Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence

Abstract

Poor prognosis and frequent relapses are major challenges for patients with high-risk neuroblastoma (NB), especially when tumors show MYCN amplification. High-dose chemotherapy triggers apoptosis, necrosis and senescence, a cellular stress response leading to permanent proliferative arrest and a typical senescence-associated secretome (SASP). SASP components reinforce growth-arrest and act immune-stimulatory, while others are tumor-promoting. We evaluated whether metronomic, i.e. long-term, repetitive low-dose, drug treatment induces senescence in vitro and in vivo. And importantly, by using the secretome as a discriminator for beneficial versus adverse effects of senescence, drugs with a tumor-inhibiting SASP were identified.We demonstrate that metronomic application of chemotherapeutic drugs induces therapy-induced senescence, characterized by cell cycle arrest, p21(WAF/CIP1) up-regulation and DNA double-strand breaks selectively in MYCN-amplified NB. Low-dose topotecan (TPT) was identified as an inducer of a favorable SASP while lacking NFKB1/p50 activation. In contrast, Bromo-deoxy-uridine induced senescent NB-cells secret a tumor-promoting SASP in a NFKB1/p50-dependent manner. Importantly, TPT-treated senescent tumor cells act growth-inhibitory in a dose-dependent manner on non-senescent tumor cells and MYCN expression is significantly reduced in vitro and in vivo. Furthermore, in a mouse xenotransplant-model for MYCN-amplified NB metronomic TPT leads to senescence selectively in tumor cells, complete or partial remission, prolonged survival and a favorable SASP.This new mode-of-action of metronomic TPT treatment, i.e. promoting a tumor-inhibiting type of senescence in MYCN-amplified tumors, is clinically relevant as metronomic regimens are increasingly implemented in therapy protocols of various cancer entities and are considered as a feasible maintenance treatment option with moderate adverse event profiles.

Keywords: MYCN-amplified neuroblastoma; NFKB1; metronomic; senescence-associated-secretory-phenotype; topotecan.

Figure 1. Therapy-induced senescence and differential secretion of tumor-promoting factors in MYCN-amplified neuroblastoma cell lines

STA-NB-10 or CLB-Ma cells were cultivated in the presence of 150 or 200 μM HU, 3 or 5 nM CPT, resp., 5 nM TPT or 15 μM BrdU for 10 or 3 weeks, respectively. After re-plating a. EdU-incorporation and b. SA-β-Gal activity was analyzed (n= 3). In (b) right panel representative images of TPT-treated and control STA-NB-10 cells after SA-β-Gal staining are depicted. The 100 μm bar refers to control (CTRL) and TPT-treated cells. c, d. Cell culture supernatants of CTRL or senescent STA-NB-10 (c, d) or CLB-Ma (d) cells were (c) analyzed by cytokine antibody array for 274 cytokines and growth factors. Secreted proteins with higher than 2-fold difference to CTRL are depicted in the heatmap. Red bar highlights a cluster of unfavorable secreted factors strongly up-regulated in BrdUsen cells. (d) Quantification of MCP-3/CCL7, MMP-9, RANTES, VEGFA and PDGF-AA by ELISA or FACS-based-assays, resp., in supernatants of STA-NB-10 and CLB-Ma treated as indicated. RPMI = complete culture medium without cells. n ≥ 3. e. PCA blot of microarray gene expression data: senescent NB-cells cluster together and are distinct from differentiatiated cells. Derived from STA-NB-10 and CLB-Ma untreated control (CTRL) cells, differentiation-inducing all-trans retinoic acid (ATRA, 5 μM) treatment for 10 d, spontaneously occurring senescent F-cells (Fsp), short term-TPT for 5 d (TPTshort) and long-term senescence-inducing CPT, TPT, BrdU or HU treatment. Colored lines represent the top 3 levels of proximity acc. to network analysis derived from Qlucore software. Asterisks indicate statistically significant differences. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.

Figure 2. Secretion of unfavorable factors by BrdU-induced senescent NB cells is dependent on NFKB1/p50

a. Representative Western blot (upper panel) of CTRL and senescent STA-NB-10 and CLB-Ma treated as indicated; densidometric quantification of NFKB1/p50 relative to GAPDH (n = 3; lower panel). b, c. Representative confocal image of NFKB1/p50 or RelA/p65, Phalloidin (F-actin) and DAPI IF-staining of STA-NB-10 cells treated as indicated. d, e. NFKB1 silencing and pharmacological inhibition in BrdUsen cells: STA-NB-10 cells were treated as indicated and transfected with 2 different siRNAs targeting NFKB1 or non-silencing control siRNA or treated for 24 h with 10 μM BMS345541 (IKKβ inhibitor). Cells and supernatants were harvested 48 h after transfection (medium change 24 h post transfection). (d) Upper panel: representative WB; cells treated as indicated. Lower panel: densidometric analysis (n = 3) (e) Quantification of MCP-3/CCL7, RANTES and VEGFA by ELISA (n = 3). Asterisks indicate statistically significant differences compared to CTRL. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; n.s. not significant.

