Abstract
Background: Neuroblastoma is an embryonal malignancy arising from the aberrant growth of neural crest progenitor cells of the sympathetic nervous system. The tachykinin receptor 1 (TACR1) – substance P complex is associated with tumoral angiogenesis and cell proliferation in a variety of cancer types. Inhibition of TACR1 was recently described to impede growth of NB cell lines. However, the relevance of TACR1 in clinical settings is unknown. Patients and Methods: We investigated gene expression levels of full-length and truncated TACR1 in 59 neuroblastomas and correlated these data with the patients' clinical parameters such as outcome, metastasis, International Neuroblastoma Staging System (INSS) status, MYCN proto-oncogene, bHLH transcription factor (MYCN) status, gender and age. Results: Our results indicated that TACR1 is ubiquitously expressed in neuroblastoma but expression levels are independent of clinical parameters. Conclusion: Our data suggest that TACR1 might serve as a potent anticancer target in a large variety of patients with neuroblastoma, independent of tumor biology and clinical stage.
Neuroblastoma (NB), an embryonal malignancy arising from the aberrant growth of neural crest progenitor cells of the sympathetic nervous system, is the most common solid extracranial tumor found in infancy and childhood (1-3). NB is characterized by its clinical heterogeneity: whereas very young children often demonstrate spontaneous tumor regression, older children frequently suffer from progressive disease with poor outcome. Although recent therapeutic approaches in multimodal therapies demonstrated better overall survival, more than 50% of children with high-risk disease do not respond to modern chemotherapy regimens, resulting in progressive disease. Overall, NB accounts for 12% of all pediatric oncological deaths (1, 3-6).
On the molecular level, several mutations have been discovered for NB. For this and other reasons, NB has served as a paradigm for biological risk assessment and treatment assignment. For example, amplification of MYCN proto-oncogene, bHLH transcription factor (MYCN) and hemizygous deletion of chromosomes 1p and 11q are found in up to 30% of patients with NB and are known to correlate with worse outcome and poor prognosis(2). However, the exact mechanisms of tumor formation and progression are still incompletely understood and other oncogenic drivers of tumorigenesis are still to be discovered (2).
Substance P (SP)–tachykinin receptor 1 (TACR1) (also named neurokinin-1 receptor) complex, which acts as a neuronal transmitter, has been linked to inflammation and cell migration (7). Furthermore, several studies have reported that activation of TACR1 (through binding of SP) is associated with tumoral angiogenesis and cell proliferation (8). Among the three subtypes of tachykinin receptors, TACR1 has the highest affinity for the ligand SP (9).
Two splice variants of TACR1 are known: a full-length variant (fl-TACR1) and a truncated variant (tr-TACR1). The latter lacks 100 amino acids at its C-terminal end, which serves as a substrate for G protein receptor kinases and has been reported to differ in its biological function in interaction with SP (10). As a consequence of splicing, the truncated variant lacks sensitivity for negative feedback inhibition, leading to constant activation of the receptor complex(11). Several studies revealed the importance of tr-TACR1 in cancer (10-13).
Importantly, the SP–TACR1 receptor system was recently described to be a potent anticancer target in NB (14). In the present study, we investigated the role and clinical relevance of fl-TACR1 and tr-TACR1 in tumor samples from children with NB.
Patients and Methods
Patients and tumor tissues. Between 2009 and 2014, 59 patients with NB underwent either biopsy or surgical resection in our Department. Patients were all treated according to the protocol of the German Society of Pediatric Oncology and Hematology (GPOH, study protocol NB2004). For this study, the patients' charts were reviewed retrospectively regarding clinical information: age, gender, International Neuroblastoma Staging System (INSS) status, outcome, metastasis, MYCN status, histology, and extent of surgical resection were obtained and correlated with gene-expression data.
Extent of surgical resection was noted as biopsy only when less than 50% of the tumor mass was removed. An incomplete resection was defined as when more than 50% but less than 89% of the tumor was macroscopically removed. Resection of more than 90% of tumor but still with visible tumor remnants (90-99%) was considered a near-complete resection. Removal of 100% macroscopical tumor mass was considered complete resection (15).
This study was approved by the Institutional Ethics Committee of the University Hospital (LMU Munich; N. 431-11). Written consent was given by the patients' parents for collection of data and laboratory analysis.
RNA extraction and reverse transcription. In essence, samples were treated with TRIzol® reagent for isolation of RNA, according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was dissolved in RNase-free water. Reverse transcription of RNA-samples (2 μg each) was performed utilizing SuperScriptTM II reverse transcriptase (Invitrogen Life Technologies), as recommended by the supplier.
