffin. Immunoreactivity of Ki-67 (myb-1) in tumors (three per time point) treated for 0, 6, or 24 hours, respectively. BV indicates blood vessel; K, kidney; T, tumor. (A) Vehicle-treated tumors under low power (4x) show extensive Ki-67 immunoreactivity, reflecting the high proliferative index of such lesions. (B, C) Ki-67 staining is maximal at 6 hours and is notably diminished at 24 hours after CPM, highlighting the responsiveness of this tumor to the drug. (D–G) Cleaved caspase-3 immunostaining in formalin-fixed paraffin-embedded tumors is minimal in vehicle-treated tumors (D), but is rapidly induced at 3 hours (E), is maximal at 6 hours (F), and largely resolves by 24 hours (G) after CPM. Caspase-3 immunoreactivity at 24 hours is greatly reduced and is colocalized to regions of cellular breakdown and nuclear atypia (G). Insets: original magnification, x20. BV indicates blood vessel; K, kidney; T, tumor. (H–I) Quantitation of Ki-67 staining (H) and cleaved caspase-3 staining (I) in the tumor sections shown in D–G at time points after CPM in five high-power fields per section from three separate tumor samples.

Figure 4

In vivo CPM treatment of murine neuroblastoma tumors induces p53 and p53 downstream targets. Tumors were harvested from transgene-positive animals at 0, 3, 6, 12, and 24 hours after therapy with CPM. Lysates were analyzed by immunoblot analysis for levels of apoptosis proteins. (A) Activation of the intrinsic (mitochondrial) apoptotic pathway occurs at 3 hours, with induction of p53 peaking at 6 hours after treatment. Activation of p53 was associated with cleavage of caspases-3 and -9, also peaking at 6 hours. PUMA was induced strongly, with a peak at 3 hours after treatment. (B) To assess the effect of CPM treatment on BH3-family proteins, lysates were probed for the activation of Bim and Bax. Rapid activations of Bax and Bim were coincident. These proteins are downstream targets of PUMA and c-myc, respectively, subject to p53 regulation of the chemotherapy-induced DNA damage response. Poly(ADP-ribose) polymerase cleavage indicates potent induction of apoptosis.

Figure 5

CPM induces apoptosis in MYCN-amplified neuroblastoma cell lines are sensitized to CPM-induced apoptosis and activate p53. Tumor cells were cultured in 10% FBS and treated with 4^OH-CPM at indicated doses for 24 hours. (A) Expression levels of Mycn and p53 protein in cultured cell lines SH-EP (MYCN diploid, no Mycn protein), SH-SY5Y (MYCN diploid, intermediate Mycn protein), and Kelly (MYCN-amplified, high Mycn protein) treated with 4^OH-CPM for 6 hours. (B) To assess the effect of CPM on proliferation, cells were analyzed at 24 hours by water-soluble tetrazolium (WST-1) assay. Kelly cells showed enhanced inhibition across the range of doses tested. Values represent means of triplicate measurements. (C) CPM induces apoptosis in human neuroblastoma cell lines. The same cells were treated for 24 hours, and apoptosis was assessed by nucleosomal DNA ELISA. Apoptosis was more readily evident in the MYCN-amplified Kelly cells. These data suggest that amplification of MYCN sensitizes human neuroblastoma cells to apoptosis induced by CPM and are consistent with observations that amplification of MYCN generally increases the susceptibility of cells to cytotoxic chemotherapy. (D) MYCN-amplified Kelly cells were treated with 20 µM 4^OH-CPM for 24 hours and analyzed by immunoblot analysis for levels of p53, phospho-p53 (S15), and caspase-3 proteins. Induction of caspase-3 cleavage occurs 6 hours after treatment with CPM coincident with maximal p53 induction and phosphorylation. This is consistent with the observations in vivo using TH-MYCN tumors.