Figure 3. Long-term low-dose TPT-treated senescent NB-cells display senescence-associated markers, down-regulation of MYCN and reduce the proliferation of non-senescent NB cells in co-culture

STA-NB-10 or CLB-Ma cells, resp., were cultivated in the absence (CTRL) or presence of 5 nM TPT for 3 weeks. a. Analysis of DDB by γH2AX IF staining on cytospin preparations of STA-NB-10 cells treated as indicated (bar: 20 μm) and b. quantification by automated imaging. Dashed line in (a) represents the cytoplasmic membrane. c. Representative image scatter plots showing clipped nuclei of p21 (red) and DAPI (blue) IF-stained STA-NB-10 cells. Cutoff MFI p21=30; Insert: Box plot showing p21 mean fluorescence intensity of CTRL and TPT-treated STA-NB-10. box plots show mean, box includes 50 percentile, whiskers 10–90 percentile; d. qRT-PCR (n = 3) and e. representative Western blot analysis for MYCN. Bar diagrams depict mean +/− SEM (n = 3); f. FACS analysis: Mean percentage of CD44+ CTRL and TPT-treated STA-NB-10 (n = 3) and CLB-Ma (n = 5). g, h. After washing, senescent cells were re-plated for co-cultures together with untreated control (CTRL) cells at indicated ratios or as pure cultures. After 7 d of co-culture EdU-incorporation was measured by FACS in the CD44− fraction. (g) Representative FACS plots showing GD2 versus CD44 staining and the gating strategy. (h) Mean % EdU incorporation +/− SEM in the CD44− fraction. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.

Figure 4. Senescence-induction, reduction of MYCN expression and prolonged survival in a xenograft mouse model for aggressive MYCN-amplified NB by continuous low-dose TPT treatment

a. Experimental setup: STA-NB-10 cells were inoculated s.c. in CD1nude mice and treatment was started at a mean tumor size of 0.53 +/− 0.2 cm³ by daily i.p. injection of vector control (CTRL), 1, 0.1, 0.03 or 0.01 mg/kg/d TPT for 2 or 15 weeks, resp. b. Kaplan-Meier graph showing survival of control animals (CTRL) (n = 15), 6 weeks (n = 6) and 15 weeks 0.1 TPT (n = 11) treated animals. p < 0.0001 (log rank test). c. Tumor size and d. SA-β-Gal activity after 5 days or 2 weeks of treatment, resp. (d) Upper panel: no/low 0 – 10%; intermediate 10 – 30%; high 30 – 100% of cells SA-β-Gal positive. Lower panel: representative SA-β-Gal and DAPI-staining of tumor touch preparations from 2 weeks treated xenotransplant tumors. e. CDKN1A (p21^WAF/CIP1) qRT-PCR analysis of CTRL (n = 5) and 0.1 mg/kg/d TPT treated (n = 4) tumors. f. TUNEL-staining on cryo-sections of fresh frozen CTRL and TPT-exposed tumors as indicated; line represents mean +/− SEM TUNEL positive cells/image counted on 5 representative areas/tumor. g. MYCN qRT-PCR analysis of CTRL (n = 6) and 0.1 mg/kg/d TPT treated (n = 7) tumors Box plots show mean relative expression values (RQ), box includes 50 percentile, whiskers 10–90 percentile. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.

Figure 5. Lack of NFKB1 up-regulation and BrdUsen specific secreted factors in low-dose TPT-exposed tumors in vivo

a. Quantification of Ki-67 IF-staining pattern and corresponding cell cycle state in untreated control (CTRL) and 0.1 mg/kg/d TPT-treated tumors (n = 5). b. mRNA expression in 0.1 mg/kg/d TPT exposed tumors over control tumors (each n = 4). Gene sets: left: senescence related genes (sen marker); middle: factors highly secreted exclusively in BrdUsen in vitro (SASP BrdU); right: in TPTsen and BrdUsen in vitro (SASP TPT) c. CD31 IF-staining on frozen tumor sections. Quantification of CD31⁺ vessel length on 30 random images/tumor (mean cumulative vessel length +/− SEM) in tumors treated as indicated. *p ≤ 0.05. d. Model: (i) Metronomic low-dose long-term TPT treatment leads to senescence via p21^WAF/CIP1 without NFKB1/p50 activation. TPTsen cells down-regulate oncogenic MYCN and act tumor-inhibiting in vitro and in vivo. (ii) In contrast, BrdU treatment leads to NFKB1/p50 activation and a tumor-promoting SASP.