Reverse transcription polymerase chain reaction (RT-PCR). Two microliters of cDNA sample were utilized in each PCR reaction with the following specific primers for fl-TACR1 and tr-TACR1 as well as TATA-box-binding-protein (TBP) housekeeping gene: For fl-TACR1: Forward, 5’-AACCCCATCATCTACTGCTGC-3’ and reverse, 5’-ATTTCCAGCCCCTCATAGTCG-3’ (NM_001058.3); for tr-TACR1 forward, 5’-GGGCCACAAGACCATCTACA-3’ and reverse, 5’-AAGTTAGCTGCAGTCCCCAC-3’ (NM_015727.2); and for TBP: forward, 5’-GCCCGAAACGCCGAATAT-3’ and reverse, 5’-CCGTGGTTCGTGGCTCTCT-3’. Samples for amplification reactions had a final volume of 20 μl. iTaq SYBR-green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) was used for amplification reactions. Samples were incubated at 95°C for 7 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with the final extension cycle performed at 72°C for 7 min. The transcript numbers were normalized according to the expression of the housekeeping gene. Relative quantification of gene expression was performed using the 2 -ΔΔCt method as described by Pfaffl (16).
Statistical analysis. Results are given as the mean±standard error of the mean. Gene-expression levels of tumor samples are displayed as dot plots for each group. Correlation analysis was performed using a Mann–Whitney U-test and a standard t-test. Differences with a p-value of less than 0.05 were considered as statistically significant. Statistical calculations were performed using biostatics software from GraphPAD Prism® (La Jolla, CA, USA).
Kaplan–Meier curves were used for demonstration of overall survival (OS), and two-sided log-rank test was utilized for comparison of survival curves.
Patient characteristics.
Results
TACR1 shows high expression in human NB. Gene-expression profiles of fl-TACR1 and tr-TACR1 were investigated in human NB samples. In general, fl-TACR1 showed a low expression profile, whereas tr-TACR1 demonstrated much higher expression levels in NB samples (Figure 1a). This difference was statistically significant (p<0.0001). However, fl-TACR1 and tr-TACR1 expression did not demonstrate a significant correlation (r=0.2201, p=0.0969) (Figure1b). Furthermore, we performed an analysis of the association between expression of each splice variant (with cutoff as the overall mean expression) and overall prognosis. There was no statistically significant difference for either splice variant (Figure 1c).
Clinical characteristics. The mean patient age at diagnosis was 40.5 months, with a range from 0 months to 19.2 years. Twenty-six patients (44.1%) had metastases at diagnosis. For further patient characteristics, see Table I.
a: Gene-expression of full-length tachykinin receptor 1 (fl)-TACR1 and truncated (tr)-TACR1 in human neuroblastoma samples. b: Correlation between fl-TACR1 and tr-TACR1. c: Association between expression of each splice variant (with cut-off as the overall mean expression) and overall prognosis.
Gene-expression levels of fl-TACR1 and tr-TACR1. Both fl-TACR1 and tr-TACR1 variants as well as their ratio were analyzed for association with clinical and biological findings of NB samples. Neither fl-TACR1 nor tr-TACR1 were significantly associated with histology, age or gender (Figure 2). Clinical parameters such as MYCN status and metastasis, which are linked with aggressive disease, also demonstrated no significant association with tr-TACR1 nor fl-TACR1. Only fl-TACR1 was significantly higher in INSS stage 4 (p=0.0164, Figure 3), but there was no association for tr-TACR1 nor their ratio. As was the case for the other clinical parameters mentioned, the analysis of gene-expression levels did not show any significant association with outcome (Figure 3).
Discussion
An abundance of studies have proven the importance of the SP–TACR1 complex in cancer and disease progression (7, 10). Moreover, during the past decade, TACR1 and its high expression in a variety of cancer types has become the focus of attention for targeted therapy. Tachykinin-receptor antagonists such as aprepitant, currently used as a clinical drug for the treatment of chemotherapy-related nausea and vomiting, in several reports demonstrated in vitro and in vivo efficacy against TACR1, resulting in a robust anticancer effect (12, 14, 17). Similar effects have been observed for other TACR1 antagonists.
Gene-expression of full-length tachykinin receptor 1 (fl)-TACR1 and truncated (tr)-TACR1, and their ratio in human neuroblastoma samples according to histology, age and gender. GN: Ganglioneuroma, G1: gangioneuroblastoma, G2: differentiated neuroblastoma, G3: undifferentiated neuroblastoma, yr: year, F: female, M: male.
Little is known about the role of TACR1 in oncogenesis and tumor progression in NB. Nowicki et al. investigated the cell immunophenotype of metastatic and primary tumors in NB and found, among other nervous tissue markers, high expression of SP in both groups (18). In 2005, Munoz et al. reported TACR1 expression in two NB cell lines (19). Perhaps the most sophisticated study regarding this topic so far was recently published by Henssen et al. The authors examined tachykinin receptors in a panel of NB cell lines. Their results suggest TACR1 expression in all tested cell lines, even though expression levels varied. Our previous studies in hepatoblastoma are consistent with this finding (12, 14). In a second step, growth inhibition was induced with targeting NB cells in vivo and in vitro (mouse xenograft model) with fosaprepitant, a tachykinin receptor antagonist similar to aprepitant. The Authors concluded that gene expression of TACR1 is increased in NB cell lines resulting in expression of this receptor, making it an attractive target for targeted therapy with TACR1 antagonists (14). Interestingly, NB is currently the only solid cancer in childhood for which a positive therapeutic effect has been proven for targeted therapy (via GD2). This understanding raises hope that there could potentially be other targets via which a similarly promising effect can be achieved.
Gene-expression of full-length tachykinin receptor 1 (fl)-TACR1 and truncated (tr)-TACR1, and their ratio in human neuroblastoma samples according to MYCN proto-oncogene, bHLH transcription factor (MYCN) status, presence of metastasis, International Neuroblastoma Staging System (INSS), and outcome. DOD: Died of disease.
In the present study, we demonstrated ubiquitous gene expression of fl-TACR1 and tr-TACR1 in a cohort of children with NB. Our results suggest increased gene expression of tr-TACR1 and low expression of fl-TACR1 in NB tumor tissue. Interestingly, gene expression levels were independent of clinical markers such as INSS stage, MYCN status and histology. These findings are in accordance with what has been found for other tumor types. As was briefly mentioned above, it is generally understood that it is the expression of the truncated splice variant that is dominantly involved in cancer formation (20, 21). This finding strongly correlates with our own data published recently for the expression profile of the SP–TACR1 receptor system in hepatoblastoma, another aggressive childhood cancer (11, 12). Compared to normal liver tissue, we found that gene expression levels of fl-TACR1 and tr-TACR1 were both increased, whereas the level of tr-TACR1 was significantly increased compared to fl-TACR1 (p=0.0301) (11). Similar to the results presented here for NB, in hepatoblastoma, we found no correlation between the expression profile of TACR1 and clinical behavior or disease stage. Yet TACR1 antagonists had a remarkable therapeutic effect on hepatoblastoma growth both in vitro and in vivo.
Our findings presented in this study are furthermore in accordance with those of other studies. Chen et al. examined expression of SP and TACR1 in tissue microarrays of colorectal cancer and adjacent healthy tissue with immunohistochemistry. Expression of SP and TACR1 were both significantly increased in samples of patients with lymph node metastasis; furthermore, high TACR1 expression was also correlated with TNM stage III and IV. Besides these significant correlations, expression of SP and TACR1 was not associated with clinical parameters such as age, sex, gender, distant metastasis and pathological grading (7).
Somewhat different results were found in a recent study in breast cancer (10). The different role of fl- and tr-TACR1 in breast cancer cell lines and breast cancer tumor samples were investigated. The authors found high gene-expression levels of tr-TACR1 in cells of advanced malignancy, whereas fl-TACR1 demonstrated mainly high gene-expression levels in normal breast and mesenchymal cells; in breast cancer cells, gene expression of fl-TACR1 was markedly reduced (10). Moreover, results suggest that fl-TACR1 was inversely correlated with invasiveness, proliferation and metastasis, whereas high expression of tr-TACR1 seemed to affect progressive disease and metastasis. Interestingly, we found similar results when investigating hepatoblastoma (12), however, as mentioned above, we did not find any correlation between disease stage or prognosis for the data presented here in NB nor for hepatoblastoma presented previously.
As all scientific studies, ours has several flaws, potentially biasing our data. Firstly, we only investigated gene-expression levels not protein-expression levels. Just as there are variations in the splice products prior to expression, there could be mRNA abrogation or other mechanisms that alter the expression profile that we did not account for. The solution to this would be immunohistochemical staining of our samples in order to correlate the protein-expression profile with the mRNA profile. Although such staining is generally feasible, it is increasingly challenging given the unavailability of appropriate antibodies against the tr-TACR1 (20). Thirdly, NB is a very heterogeneous tumor. By taking one sample per tumor, the examined areas might not be representative of the whole specimen. Furthermore, we lacked a control group with healthy tissue to compare gene-expression levels simply due to the nature of the tumor type we investigated.
Nevertheless, despite these limitations, we feel that our findings support evidence that NB tissue, similarly to other cancer types, ubiquitously expresses TACR1. This understanding could hold tremendous clinical relevance and make TACR1 an attractive therapeutic target for a large variety of clinical and biological NB subsets. Further research is needed to clarify the exact clinical and biological role of TACR1 in the oncogenesis of childhood NB.
Acknowledgements
The Authors are grateful to Fatemeh Promoli for technical assistance.
- Received August 6, 2017.
- Revision received September 15, 2017.
- Accepted September 19, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
